MXPA00005122A

MXPA00005122A - Virulence-associated nucleic acid sequences and uses thereof

Info

Publication number: MXPA00005122A
Application number: MXPA/A/2000/005122A
Authority: MX
Inventors: Frederick Ausubel; Howard M Goodman; Laurence G Rahme; Shalina Mahajanmiklos; Manwah Tan; Hui Cao; Eliana Drenkard; John Tsongalis
Original assignee: The General Hospital Corporation
Priority date: 1997-11-25
Filing date: 2000-05-24
Publication date: 2001-07-03

Abstract

Disclosed are bacterial virulence polypeptides and nucleic acid sequences (e.g., DNA) encoding such polypeptides, and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing such polypeptides to screen for antibacterial or bacteriostatic compounds

Description

SEQUENCES OF NUCLEIC ACIDS ASSOCIATED WITH VIRULENCE, AND THEIR USES BACKGROUND OF THE INVENTION This invention relates to nucleic acid molecules, genes and polypeptides that are related to microbial pathogenicity. Pathogens employ a number of genetic strategies to cause infections and, occasionally, diseases in their hosts. The expression of microbial pathogenicity depends on complex genetic regulatory circuits. Knowledge of microbial pathogenicity issues is necessary to understand the mechanisms of pathogenic virulence and the development of new "anti-virulence or anti-pathogenic" agents, which are necessary to combat infection and disease. In a particular example, the opportunistic human pathogen, Pseudomonas aeruginosa, is a ubiquitous, gram-negative bacterium from soil, water and plants (Palleroni, JN In: Bergey's Manual of Systema tic Bacteriology, ed. ., JG Holt, Williams &Wilkins, Baltimore, MD, pp. 141-172, 1984). A variety of P. aeruginosa factors have been described and most of these, such as exotoxin A, elastase, and phospholipase C, were first detected biochemically on the basis of their cytotoxic activity (Fink, RB, Pseudomonas aeruginosa the Opportunist: Pa thogenesis and Disease, Boca Raton, CRC Press Inc., 1993). Subsequently, the genes that correspond to these factors or genes that regulate the expression of these factors were identified. In general, most of the genes related to pathogenicity in mammalian bacterial pathogens were first detected using a bioassay. In contrast to mammalian pathogens, simple systematic genetic strategies have been systematically employed to identify genes related to pathogenicity in plant pathogens. Following the random transposon-mediated mutagenesis, thousands of mutant clones of the phytopathogen within the plants are inoculated separately to determine if they contain a mutation that affects the pathogenic interaction with the host (Bouer et al., J. Bacteriol. 5626-5623, 1987, Comai and Kosuge, J. Bacteriol 149: 40-46, 1982, Lindgren et al, J. Bacteriol 168: 512-522, Rahme et al., J. Bacteriol 173: 575-586, 1991; Willis et al., Mol. Plant-Microbe Interact., 3: 149-156, 1990). Affordable experiments using models of mammalian pathogenicity in the whole animal are not feasible, due to the vast number of animals that must undergo pathogenic attack. SUMMARY OF THE INVENTION We have identified and characterized a number of nucleic acid molecules, polypeptides, and small molecules (e.g., phenazines), which is involved in the conference of pathogenicity and virulence to a pathogen. This discovery therefore provides a basis for drug screening assays that address the evaluation and identification of "anti-virulence" agents, which can block the pathogenicity and virulence of a pathogen, for example, by means of selectively connecting or disconnecting the expression of genes from the pathogen, or deactivating or inhibiting the activity of a polypeptide that is involved in the pathogenicity of a microbe. Drugs that target these molecules are useful as these anti-virulence agents. In one aspect, the invention features an isolated nucleic acid molecule that includes a sequence substantially identical to any of BI48 (SEQ ID NO: 1), ORF2 (SEQ ID NO: 2), ORF3 (SEQ ID NO: 4), ORF602c (SEQ ID NO: 6), ORF214 (SEQ ID NO: 8), ORFI242c (SEQ ID NO: 10), ORF594 (SEQ ID NO: 12), ORFI040 (SEQ ID NO: 14), ORFl640c (SEQ ID NO: 16), ORF2228c (SEQ ID NO: 18), ORF2068C (SEQ ID NO: 20), ORF1997 (SEQ ID NO: 22), ORF2558c (SEQ ID NO: 24), ORF2929c (SEQ ID NO: 26), ORF3965c (SEQ ID NO: 28), ORF3218 (SEQ ID NO: 30), ORF3568 (SEQ ID NO: 32), ORF4506c (SEQ ID NO: 34), ORF3973 (SEQ ID NO: 36), ORF4271 (SEQ ID NO: 38), ORF4698 (SEQ ID NO: 40), ORF5028 (SEQ ID NO: 42), ORF5080 (SEQ ID NO: 44), ORF6479c (SEQ ID NO: 46), ORF5496 (SEQ ID NO: 48), ORF5840 (SEQ ID NO: 50), ORF5899 (SEQ ID NO: 52), ORF6325 (SEQ ID NO: 54), ORF7567c (SEQ ID NO: 56), ORF7180 (SEQ ID NO: 58), ORF7501 (SEQ ID NO: 60), ORF7584 (SEQ ID NO: 62), ORF8208c (SEQ ID NO: 64), ORF8109 (SEQ ID NO: 66), ORF9005c (SEQ ID NO: 68), ORF8222 (SEQ ID NO: 7 0), ORF8755c (SEQ ID NO: 72), ORF9431c (SEQ ID NO: 74), ORF9158 (SEQ ID NO: 76), ORFI0125C (SEQ ID NO: 78), ORF9770 (SEQ ID NO: 80), ORF9991 (SEQ ID NO: 82), ORF1 0765c (SEQ ID NO: 84), ORF10475 (SEQ ID NO: 86), ORF11095C (SEQ ID NO: 88), ORF11264 (SEQ ID NO: 90), ORF11738 (SEQ ID NO. : 92), ORFl2348c (SEQ ID NO: 94), ORF12314c (SEQ ID NO: 96), ORF13156c (SEQ ID NO: 98), ORF12795 (SEQ ID NO.100), ORF13755c (SEQ ID NO: 210), OR .F13795c (SEQ ID NO: 212), ORFl4727c (SEQ ID N0.214), ORF13779 (SEQ ID NO: 216), ORFl4293c (SEQ ID NO: 218), ORF14155 (SEQ ID NO: 220), ORF14360 (SEQ ID NO: 222), ORF15342c (SEQ ID NO: 224), ORFl5260c (SEQ ID NO: 226), ORF14991 (SEQ ID NO: 228), ORF15590c (SEQ ID NO: 230), ORF15675c (SEQ ID NO: 232), ORF16405 (SEQ ID NO: 234), ORF16925 (SEQ ID NO: 236), ORF17793c (SEQ ID NO: 238), ORFl8548c (SEQ ID NO: 240), ORF17875 (SEQ ID NO: 242), ORF18479 (SEQ ID NO: 244), ORF19027c (SEQ ID NO: 246), ORFI9305 (SEQ ID NO: 248), ORF19519 (SEQ ID NO: 250), ORF19544 (SEQ ID NO: 252), ORF20008 (SEQ ID NO: 254), ORF20623c (SEQ ID NO: 256), ORF21210c (SEQ ID NO: 258), ORF21493c (SEQ ID NO: 260), ORF21333 (SEQ ID NO: 262), ORF22074c (SEQ ID NO: 264), ORF21421 (SEQ ID NO: 266), ORF22608c (SEQ ID NO: 268), ORF22626 (SEQ ID NO: 270), ORF23228 (SEQ ID NO: 272), ORF23367 (SEQ ID NO: 274), ORF25103c (SEQ ID NO: 276), ORF23556 (SEQ ID NO: 278), ORF26191c (SEQ ID NO: 280), ORF23751 (SEQ ID NO: 282), ORF24222 (SEQ ID NO: 284), ORF24368 (SEQ ID NO: 286), ORF24888c (SEQ ID NO: 288), ORF25398c (SEQ ID NO: 290), ORF25892c (SEQ ID NO: 292), ORF25110 (SEQ ID NO: 294), ORF25510 (SEQ ID NO: 296), ORF26762c (SEQ ID NO: 298), ORF26257 (SEQ ID NO: 300), ORF26844c (SEQ ID NO: 302), ORF26486 (SEQ ID NO: 304), ORF26857c (SEQ ID NO: 306), ORF27314c (SEQ ID NO: 308), ORF27730c (SEQ ID NO: 310), ORF26983 (SEQ ID NO: 312), ORF28068c (SEQ ID NO: 314), ORF27522 (SEQ ID NO: 316), ORF28033C (SEQ ID NO: 318), ORF29701c (SEQ ID NO: 320), ORF28118 (SEQ ID NO: 322), ORF28129 (SEQ ID NO: 324), ORF29709c (SEQ ID NO: 326), ORF29189 (SEQ ID NO: 328), ORF29382 (SEQ ID NO: 330), ORF30590C (SEQ ID NO: 332), ORF29729 (SEQ ID NO: 334), ORF30221 (SEQ ID NO: 336), ORF30736c (SEQ ID NO: 338), ORF30539 (SEQ ID NO: 340), ORF31247c (SEQ ID NO: 342), ORF31539c (SEQ ID NO: 346), ORF31222 (SEQ ID NO: 348), ORF31266 (SEQ ID NO: 350), ORF31661C (SEQ ID NO: 352), ORF32061c (SEQ ID NO: 354), ORF32072c (SEQ ID NO: 356), ORF31784 (SEQ ID NO: 358), ORF32568c (SEQ ID NO: 360), ORF33157C (SEQ ID NO: 362), ORF32539 (SEQ ID NO: 364), ORF33705C (SEQ ID NO: 366), ORF32832 (SEQ ID NO: 368), ORF33547c (SEQ ID NO: 370), ORF33205 (SEQ ID NO: 372), ORF33512 (SEQ ID NO: 374), ORF33771 (SEQ ID NO: 376), ORF34385c (SEQ ID NO: 378), ORF33988 (SEQ ID NO: 380), ORF34274 (SEQ ID NO: 382), ORF34726c (SEQ ID NO: 384), ORF34916 (SEQ ID NO: 386), ORF35464c (SEQ ID NO: 388), ORF35289 (SEQ ID NO: 390), ORF35410 (SEQ ID NO: 392), ORF35907C (SEQ ID NO: 394), ORF35534 (SEQ ID NO: 396), ORF35930 (SEQ ID NO: 398), ORF36246 (SEQ ID NO: 400), ORF26640c (SEQ ID NO: 402), ORF36739 (SEQ ID NO: 404), ORF37932c (SEQ ID NO: 406), ORF38640c (SEQ ID NO: 408), ORF39309c (SEQ ID NO: 410), ORF38768 (SEQ ID NO: 412), ORF40047c (SEQ ID NO: 414), ORF40560c (SEQ ID NO: 416), ORF40238 (SEQ ID NO: 418), ORF40329 (SEQ ID NO: 420), ORF40709c (SEQ ID NO: 422), ORF40507 (SEQ ID NO: 424), ORF41275c (SEQ ID NO: 426), ORF42234c (SEQ ID NO: 428), ORF41764c (SEQ ID NO: 430), ORF41284 (SEQ ID NO: 432), ORF41598 (SEQ ID NO: 434), ORF42172c (SEQ ID NO: 436), ORF42233c (SEQ ID NO: 438), 33A9 (SEQ ID NO: 102,189,190,191, 192,193,194,195,196,197, and 198), 34B12 (SEQ ID NO: 104), 34BI2-ORF1 (SEQ ID NO: 105), 34B12-ORF2 (SEQ ID NO: 106";, 36A4 (SEQ ID NO: 109), 36A4 contig (SEQ ID NO: lli;, 23A2 (SEQ ID NO: 112, 3E8 phn (-) (SEQ ID NO: 114), 3E8 with tigPAOl (SEQ ID NO: 115;, 34H4 (SEQ ID NO: 118;, 33C7 (SEQ ID N0.119), 25 to 2.

(SEQ ID NO: 120;, 8C12 (SEQ ID NO: 121), 2A8 (SEQ ID NO: 122, 41A5 (SEQ ID N0.123), 50E12 (SEQ ID NO: 124), 35A9 (SEQ ID NO: 125), ph? 23 (SEQ ID NO: 126), 16G12 (SEQ ID NO: 127), 25F1 (SEQ ID NO: 128), PA14 degP (SEQ ID NO: 131), 1126 contig (SEQ ID NO: 135), contig 1344 (SEQ ID N0.136), ORFA (SEQ ID NO: 440), ORFB (SEQ ID NO: 441), ORFC (SEQ ID NO: 442), phzR (SEQ ID NO: 164, and 1G2 (SEQ ID NO: 137). Preferably, the isolated nucleic acid molecule includes any of the sequences described above or a fragment thereof; and is derived from a pathogen (e.g., a bacterial pathogen such as Pseudomonas aeruginosa). Additionally, the invention includes a vector and a cell, each of which includes at least one of the isolated nucleic acid molecules of the invention; and a method for producing a recombinant polypeptide that includes providing a cell that was transformed with a nucleic acid molecule of the invention positioned for expression in the cells, culturing the transformed cell under conditions to express the nucleic acid molecule, and isolating a recombinant polypeptide. The invention further presents recombinant polypeptides that were produced by the expression of an isolated nucleic acid molecule of the invention, and substantially pure antibodies that specifically recognize and bind to those recombinant polypeptides. In another aspect, the invention features a substantially pure polypeptide that includes an amino acid sequence that is substantially identical to the amino acid sequence of any of ORF2 (SEQ ID NO: 3), ORF3 (SEQ ID NO: 5), ORF602c (SEQ ID NO: 7), ORF214 (SEQ ID NO: 9), OR. F1242c (SEQ ID NO: li;, ORF594 (SEQ ID NO: 13), ORF1040 (SEQ ID NO: 15), ORF1640c (SEQ ID NO: 17), ORF2228c (SEQ ID NO: 19), ORF2068c (SEQ ID NO. : 2 1), ORF1997 (SEQ ID NO: 23), ORF2558c (SEQ ID NO: 25), ORF2929c (SEQ ID NO: 27), ORF3965C (SEQ ID NO: 29), ORF3218 (SEQ ID NO: 31), ORF3568 (SEQ ID NO: 33), ORF4506c (SEQ ID NO: 35), ORF3973 (SEQ ID NO: 37), ORF4271 (SEQ ID NO: 39), ORF4698 (SEQ ID NO: 41), ORF5028 (SEQ ID NO: 43), ORF5080 (SEQ ID NO: 45), ORF6479c (SEQ ID NO: 47), ORF5496 (SEQ ID NO: 49), ORF5840 (SEQ ID NO: 51), ORF5899 (SEQ ID NO: 53), ORF6325 (SEQ ID NO: 55), ORF7567c (SEQ ID NO: 57), ORF7180 (SEQ ID NO. -59), ORF7501 (SEQ ID NO: 61), ORF7584 (SEQ ID NO: 63), ORF8208c (SEQ ID NO: 65), ORF8109 (SEQ ID NO: 67), ORF9005c (SEQ ID NO: 69), ORF8222 (SEQ ID NO: 71), ORF8755c (SEQ ID NO: 73), 0RF9431c (SEQ ID NO: 75), ORF9158 (SEQ ID NO: 77), ORF10125c (SEQ ID NO: 79), ORF9770 (SEQ ID NO: 81), ORF9991 (SEQ ID NO: 83), ORF10765c (SEQ ID NO: 85), ORF10475 (SEQ ID NO: 87), ORFll095c (SEQ ID NO: 89), ORF11264 (SEQ ID NO: 91), ORF11738 (SEQ ID NO: 93), ORF12348c (SEQ ID NO: 95), ORF12314C (SEQ ID NO: 97), ORF13156c (SEQ ID NO: 99), ORF12795 (SEQ ID NO: 101), ORF13755c ( SEQ ID NO: 211), ORF13795C (SEQ ID NO: 213), ORF14727c (SEQ ID N0.215), ORF13779 (SEQ ID NO: 217), ORF14293c (SEQ ID NO: 219), ORF14155 (SEQ ID NO: 221), ORF14360 (SEQ ID NO: 223), ORF15342c (SEQ ID NO: 225), ORF15260c (SEQ ID NO: 227), ORF14991 (SEQ ID NO: 229), ORFl5590c (SEQ ID NO: 23 1), ORFl5675c (SEQ ID NO: 233), ORF16405 (SEQ ID NO: 235), ORF16925 (SEQ ID NO: 237), ORF17793c (SEQ ID NO: 239), ORF18548c (SEQ ID NO: 241), ORF17875 (SEQ ID NO: 243), ORF18479 (SEQ ID NO: 245), ORF19027c (SEQ ID NO: 247), ORF19305 (SEQ ID NO: 249), ORF19519 (SEQ ID NO: 251), ORF19544 (SEQ ID NO: 253), ORF20008 (SEQ ID NO: 255), ORF20623C (SEQ ID NO: 257), ORF21210c (SEQ ID NO: 259), ORF21493c (SEQ ID NO: 26 1), ORF21333 (SEQ ID NO: 263), ORF22074c (SEQ ID NO: 265), ORF21421 (SEQ ID NO: 267), ORF22608c (SEQ ID NO: 269), ORF22626 (SEQ ID NO. : 271), ORF23228 (SEQ ID NO: 273), ORF23367 (SEQ ID NO: 275), ORF25103c (SEQ ID NO: 277), ORF23556 (SEQ ID NO: 279), ORF26191C (SEQ ID N0.281), ORF23751 (SEQ ID NO: 283), ORF24222 (SEQ ID NO: 285), ORF24368 (SEQ ID NO: 287), ORF24888c (SEQ ID NO: 289), 0RF25398c (SEQ ID NO: 291), ORF25892c (SEQ ID NO: 293), ORF25110 (SEQ ID NO: 295), ORF25510 (SEQ ID NO: 297), ORF26762c (SEQ ID NO: 299), ORF26257 (SEQ ID NO: 301), ORF26844c (SEQ ID NO: 303), ORF26486 (SEQ ID NO: 305), ORF26857c (SEQ ID NO: 307), ORF27314c (SEQ ID NO: 309), ORF27730c (SEQ ID NO: 311), ORF26983 (SEQ ID N0.313), ORF28068c (SEQ ID NO: 315), 0RF27522 (SEQ ID N0.317), ORF28033c (SEQ ID NO: 319), ORF29701c (SEQ ID NO: 321), ORF28118 (SEQ ID NO: 323), ORF28129 (SEQ ID NO: 325), ORF29709c (SEQ ID NO: 327), ORF29189 (SEQ ID NO: 329), ORF29382 (SEQ ID NO: 331), ORF30590C (SEQ ID NO: 333), ORF29729 (SEQ ID NO: 335), ORF30221 (SEQ ID NO: 337), ORF30736c (SEQ ID NO: 339), ORF30539 (SEQ ID NO: 341), ORF31247c (SEQ ID NO: 343), ORF30963c (SEQ ID NO: 345), ORF31539c (SEQ ID NO: 347), ORF31222 (SEQ ID NO: 349), ORF31266 (SEQ ID NO: 351), ORF31661c (SEQ ID NO: 353), ORF32061c (SEQ ID NO: 355), ORF32072c (SEQ ID NO: 357), ORF31784 (SEQ ID NO: 359), ORF32568c (SEQ ID NO: 361), ORF33157c (SEQ ID NO: 363), ORF32530 (SEQ ID NO: 365), ORF33705c (SEQ ID NO: 367), ORF32832 (SEQ ID NO: 369), ORF33547c (SEQ ID NO: 371), ORF33205 (SEQ ID NO: 373), ORF33512 (SEQ ID NO: 375), ORF33771 (SEQ ID NO: 377), ORF34385c (SEQ ID NO: 379), ORF33988 (SEQ ID NO: 381), ORF34274 (SEQ ID NO: 383), ORF34726c (SEQ ID NO: 385), ORF34916 (SEQ ID NO: 387), ORF35464c (SEQ ID NO: 389), ORF35289 (SEQ ID NO: 391), ORF35410 (SEQ ID NO: 393), ORF35907c (SEQ ID NO: 395), ORF35534 (SEQ ID NO: 397), ORF35930 (SEQ ID NO: 399), ORF36246 (SEQ ID NO: 401), ORF26640C (SEQ ID NO: 403), ORF36769 (SEQ ID NO: 405), 0RF37932c (SEQ ID NO: 407), ORF38640c (SEQ ID NO: 409), ORF39309c (SEQ ID NO: 41 1), ORF38768 (SEQ ID N0.413), ORF40047c (SEQ ID NO: 415), ORF40560c (SEQ ID NO: 417), ORF40238 (SEQ ID NO: 419), ORF40329 (SEQ ID NO: 421), ORF40709c (SEQ ID NO: 423), ORF40507 (SEQ ID NO: 425), ORF41275c (SEQ ID NO: 427), ORF42234c (SEQ ID NO: 429), ORF41764c (SEQ ID NO: 43 1), ORF41284 (SEQ ID NO: 433), ORF41598 (SEQ ID NO: 435), ORF42172c (SEQ ID NO: 437), ORF42233c (SEQ ID NO.- 439), 33A9 (SEQ ID NOS: 103, 199, 200, 201, 202, 203, 204, 205, 206, 207, and 208), 34BI2-ORF1 (SEQ ID NO: 207;, 34B12-0RF2 (SEQ ID NO: 108), 36A4 (SEQ ID NO: 110), 3E8phzA (SEQ ID NO .- 11 6), 3E8phzB (SEQ ID NO: 117), PhzR (SEQ ID NO: 165), ORFA (SEQ ID NO: 443), ORFB (SEQ ID NO: 444), ORFC (SEQ ID NO: 445), and PA14degP (SEQ ID NO: 132). Preferably, the substantially pure polypeptide includes any of the sequences described above or a fragment thereof; and is derived from a pathogen (e.g., from a bacterial pathogen such as Pseudomonas aeruginosa). In yet another related aspect, the invention features a method for identifying a compound that can decrease the expression of a pathogenic virulence factor (e.g., at transcriptional or post-transcriptional levels), which includes (a) providing a pathogenic cell that expressing any of the isolated nucleic acid molecules of the invention; and (b) contacting the pathogenic cell with a candidate compound, a decrease in the expression of the nucleic acid molecule after contact with the candidate compound that identifies a compound, which decreases the expression of a pathogenic virulence factor. In preferred embodiments, the pathogenic cell infects a mammal (e.g., a human) or a plant. In yet another related aspect, the invention features a method for identifying a compound which binds a polypeptide, which includes (a) contacting a candidate compound with a substantially pure polypeptide that includes any of the amino acid sequences of the invention, conditions that allow fixation; and (b) detecting the binding of the candidate compound to the polypeptide. In addition, the invention provides a method for treating a pathogenic infection in a mammal, which includes (a) identifying a mammal that has a pathogenic infection.; and (b) administering to the mammal a therapeutically effective amount of a composition that inhibits the expression of a polypeptide encoded by any of the nucleic acid molecules of the invention. In the preferred embodiments, the pathogen is Pseudomonas aeruginosa. In yet another aspect, the invention features a method for treating a pathogenic infection in a mammal, which includes (a) identifying a mammal having a pathogenic infection; and (b) administering to the mammal a therapeutically effective amount of a composition that binds and inhibits a polypeptide encoded by any of the amino acid sequences of the invention. In the preferred embodiments, the pathogenic infection is caused by Pseudomonas aeruginosa. In addition, the invention features a method for identifying a compound which inhibits the virulence of a Pseudomonas cell that includes (a) providing a Pseudomonas cell; (b) contacting the cell with a candidate compound; and (c) detecting the presence of a phenazine, wherein a decrease in phenazine relative to the untreated control culture, is an indication that the compound inhibits the virulence of the Pseudomonas cell. In the preferred embodiments, the cell is Pseudomonas aeruginosa; the cell is present in a cell culture, and phenazine is detected by spectroscopy (for example, pyocyanin is detected at an absorbance of 520 nm). The pyocyanin is generally detected in accordance with any standard method, for example, those described herein. By "isolated nucleic acid molecule" is meant a nucleic acid (eg, a DNA) that is free of genes which, in the naturally occurring genome of the organism from which the nucleic acid molecule is derived from invention, flank the gene. Therefore, the term includes, for example, a recombinant DNA that is incorporated within a vector; within a plasmid or virus that doubles autonomously; or within a genomic DNA of a prokaryote or a eukaryote; or that it exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment that is produced by polymerase chain reaction or restriction endonuclease digestion), independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA which is part of an additional polypeptide sequence encoding a hybrid gene. By "polypeptide" is meant any chain of amino acids, regardless of length or posttranslational modification (eg, glycosylation or phosphorylation). By "substantially pure polypeptide" is meant a polypeptide of the invention that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it has at least 60 weight percent, free of the naturally occurring proteins and organic molecules with which it is naturally associated. Preferably, the preparation is at least 75 percent, preferably 90 percent, and most preferably at least 99 percent by weight, a polypeptide of the invention. A substantially pure polypeptide of the invention can be obtained, for example, by extraction from a natural source (e.g., a pathogen); by expressing a recombinant nucleic acid encoding that polypeptide; or by means of chemically synthesizing the protein. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by high performance liquid chromatography analysis. By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 25 percent identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, this sequence is at least 30 percent, 40 percent, 50 percent, 60 percent, more preferably 80 percent and most preferably 90 percent or up to 95 percent identical to the amino acid or acid level nucleic with the sequence used for the comparison. Sequence identity is typically measured using sequence analysis software (eg, Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705, programs CLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX). This software matches identical or similar sequences by assigning degrees of homology to different substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, theonin; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability hit between e-3 and e-100 indicating a closely related sequence. By "transformed cell" is meant a cell within which (or within whose ancestor) a molecule encoding (as used herein) has been introduced, by means of recombinant DNA techniques, a polypeptide of the invention . By "set for expression" it is meant that the DNA molecule is placed adjacent to a DNA sequence which directs the transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention , or an RNA molecule). By "purified antibody" is meant the antibody that is at least 60 percent by free weight of naturally occurring proteins and organic molecules with which it is naturally associated. Preferably, the preparation is at least 75 percent, more preferably 90 percent, and most preferably at least 99 percent, by weight, antibody. A purified antibody of the invention can be obtained, for example, by affinity chromatography using a recombinantly produced polypeptide of the invention and standard techniques. By "specifically fixed" is meant a compound or antibody, which recognizes and binds a polypeptide of the invention, but which does not substantially recognize or bind other molecules in a sample, eg, a biological sample, which includes natural a polypeptide of the invention. By "derived form" is meant isolated from, or having the sequence of a sequence that occurs naturally (e.g., a cDNA, genomic, synthetic, or a combination thereof). By "inhibition of a pathogen" is meant the ability of a candidate compound to decrease, suppress, attenuate, diminish, or arrest the development or progress of a pathogen-mediated disease or an infection in a eukaryotic host organism. Preferably, this inhibition decreases pathogenicity by at least 5 percent, more preferably by at least 25 percent, and more preferred by at least 50 percent, compared to symptoms in the absence of the candidate compound in any appropriate pathogenicity assay (e.g., those assays described herein). In a particular example, inhibition can be measured by monitoring pathogenic symptoms in a host organism that is exposed to a candidate compound or extract, a decrease in the level of symptoms relative to the level of pathogenic symptoms in an organism. host that was not exposed to the compound indicating the inhibition mediated by the compound of the pathogen. By "pathogenic virulence factor" is meant a cellular component (e.g., a protein such as a transcription factor, as well as the gene encoding that protein), without which the pathogen is unable to cause the disease or infection in a eukaryotic host organism. The invention provides a number of targets that are useful for the development of drugs that specifically block the pathogenicity of a microbe. In addition, the methods of the invention provide an easy means to identify compounds that are safe for use in eukaryotic host organisms (i.e., compounds that do not adversely affect the normal development and physiology of the organism), and effective against microbes pathogenic (ie, by suppressing the virulence of a pathogen). In addition, the methods of the invention provide a route to analyze virtually any number of compounds for an anti-virulent effect with a high volume yield, high sensitivity, and low complexity. The methods are also relatively inexpensive to perform and facilitate the analysis of small amounts of active substances that are in the form of either purified or crude extract. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Detailed Description First, the drawings will be described. Drawings Figure 1 is a schematic diagram showing the physical map of cosmid BI48 (SEQ ID NO: l) and the orientation of identified open reading frames (ORFs). Figure 2 shows the nucleotide sequence of cosmid BI48 (SEQ ID NO: 1). Figure 3 shows the nucleotide sequences for ORF2 (SEQ ID NO: 2), ORF3 (SEQ ID NO: 4), ORF602c (SEQ ID NO: 6), ORF214 (SEQ ID NO: 8), ORFI242c (SEQ ID NO: 10), ORF594 (SEQ ID NO: 12), ORF1040 (SEQ ID NO: 14), ORF1640c (SEQ ID NO: 16), ORF2228c (SEQ ID NO: 18), ORF2068c (SEQ ID NO: 20), ORF1997 (SEQ ID NO: 22), ORF2558c (SEQ ID NO: 24), ORF2929c (SEQ ID NO: 26), ORF3965c (SEQ ID NO: 28), ORF3218 (SEQ ID NO: 30), ORF3568 (SEQ ID NO: 32), ORF4506c (SEQ ID NO: 34), ORF3973 (SEQ ID NO: 36), ORF4271 (SEQ ID NO: 38), ORF4698 (SEQ ID NO: 40), ORF5028 (SEQ ID NO: 42), ORF5080 (SEQ ID NO: 44), ORF6479c (SEQ ID NO: 46), ORF5496 (SEQ ID NO: 48), ORF5840 (SEQ ID NO: 50), ORF5899 (SEQ ID NO: 52), ORF6325 (SEQ ID NO: 54), ORF7567c (SEQ ID NO: 56), ORF7180 (SEQ ID NO: 58), ORF7501 (SEQ ID NO: 60), ORF7584 (SEQ ID NO: 62), ORF8208c (SEQ ID NO: 64), ORF8109 (SEQ ID NO: 66), ORF9005c (SEQ ID NO: 68), ORF8222 (SEQ ID NO: 70), ORF8755c (SEQ ID NO: 72), ORF9431c (SEQ ID NO: 74), ORF9158 (SEQ ID NO: 76), ORF10125c (SEQ ID NO: 78), ORF9770 (SEQ ID NO: 80), ORF9991 (SEQ ID NO: 82), ORF10765c (SEQ ID NO: 84), ORF10475 (SEQ ID NO: 86), ORF11095c (SEQ ID NO: 88), ORF11264 (SEQ ID NO: 90), ORF11738 (SEQ ID NO: 92), ORF12348c (SEQ ID NO: 94), ORF12314c (SEQ ID NO: 96), ORF13156c ( SEQ ID NO: 98), ORF12795 (SEQ ID NO: 100), ORF13755c (SEQ ID NO: 210), ORF13795c (SEQ ID NO: 212), ORFl4727c (SEQ ID NO: 214), ORF13779 (SEQ ID NO: 216), ORF14293c (SEQ ID N0.218), ORF14155 (SEQ ID NO: 220), ORF14360 (SEQ ID NO: 222), ORF15342c (SEQ ID NO: 224), ORF15260c (SEQ ID NO: 226), ORF14991 (SEQ ID NO: 228), ORF15590c (SEQ ID NO: 230), ORF15675c (SEQ ID NO: 232), ORF16405 (SEQ ID NO: 234), ORF16925 (SEQ ID NO: 236), ORF17793c (SEQ ID NO: 238), ORF18548c (SEQ ID NO: 240), ORF17875 (SEQ ID NO: 242), ORF18479 (SEQ ID NO: 244), ORF19027c (SEQ ID NO: 246), ORF19305 (SEQ ID NO: 248), ORF19519 (SEQ ID NO: 250), ORF19544 (SEQ ID NO: 252), ORF20008 (SEQ ID NO: 254), ORF20623c (SEQ ID NO: 256), ORF21210c (SEQ ID NO: 258), ORF21493c (SEQ ID NO: 260), ORF21333 (SEQ ID NO: 262), ORF22074c (SEQ ID NO: 264), ORF21421 (SEQ ID NO: 266), ORF22608c (SEQ ID NO: 268), ORF22626 (SEQ ID NO: 270), ORF23228 (SEQ ID NO: 272), ORF23367 (SEQ ID NO: 274), ORF25103c (SEQ ID NO: 276), ORF23556 (SEQ ID NO: 278), ORF26191c (SEQ ID NO: 280), ORF23751 (SEQ ID NO: 282), ORF24222 (SEQ ID NO: 284), ORF24368 (SEQ ID NO: 286), ORF24888c (SEQ ID NO: 288), ORF25398c (SEQ ID NO: 290), ORF25892c (SEQ ID NO: 292), ORF25110 (SEQ ID NO: 294), ORF25510 (SEQ ID NO: 296), ORF26762c (SEQ ID NO.-298), ORF26257 (SEQ ID NO: 300), ORF26844c (SEQ ID NO: 302), ORF26486 (SEQ ID NO: 304), ORF26857c (SEQ ID NO: 306), ORF27314c (SEQ ID NO: 308), ORF27730c (SEQ ID NO.310), ORF26983 (SEQ ID TsTO: 312), ORF28068c (SEQ ID NO: 314), ORF27522 (SEQ ID N0.316), ORF28033C (SEQ ID NO: 318), ORF29701c (SEQ ID NO: 320), ORF28118 (SEQ ID NO: 322), ORF28129 (SEQ ID NO: 324), ORF29709c (SEQ ID NO: 326), ORF29189 (SEQ ID NO: 328), ORF29382 (SEQ ID NO: 330), ORF30590c (SEQ ID NO: 332), ORF29729 (SEQ ID NO: 334), ORF30221 (SEQ ID NO: 336), ORF30736c (SEQ ID NO: 338), ORF30539 (SEQ ID NO: 340), ORF31247c (SEQ ID NO: 342), ORF31539c (SEQ ID NO: 346), ORF31222 (SEQ ID NO: 348), ORF31266 (SEQ ID NO: 350), ORF31661c (SEQ ID NO: 352), ORF32061c (SEQ ID NO: 354), ORF32072c (SEQ ID NO: 356), ORF31784 (SEQ ID NO: 358), ORF32568c (SEQ ID NO: 360), ORF33157C (SEQ ID NO: 362), ORF32539 (SEQ ID NO: 364), ORF33705c (SEQ ID NO: 366), ORF32832 (SEQ ID NO: 368), ORF33547c (SEQ ID NO: 370), ORF33205 (SEQ ID NO: 372), ORF33512 (SEQ ID NO: 374), ORF33771 (SEQ ID NO: 376), ORF34385c (SEQ ID NO: 378), ORF33988 (SEQ ID NO: 380), ORF34274 (SEQ ID NO: 382), ORF34726c (SEQ ID NO: 384), ORF34916 (SEQ ID NO: 386), ORF35464c (SEQ ID NO: 388), ORF35289 (SEQ ID NO: 390), ORF35410 (SEQ ID NO: 392), ORF35907C (SEQ ID NO: 394), ORF35534 (SEQ ID NO: 396), ORF35930 (SEQ ID NO: 398), ORF36246 (SEQ ID NO: 400), ORF26640c (SEQ ID NO: 402), ORF36739 (SEQ ID NO: 404), ORF37932c (SEQ ID NO: 406), ORF38640c (SEQ ID NO: 408), ORF39309c (SEQ ID NO: 410), ORF38768 (SEQ ID NO: 412), ORF40047c (SEQ ID NO: 414), ORF40560c (SEQ ID NO: 416), ORF40238 (SEQ ID NO: 418), ORF40329 (SEQ ID NO: 420), ORF40709c (SEQ ID NO: 422), ORF40507 (SEQ ID NO: 424), ORF41275c (SEQ ID NO: 426), ORF42234c (SEQ ID NO: 428), ORF41764c (SEQ ID NO: 430), ORF41284 (SEQ ID NO: 432), ORF41598 ( SEQ ID NO: 434), ORF42172c (SEQ ID NO: 436), and ORF42233c (SEQ ID NO: 438). Figure 4 shows the deduced amino acid sequences for ORF2 (SEQ ID NO: 3), ORF3 (SEQ ID NO: 5), ORF602c (SEQ ID NO: 7), ORF214 (SEQ ID NO: 9), ORF1242c (SEQ ID NO: ll), OR594 (SEQ ID NO: 13), ORF1040 (SEQ ID NO: 15), ORF1640c (SEQ ID NO: 17), ORF2228C (SEQ ID NO: 19), ORF2068c (SEQ ID NO: 21), ORF1997 (SEQ ID NO: 23), ORF2558c (SEQ ID NO: 25), ORF2929c (SEQ ID NO: 27), ORF3965c (SEQ ID NO: 29), ORF3218 (SEQ ID NO: 31), ORF3568 (SEQ ID NO: 33), ORF4506c (SEQ ID NO: 35), ORF3973 (SEQ ID NO: 37), ORF4271 (SEQ ID NO: 39), ORF4698 (SEQ ID NO: 41), ORF5028 (SEQ ID NO: 43), ORF5080 (SEQ ID NO: 45), ORF6479c (SEQ ID N0: 47), ORF5496 (SEQ ID NO: 49), ORF5840 (SEQ ID NO: 51), ORF5899 (SEQ ID NO: 53), ORF6325 (SEQ ID NO: 55), ORF7567c (SEQ ID NO: 57), ORF7180 (SEQ ID NO: 59), ORF7501 (SEQ ID NO: 61), ORF7584 (SEQ ID NO: 63), ORF8208c (SEQ ID NO: 65), ORF8109 (SEQ ID NO: 67), ORF9005c (SEQ ID NO: 69), ORF8222 (SEQ ID NO.71), ORF8755c (SEQ ID NO: 73), ORF9431c (SEQ ID NO: 75), ORF9158 (SEQ ID NO: 77), ORF10125c (SEQ ID NO: 79), ORF9770 (SEQ ID NO: 81), ORF9991 (SEQ ID NO: 83), ORFl0765c (SEQ ID NO: 85), ORF10475 (SEQ ID NO: 87), ORF11095c (SEQ ID NO: 89), ORF11264 (SEQ ID NO: 91), ORF11738 (SEQ ID NO: 93), ORF12348C (SEQ ID NO: 95), ORF12314c (SEQ ID NO: 97), ORF13156c (SEQ ID NO: 99), ORF12795 (SEQ IDNO.101), ORF13755c (SEQ ID NO: 211), ORF13795c (SEQ ID NO: 213), ORF14727c (SEQ ID NO: 215), ORF13779 (SEQ ID NO: 217), ORF14293c (SEQ ID NO: 219), ORF14155 (SEQ ID N0.221), ORF14360 (SEQ ID NO: 223), 0RF15342c (SEQ ID NO: 225), ORFl5260c (SEQ ID NO: 227), ORF14991 (SEQ ID NO: 229), ORF15590c (SEQ ID NO: 231), ORF15675c (SEQ ID NO: 233), ORF16405 (SEQ ID NO: 235), ORF16925 (SEQ ID NO.-237), ORF17793C (SEQ ID NO: 239), ORF18548c (SEQ ID NO: 241), ORF17875 (SEQ ID243), ORF18479 (SEQ ID NO: 245), ORF19027c (SEQ ID NO: 247), ORF19305 (SEQ ID NO: 249), ORF19519 (SEQ ID NO: 251), ORF19544 (SEQ ID NO: 253), ORF20008 (SEQ ID NO: 255), ORF20623c (SEQ ID NO: 257), ORF21210c (SEQ ID NO: 259), ORF21493c (SEQ ID NO: 261), ORF21333 (SEQ ID NO: 263), ORF22074c (SEQ ID NO: 265), ORF21421 (SEQ ID NO: 267), ORF22608c (SEQ ID NO: 269), ORF22626 (SEQ ID N0.271), ORF23228 (SEQ ID NO: 273), ORF23367 (SEQ ID NO: 275), ORF25103c (SEQ ID NO: 277), ORF23556 (SEQ ID NO: 279), ORF26191C (SEQ ID NO: 281), ORF23751 (SEQ ID NO: 283), ORF24222 (SEQ ID NO: 285), ORF24368 (SEQ ID NO: 287), ORF24888c (SEQ ID NO.-289), ORF25398c (SEQ ID NO: 291), ORF25892c (SEQ ID NO: 293), ORF25110 (SEQ ID NO: 295), ORF25510 (SEQ ID NO: 297), ORF26762c (SEQ ID NO: 299), ORF26257 (SEQ ID NO: 301), ORF26844c (SEQ ID NO: 303), ORF26486 (SEQ ID NO: 305), ORF26857c (SEQ ID NO: 307), ORF27314c (SEQ ID NO: 309), ORF27730c (SEQ ID NO: 311), ORF26983 (SEQ ID NO: 313), ORF28068c (SEQ ID NO: 315), ORF27522 (SEQ ID N0.317), ORF28033c (SEQ ID NO: 319), ORF29701c (SEQ ID NO: 321), ORF28118 (SEQ ID NO: 323), ORF28129 (SEQ ID NO: 325), ORF29709c (SEQ ID NO: 327), ORF29189 (SEQ ID NO: 329), ORF29382 (SEQ ID NO: 331), ORF30590C (SEQ ID NO. : 333), ORF29729 (SEQ ID NO: 335), ORF30221 (SEQ ID NO: 337), ORF30736c (SEQ ID NO: 339), ORF30539 (SEQ ID NO: 341), ORF31247c (SEQ ID NO: 343), ORF30963c (SEQ ID NO.-345), ORF31539C (SEQ ID NO: 347), ORF31222 (SEQ ID NO: 349), ORF31266 (SEQ ID NO. : 35 1), ORF31661c (SEQ ID NO: 353), ORF32061c (SEQ ID NO: 355), ORF32072c (SEQ ID NO: 357), ORF31784 (SEQ ID NO: 359), ORF32568c (SEQ ID N0.361), ORF33157c (SEQ ID NO: 363), ORF32530 (SEQ ID NO: 365), ORF33705c (SEQ ID NO: 367), ORF32832 (SEQ ID NO: 369), ORF33547c (SEQ ID NO: 371), ORF33205 (SEQ ID NO: 373), ORF33512 (SEQ ID NO: 375), ORF33771 (SEQ ID NO: 377), ORF34385c (SEQ ID NO: 379), ORF33988 (SEQ ID N0.381), ORF34274 (SEQ ID NO: 383), ORF34726c (SEQ ID NO: 385), ORF34916 (SEQ ID NO: 387), ORF35464c (SEQ ID NO: 389), ORF35289 (SEQ ID NO: 391), ORF35410 (SEQ ID NO: 393), ORF35907c (SEQ ID NO: 395), ORF35534 (SEQ ID NO: 397), ORF35930 (SEQ ID NO: 399), ORF36246 (SEQ ID NO.401), ORF26640C (SEQ ID NO: 403), ORF36769 (SEQ ID NO: 405), ORF37932c (SEQ ID NO: 407), ORF38640c (SEQ ID NO: 409), ORF39309c (SEQ ID NO: 411), ORF38768 (SEQ ID NO: 413), ORF40047c (SEQ ID NO: 415), ORF40560c (SEQ ID NO: 417), ORF40238 (SEQ ID NO: 419), ORF40329 (SEQ ID N0. 421), ORF40709c (SEQ ID NO: 423), ORF40507 (SEQ ID NO: 425), ORF41275c (SEQ ID NO: 427), ORF42234c (SEQ ID NO: 429), ORF41764c (SEQ ID NO: 431), ORF41284 (SEQ ID NO: 433), ORF41598 (SEQ ID NO: 435), ORF42172c (SEQ ID NO: 437), and ORF42233c (SEQ ID NO: 439).

Figure 5 shows the nucleotide sequence (SEQ ID NO: 102) encoding a protein encoded by the sequence 33A9. Figure 6A shows the deduced amino acid sequence (SEQ ID NO.103), a protein encoding the 33A9 sequence. Figure 6B shows the nucleotide sequences of the different ORFsl-10 (SEQ IDNOS: 189, 190, 191, 192, 193, 194, 195, 196, 197, and 198) which is identified in sequence 33A9 in their sequences respective amino acids (ORFsl-10; SEQ ID NOS: 199, 200, 201, 202, 203, 204, 205, 206, 207, and 208). Figure 7 shows the physical map of the 34B12 EcoRl fragment map that identifies the positions of three ORFs: ORF1 (L-S), ORF2, and ORF ÍS. Also shown is the nucleotide sequence corresponding to the insertion pho34Bl2 (SEQ ID NO: 104) containing the ORFl (LS) (SEQ ID NOS: 105 and 107), ORF2 (SEQ ID NOS: 106 and 108), and ORF1 -S (SEQ ID NOS: 208 and 209). Figure 8 shows the deduced amino acid sequence of ORF1 (LS) (SEQ ID NO: 107), which is described in Figure 7. Figure 9 shows the deduced amino acid sequence of ORF2 (SEQ ID NO: 108), which is described in Figure 7. Figure 10 shows the nucleotide sequence (SEQ ID NO: 109), which corresponds to insertion 36A4.

Figure 11 shows the deduced amino acid sequence of the peptide (SEQ ID NO: 110) that encoded the 36A4 sequence. The previous peptide that encoded the 36A4 sequence has homology to the hrpM gene of Pseudomonas syringae (Loubens, et al., Mol.Mibriol.10: 329-340, 1993). Figure 12 shows the nucleotide sequence (SEQ ID NO: 111) of contig 2507 which is identified using the nucleotide sequence 36A4. Figure 13 shows the nucleotide sequence (SEQ ID NO: 112) that corresponds to the 23A2 insert. Figure 14A shows the deduced amino acid sequence of the peptide (SEQ ID NO: 113) that encoded the 23A2 sequence. The peptide hosted by the sequence 23A2 is homologous to a protein known in Pseudomonas aeruginosa (strain CD10): the mexA gene. This gene is part of an operon that also contains two other genes: mexB and oprM (Poole et al., Mol.Microbiol.10: 529-544, 1993); presentation of GenBank: L11616. Figure 14B shows the nucleotide sequence (SEQ ID NO: 148) and the previous partial amino acid sequences of PA14 mexA and mexB (SEQ ID NOS: 149 and 150, respectively). Figure 15 shows the nucleotide sequence (SEQ ID NO: 114) of the phenazine operon PAOl that was identified using the 3E8 sequence tag. Figure 16A shows the nucleotide sequence (SEQ ID NO: 115) of the 3E8 sequence tag.

Figure 16B shows the nucleotide sequence flanking the 3E8 sequence tag (SEQ ID NO: 160). Figure 17 shows the amino acid sequence 3E8 deduced PHZA (SEQ ID NO: 116). Figure 18A shows the deduced amino acid sequence 3E8 PHZB (SEQ ID NO: 117). Figure 18B shows the partial amino acid sequence 3E8 deduced PHZA (SEQ ID NO: 161). Figure 18C shows the partial amino acid sequence 3E8 deduced PHZB (SEQ ID NO: 162). Figure 18D shows the partial amino acid sequence 3E8 PHZC deduced (SEQ ID NO: 163). Figure 18E shows the nucleotide sequence (SEQ ID NO: 164) and the previous partial amino acid sequence (SEQ ID NO: 165) of PA14 phzR. Figure 19 shows the nucleotide sequence (SEQ ID NO: 118) of the sequence tag 34H4. Figure 20 shows the nucleotide sequence (SEQ ID NO: 119) of the sequence tag 33C7. Figure 21 shows the nucleotide sequence (SEQ ID NO: 120) of the 25al2.3 sequence tag. Figure 22 shows the nucleotide sequence (SEQ ID NO: 121) of the sequence tag 8C12. Figure 23 shows the nucleotide sequence (SEQ ID NO: 122) of the sequence tag 2A8.

Figure 24A shows the nucleotide sequences (SEQ ID NOS: 123, 124, 125, 126, 127, and 128) of the sequence tags 41A5, 50E12, 35A9, pho23, 16G12, and 25F1 TnphoA, respectively. Figure 24B shows the nucleotide sequence (SEQ ID NO: 166) and the previous amino acid sequence (SEQ ID NO: 167) of PA14 pho! 5. Figure 24C shows the nucleotide sequence (SEQ ID NO: 168) of PA14 50E12 encoding YgdPPa (SEQ ID NO: 169) and PtsPPa (SEQ ID NO: 170). Figure 24D shows the nucleotide sequence (SEQ ID NO: 171) of PA14 35A9 encoding mtrRPa (SEQ ID NO: 172). Figure 24E shows the nucleotide sequence (SEQ ID NO: 173) of PA14 25F1 encoding the ORFT (SEQ ID NO: 174), ORFU (SEQ ID NO: 175), and Dj lAPa (SEQ ID NO: 176). Figure 25 shows the nucleotide sequence (SEQ ID NO: 129) of the phnA and phnB genes of Pseudomonas aeruginosa of PAO1 and PA14, respectively. Figure 26 shows the deduced amino acid sequence (SEQ ID NO: 130) of PHNA. Figure 27 shows the nucleotide sequence (SEQ ID NO: 131) of the PA14 degP gene. Figure 28 shows the deduced amino acid sequence (SEQ ID NO: 132) of the PA14 degP gene. Figure 29 shows the nucleotide sequence (SEQ ID NO: 133) of the algD gene of strain 8830 of Pseudomonas aeruginosa. Figure 30 shows the deduced amino acid sequence (SEQ ID NO: 134) of the algD gene of strain 8830 of Pseudomonas aeruginosa. Figure 31 shows the nucleotide sequence (SEQ ID NO: 135) of contig 1126 that was identified using 25A12. Figure 32 shows the physical map of contig 1344 (SEQ ID NO: 136) that was identified using 33C7 illustrating the three identified ORFs: ORFA (SEQ ID NO: 440), ORFB (SEQ ID NO: 441), and ORFC (SEQ ID NO: 442). Also shown are the amino acid sequences of ORFA (SEQ ID NO: 443), ORFB (SEQ ID NO: 444), and ORFC (SEQ ID NO: 445) encoded by their respective ORF. Figure 33 shows the nucleotide sequence (SEQ ID NO: 137) of the 1G2 sequence tag. Figures 34A-D are graphs showing complementation of the pathogenicity phenotype of the mutant worm .4 Tnp-ho? who use the C. elegans slow elimination test. Figure 34A is a graph showing that the non-pathogenic phenotype of the 12 Al mutant (open diamonds) can be fully complemented to the wild-type PA14 levels (filled squares) by the lasR gene of the PAOl under the control of the constitutive lacZ promoter in trans in strain 12 Al (pKDT17) (open circles). The reconstructed lasR mutant, PA14 lasR-G (open squares) is as nonpathogenic as 12A1 (open diamonds). The results of an experiment using one-day-old adults are shown. Figure 34B is a graph showing the complementation of the phol5 slow elimination phenotype (pPAdsbA), which carries the dsbA gene of E. coli and P. aeruginosa, respectively, in trans under the control of the constitutive lacZ promoter. Figure 34C is a graph showing that the slow elimination phenotype of 25F1 was only partially restored by strains 25F1 (pORF338) and 25F1 (p3-ORFs) carrying the plasmids containing the orf338-orf224-djlAPa, respectively . Figure 34D is a graph showing the complementation of 50E12 by the orfl59-ptsPPa operon. The 50el2 strain (pUCPld), like the 12A1 mutant, does not eliminate the worms even after 63 hours. The two strains, 50E12 (pMT205-lac) and 50E12 (pMT206-nat), which express the putative orfl59-ptsPPa operon, were able to eliminate C. elegans. In 50E12 (pMT205-lac), the transcription of orfl59-ptsPPa is under the control of the constitutive lacZ promoter, whereas in 50E12 (pMT206-nat), the operon is controlled by its native promoter. Each data point represents mid-term replicas ± SD of 3-4. Unless otherwise indicated, synchronized L4 coils were used in the experiment. At least two independent experiments were performed for each complementation analysis. Figure 35A is a schematic illustration showing the complex of anthranilate synthase that is encoded by the phnA and phnB genes, which synthesizes the conversion of corismato to anthranilate. The anthranilate serves as a precursor for the production of pyocyanin in P. aeruginosa, strain PAOl (Essar et al, J. Bacteriol 172: 884-900, 1990). The double arrow indicates the inclusion of multiple, undefined steps leading from the conversion of anthranilate to pyocyanin. Figure 35B is a schematic illustration showing the generation of the mutant AphnAphnB by deletion within the 1602 bp within the phna and phnB genes. Figure 35C is a graph showing the effect of mutant AphnAphnB on rapid elimination in C. elegans. Rapid elimination assays were conducted using the wild-type PA14 strain, the 3E8 mutant of the? Nphoa or the AphnAphnB strain. The mortality of the worm was monitored 3 hours after the initial exposure to the bacteria and the rapid elimination defect seen with the AphnAphnB strain was comparable with that of another phenazine mutant, 3E8. Identification v Characterization of the Virulence Factor As described herein, plants were used as a pathogenic model in vivo for the identification of the virulence factors of the human opportunistic pathogen, Pseudomonas aeruginosa. Nine of the nine derivatives of the TnphoA mutant of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for fewer pathogenic mutants, also exhibited a significantly reduced pathogenicity in a mouse burn assay, which suggests that P. aeruginosa used more common strategies to infect the two hosts. Seven of these nine mutants contained inserts of TnphoA in previously unknown genes. These results demonstrated that an alternative invertebrate host of a human bacterial pathogen could be used in a high throughput screen in vivo to identify the virulence factors of novel bacteria involved in the pathogenesis of mammals. It is intended that these experimental examples illustrate, not limit, the scope of the claimed invention. These experiments were performed using the following techniques. • Strains, Culture Conditions and Plasmids. The strain UCBPP-PA14 of P. aeruginosa is a human clinical isolate that was used in these experiments for the identification of genes related to novel virulence (Ausubel et al., Methods of Screening Compounds Useful for Prevention of Infection or Pathogenici ty, USSNs 08 / 411,560, 08 / 852,927, and 08 / 962,750, filed on March 25, 1995; May 7, 1997; and November 3, 1997, respectively; Rahme et al., Science 268: 1899-1902, 1995), and PAK strains of P. aeruginosa (Ishimoto and Lory, Proc. Na ti. Acad. Sci. USA 86: 1954-1957, 1989) and PAOl (Holloway et al., Microbiol. Rev. 43: 73-102, 1979) have been studied extensively in many laboratories The broth and agar-agar of Luria Bertani were used for the cultivation of the strains of P. aeruginosa and the Escherichia coli at 37 ° C. Also, he used the minimum medium (M9) for the cultivation of P. aeruginosa. Mutacrénesis by Transposón. Transposon-mediated mutagenesis of UCBPP-PA14 was performed using the TnphoA carrying the suicide pRT731 plasmid in the SM10? Pir strain of E. coli (Taylor et al, J. Bacteriol 171: 1870-1878, 1989). The donor and recipient cells that were cultured in this medium were placed together in plates on Luria Bertani agar plates and incubated at 37 ° C for eight to ten hours and subsequently plated on Luria Bertani plates. contained rifampicin (100 μg / milliliter) (to be screened against E. coli donor cells) and kanamycin (200 μg / milliliter) (to select for P. aeruginosa cells containing TnphoA). The colonies that grew in the rifampicin and kanamycin media were reapplied to Luria Bertani containing ampicillin (300 μg / milliliter); the ampicillin-resistant colonies indicated the integration of pRT731 into the genome of UCBPP-PA14 and were desired. Activity of Alkaline Phosphatase. Two thousand five hundred (2,500) prototrophic TnphoA mutants of UCBPP-PA14 were selected on peptone glucose agar-agar plates (Ostroff et al, J, Bacteriol 172: 5915-5923, 1990) containing 40 μg / milliliter of phosphate of 5-bromo-4-chloro-3-indolyl (XP). The peptone medium was selected because it suppressed the production of the pyomycin of the endogenous blue-green pigment and the pioverdin of the fluorescent yellow pigment, allowing the visualization of the blue color that resulted from the dephosphorylation of the XP by the periplasmic alkaline phosphatase that was generated using the PhoA + mutants. Cultivation Conditions and Mutant Isolation Strategy. The strains of P. aeruginosa that were cultured until saturation in the L-broth at 37 ° C in lOmM of MgSO4 were washed, resuspended at an optical density of 0.2 (OD600 = 0.2) in lOmM of MgSO4 and diluted to 1: 100 and 1: 1000 (corresponding to a bacterial density of approximately 10d and 105 cfu / milliliter, respectively). Approximately 10 milliliters of the diluted cells were inoculated with a pipette into the stems of approximately 12 week old lettuce plants (Roman variety or Big lake) that were grown in MetroMix pot soil in a greenhouse (26 ° C). The stems were washed with 0.1 percent bleach and placed in 15-centimeter-diameter petri dishes containing a Whatman filter (Whatman # 1) that was impregnated with 10mM MgSO4. The midrib of each lettuce leaf was inoculated with three different mutants of P. aeruginosa generated by TnphoA to be tested and the wild-type strain UCBPP-PA14 as a control. Plates were maintained in a culture chamber during the course of the experiment at 28-30 ° C and 90-100 percent relative humidity. The symptoms were monitored daily for 5 days. In the Arabidopsis leaf infiltration model, strains of P. aeruginosa that were cultured and washed were diluted as described above at 1: 100 in 10 mM MgSO4 (corresponding to a bacterial density of 103 / cm 2). of leaf disk area) and injected into the leaves of six-week-old Arabidopsis plants, as described for the infiltration of Pseudomonas syringae (Ausubel et al., Methods of Screening Compounds Useful for Prevention of Infection or Pa thogenicity, USSNs 08 / 411,560; 08 / 852,927, and 08 / 962,750, which were filed on March 25, 1995, May 7, 1997 and November 3, 1997, respectively; Rahme et al., Science 268: 1899-1902, 1995; Dong et al., Plant Cell 3: 61-72, 1991). The incubation conditions and the monitoring of the symptoms were the same as in the experiments with lettuce. The intercellular fluid of the leaf containing bacteria was harvested, and the bacterial counts were determined as described (Rahme et al., Science 268: 1899-1902, 1995; Dong et al., Plant Cell 3: 61-72, 1991). Four different samples were taken using two leaf disks per sample. The control plants that were inoculated with 10 M MgSO4 did not show any development of symptoms. Mortality Studies in Mice. A burn area of a total area of 5 percent was adjusted in the stretched abdominal skin of six-week-old male AKR / J mice (Jackson Laboratories) weighing between 25 and 30 grams as described previously (Ausubel et al. Methods of Screening Compounds Useful for Prevention of Infection or Pathogenicity, USSNs 08 / 411,560; 08 / 852,927, and 08 / 962,750, filed on March 25, 1995, May 7, 1997, and November 3, 1997 , respectively, Rahme et al., Science 268: 1899-1902, 1995; Stevens, J. Burn Care Rehabil., 15: 232-235, 1994). Immediately after the burn, the mice were injected with 5 x 10 3 or 5 x 10 5 cells of P. aeruginosa, and the number of animals that died of sepsis daily for 10 days was monitored. Animal study protocols were reviewed and approved by the Subcommittee on Animal Studies at Massachusetts General Hospital. Statistical significance for mortality data was determined using a? 2 test with Yates' correction or Fisher's exact test. The differences between the groups were considered statistically significant at P = 0.05. DNA Manipulation, Molecular Cloning, and Sequence Analysis of TnphoA Mutants. Chromosomal DNA from P. aeruginosa was isolated by phenol extraction (Strom and Lory, J. Bacteriol, 165: 367-372, 1986), and DNA staining hybridization studies were performed as described by Ausubel et al. Protocols in Molecular Biology, Wiley, New York, 1996). The oligonucleotides 5 '-AATATCGCCCTGAGCAGC-3' (LGR1) (SEQ ID NO: 138) and 5 '-AATACACTCACTATGCGCTG-3' (LGR2) (SEQ ID NO: 139) corresponded to the sequences in the opposite strands at the 5 'end of TnphoA. The oligonucleotides 5'-CCATCTCATCAGAGGGTA-3 '(LGR3) (SEQ ID NO: 140) and 5' -CGTTACCAT GTTAGGAGGTC-3 '(LGR4) (SEQ ID NO: 141) corresponded to the sequences in the opposite chains of the 3' end of TnphoA. LGR1 + LGR2 or LGR3 + LGR4 were used to amplify by the DNA sequences of the reverse polymerase chain reaction (IPCR) adjacent to the sites of the TnphoA insertion as described (Ochman et al., 1993, A Guide to Methods and Applications, eds., Innis, MA States, DJ, 1990). The amplified DNA fragments were cloned from 350 to 650 base pairs within the EcoRV site of pBlueScript SK +/-. To determine the sequence of the products amplified by the IPCR, double-stranded DNA sequencing was performed using the Sequenase 2.0 kit (U.S. Biochemical, Inc.). The sequences obtained with the non-redundant peptide sequence databases were compared at the National Center for Biotechnology Information (NCBI), using the BLAST program (Gish and States, Na t. 3: 266-272, 1993) Isolation and Manipulation of Wild-type Clone DNA Containing the Gene Corresponding to the pho34B12 Mutation from the Genomic Library of the UCBPP-PA14. The IPCR product that was generated from the TnphoA mutant of UCBPP-PA14 was labeled using a randomly primed DNA tagging kit (Boehringer Mannheim, Indianapolis, IN). and was used to probe a genomic genomic DNA library of UCBPP-PA14 in pJSR1 (Rahme et al., Science 268: 1899-1902, 1995) for a clone containing the gene corresponding to the pho34B12 mutation. A 37 kb EcoRI fragment, which was identified in the cosmid clone pLGR34B12 which corresponded to the pho34B12 mutation, was subcloned into the EcoRI site of pRR54 (Roberts et al., J. Bacteriol 172: 6204-6216, 1990). The same fragment (which was made with one end) was subcloned into the Smal site of pCVD (Donnenberg and Kaper, Infect, Immun, 59: 4310-4317, 1991), to construct pLGR34. PLGR34 was used to replace the mutated pho34B12 gene with a wild-type copy as described (Donnenberg and Kaper, Infect Immun 59: 4310-4317, 1991). The 3.7 kb EcoRI fragment was also subcloned into the EcoRI site of pBlueScript SK +/-, to construct pBSR34B12 and used for DNA sequence analysis. A sequence of 1,659 base pairs corresponding to the insertion pho34B12 containing two overlapping open reading frames (ORF1 and ORF2) in the chains opposite GenBank was assigned and assigned access number AF031571. The ORFl has 1,148 bp (nucleotides 361 to 1509) and the ORF2 has 1,022 bp (nucleotides 1458 to 436). The overlap of the two ORFs is from nucleotide 436 to 1458. The ORF1 contains a second putative translational start site at nucleotide 751 which corresponds to a coding region of 758 bp. The primers of the oligonucleotide 5 '-CGCATCGTC GAAACGCTGGCGGCC-3' (SE ID NO: 142) and of the 5'-GCCGATGGCGAGATCATGGC GATG-3 '(SEQ ID NO: 143) were used to amplify a 1100 bp fragment from the ORFl which contained pBSR34B12. Due to the two putative initiation sites present in the ORF1, primers of the 5'-oligonucleotide -TGCGCAACGATACGCCGT TGCCGACGATC-3 '(SEQ ID NO: 144) and of the 5'-GATTTCCACCTTCGCAGCGCA GCCC-3' (Reg3) (SEQ ID NO: 145) to amplify one of 1659 bp from the ORFÍ containing pBSR34B12. The oligonucleotide primers 5'-GATTCCACCTTCGCAGCGCAGCCC-3 '(SEQ ID NO: 146) and 5'-GCCGATGGCGAGATCATGGCGATG-3' (SEQ ID NO: 147) were used to amplify a 1302 bp fragment from the ORF2 containing the pBSR34B12. All combinations of primers were designed to contain the putative upstream regulatory elements of each ORF. The PCR products that were obtained (1100, 1659, and 1302 bp) were cloned into pCR2.1 (Invitrogen Inc.) to constitute pLEl5, PLE1, and pLE2, respectively. All the products of the polymerase chain reaction were subcloned into pRR54 to construct pRRLEl5, pRRLEl, and pRRLE2, respectively. Enzymatic Activities of the TnphoA Mutants. Strains of P. aeruginosa that were cultured for eighteen hours in the medium of LB were used for assays of enzymatic activities. The proteolytic and elastolytic activities were determined as described previously (Toder et al., Mol.Microbiol.5: 2003-2010, 1991). The quantification of pyocyanin was determined as described (Essar et al, J. Bact, 172: 884-900, 1990). Hemolytic activity was detected after incubation on plates containing trypticase soy agar (BBL) supplemented with 5 percent sheep red blood cells (Ostroff and Vasil, J. Bacteriol, 169: 4957-4601, 1987). . Generation of a Non Polar GacA Mutation. A nonpolar gacA mutation was constructed in the UC BPP-PA14 by cloning a 3.5 kb PstI fragment containing the gacA gene from the cosmid pLGR43 (Rahme et al., Science 268: 1899-1902, 1995) within the site BamHI restriction site in the suicide vector pEGBR (Akerley et al., Cell 80: 611-620, 1995), using BamHI linkers. A filled fragment at the EcoRI-HincII Klenow end of 950 bp containing the cartridge of the kanamycin resistance gene was then cloned from pUC18K (Menard et al., J. Bacteriol. 175: 5899-5906, 1993) within the unique restriction site BamHI (which was made blunt end) in gacA, so that transcription was maintained and the translation of the downstream portion of the gacA at the 3 'end was restarted of the kanamycin cartridge. The resulting construct, SW 7-4, containing the cartridge of the kanamycin gene within the gacA gene and in the orientation of its transcription, was used to exchange by marker by homologous recombination, the interrupted gacA gene within the UCBPP-PA14 genome. of wild type. Isolation v Characterization of the Virulence Factors of P. aerusinosa. Using the procedures described above, the UCBPP-PA14 genome of P. aeruginosa was mutagenized with the TnphoA transposon, and 2,5000 prototrophic mutants were selected for impaired pathogenicity in the lettuce trunk assay. This lettuce trial allowed the testing of different mutants on a single lettuce stalk. Interestingly, we found that lettuce was not only susceptible to infection by UCBPP-PA14 but was also susceptible to the well-characterized PAK strains of P. aeruginosa (Ishimoto and Lory, Proc. Na ti. Acad. Sci USA). 86: 1954-1957, 1989) and PAOl (Holloway et al., Microbiol., Rev. 43:73, 1979). These last two strains proliferated on the leaves of lettuce and produced disease symptoms similar to those produced by UCBPP-PA14, characterized by being soaked in water followed by soft putrefaction four to five days after infection. In the late stages of infection, the three strains of P. aeruginosa invaded the entire midrib of a lettuce leaf, resulting in complete maceration and tissue collapse. As summarized in Table 1, we identified nine mutants generated by TnphoA from UCBPP-PA14 among the 2,500 tracked prototrophs that produced either null, weak, or moderate putrefactive symptoms on the lettuce stems compared to the wild-type strain. Table 1 a Four different samples were taken using two discs / sheet samples. The control plants that were inoculated with 10 mM MgSO4 showed no symptoms during the course of the experiments. Three independent experiments gave similar results. b Symptoms observed four to five days after infection. None, without symptoms; chlorosis, chlorosis that is confined to the site of inoculation; weak, wet in localized water and chlorosis of the tissue that is circumscribed to the site of inoculation; moderate, wet in moderate water and chlorosis with most of the tissue softened around the inoculation site; severe, severe soft rot of the whole leaf characterized by a reaction zone soaked in water and chlorosis around the inoculation site two to three days after infection. c All animal experiments were conducted at least twice using 8-10 animals / experiment. Independent experiments showed mortality proportions of similar percentages. The mice were injected with - 5 x 10 3 or 5 x 10 5 cells. d BLAST analysis did not produce encoded proteins with significant homology. No severe maceration of the leaf was observed with any of the mutants. DNA staining analysis showed that each of the nine mutants contained a single TnphoA insert, using as a probe a 1542 base pair BglI-BamHI fragment containing the tnphoA kanamycin resistance conference gene (Taylor et al. collaborators, J. Bact. 171: 1870-1878, 1989). Two of the nine TnphoA mutants of UCBPP-PA14, pho34Bl, and phol 5, expressed the activity of alkaline phosphatase, suggesting that genes containing these TnphoA inserts encoded membrane-secreted or secreted proteins (Taylor et al. J. Bact., 171: 1870-1878, 1989, Manoil and Beckwith, Proc.Nat.Acid.Sci.A. USA 82: 5117, 1985). The nine TnphoA mutants were further tested by measuring their percentage of culture over the course of four days in Arabidopsis leaves as a quantitative measure of pathogenicity (Rahme et al., Science 268: 1899-1902, 1995; Dong et al., Plant Cell 3: 61-72, 1991). Although none of the mutants showed any significant difference in their growth percentages compared to the wild-type strain in both rich and minimal media, the growth rates over time of the nine mutants in the Arabidopsis leaves were lower than in the wild-type strain. Table 1 lists the maximum levels of growth achieved by each mutant on the fourth day after infection. In the case of the nine mutants, the development of less severe symptoms reflected the reduced bacterial counts in the leaves. All mutants, except 33C7, produced either weak or moderate putrefaction and soaking symptoms with varying amounts of chlorosis (yellowing) (Table 1). Interestingly, however, as summarized in Table 1, the proliferation levels of the individual mutants did not correlate directly with the severity of the symptoms they produced. For example, although mutant 25A12 (Figure 21) grew to similar levels as mutants 33A9 (Figures 5 and 6A-B), pho34B12 (Figures 7, 8, and 9), and 34H4 (Figure 19), and only ten times less than UCBPP-PA14, mutant 25A12 produced very weak symptoms. Similarly, the mutants 33C7 (Figure 20), phol5 (Figure 24B), and 25F1 (Figure 24A), all reached similar maximum growth levels approximately 103 times less than the wild-type culture); however, only mutant 33C7 failed to cause any symptoms of the disease (Table 1). The differences that were observed in the degree of symptoms and the levels of proliferation among the ten mutants, suggested that these mutants probably carried insertions in the genes that are involved in different stages of the infection process of the plant. The pathogenicity of each of the nine mutants generated by TnphoA that were less pathogenic in the plant leaf assay in a mouse model of full-thickness skin thermal burn (Rahme et al., Science 268: 1899-) was measured. 1902, 1995; Stevens et al., J. of Burn Care and Rehabil., 15: 232-235, 1994). As shown in Table 1, all mutants were significantly different from the wild type with P = 0.05 in the two doses, except for 25A12 and 16G12 (Figure 24A), which were not significantly different from the wild type to the highest dose of 5 x 105 cells. In addition to the data shown in Table 1, mutant 33A9 did not cause mortality even at the highest dose of 5 x 106. We used DNA staining analysis and DNA sequence analysis to determine whether TnphoA in the nine less pathogenic mutants had been inserted into known genes. DNA staining analysis revealed that the 1D7 mutant contained a TnphoA insert in the gacA gene (Laville et al, Proc.Na ti.Acid.Sci.USA 89: 1562-1566, 1992, Gaffney et al., Mol. Microbe Interact 7: 455-463, 1994), which we have previously shown to be an important pathogenicity factor for P. aeruginosa in both plants and animals (Ausubel et al., Methods of Screening Compounds Useful for Prevention of Inf ction or Pa thogenicity, USSNs 08 / 411,560, 08 / 852,927, and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, respectively, Rahme et al., Science 268: 1899- 1902, 1995). For the other eight mutants, we used the reverse polymerase chain reaction (IPCR) to generate amplified products corresponding to the DNA sequences adjacent to the TnphoA insert sites (Ochman et al., 1993, A Guide to Methods and Applications, eds., Innis, MA States, DJ, 1990). The products of the IPCR were cloned and then subjected to DNA sequence analysis. The phold mutant contained the TnphoA inserted into a P. aeruginosa gene (from the PAO1 strain) that was previously deposited in GenBank (Accession Number U84726) showing a high degree of similarity to the dsbA gene of Azotobacter vinelandii, which encodes a periplasmic disulfide bond that forms the enzyme (Bardwell et al., Cell 67: 581-589, 1991). Homologs are required in the bacterial pathogen Erwinia chrysanthemi and the human pathogens Shigella flexneri and Vibrio cholera for pathogenesis (Shevchik et al., Mol.Microbiol.16: 745-753, 1995; Peek and Taylor, Proc. Nati Acad. Sci. USA 89: 6210-6214, 1992; Watarie et al., Proc. Na ti. Acad. Sci. USA 92: 4927-4931, 1995). The computer analysis that used the BLASTX program showed that when the DNA sequences that correspond to the other seven TnphoA insertions were translated into all possible reading frames, no significant similarity was found with any of the known genes (Table 1) . We performed a variety of biochemical tests to list the nine least pathogenic UCBPP-PA14 mutants on the basis of whether they carried defects in the primary virulence factors previously described and / or in the metabolic trajectories. All mutants were tested for the presence of protease, elastase, and phospholipase activities and for their ability to secrete pyocyanin from the secondary metabolite (Toder et al., Mol. Microbiol. : 2003-2010, 1991; Essar et al., J. Bact. 172: 884-900, 1990; Ostroff and Vasil, J. Bacteriol. 169: 4597-4601, 1987). Piocyanine is a redox-active phenazine compound that excretes most of the clinical strains of P. aeruginosa that kill mammalian and bacterial cells through the generation of reactive oxygen intermediates and which has been implicated as a factor of virulence of P. aeruginosa (Hassett et al., Infect. Immun.60: 328-336, 1992; Kanthakumar et al., Infect. Immun. 61: 2848-2853, 1993; Miller et al., Infect. Immun. 182, 1996). The mutants 33C7, 33A9, 25F1, and 1 6G12 showed no defects in any of the biochemical assays that were used. Mutant pho34B12 showed decreased hemoc activity in blood agar plates, reduced elastase activity (-50 percent), and no detectable pyocyanin production. The phold mutant only showed traces of elastase activity and a decrease in proteoc activity (60-70 percent), compared to the wild type. Mutant 25A12 showed a 50 percent decrease in elastoc activity. Finally, the 1D7 mutant containing an insert in the gacA showed reduced levels of pyocyanin (-50 percent) compared to the wild type. In addition to mutant 1D7, a second independent gacA:: TnphoA mutant was identified from our selection of the plant, mutant 33D11. This last mutant also exhibited a similar reduction in pyocyanin production and reduced virulence in both plants and mice. On the basis of the sequence analysis of the DNA and the biochemical test of the mutants, the genes that were targets of the TnphoA insertions in the mutants 1D7 and phoA34Bl2 were selected for further analysis. As discussed above, 1D7 contained an insertion in gacA, which we had previously shown to encode a virulence factor in P. aeruginosa (Rahme et al., Science 268: 1899-1902, 1995). Recently, it has also been shown that a gacA-like gene is a major virulence factor for Salmonella typhimurium (Johnston et al., Mol.Microbiol., 22: 715, 1996). However, the two gacA insertions: TnphoA (1D7 and 33D11), the gacA insertion mutant that we previously constructed (Ausubel et al., Methods of Screening Compounds Useful for Prevention of Infection or Pa thogenicity, USSNs 08 / 411,560. 852,927, and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, respectively, Rahme et al., Science 268: 1899-1902, 1995), and a gacA mutation of the P. aeruginosa, which was constructed independently, which affects the production of different known virulence factors (Hassett et al., Infect.Immun.60: 328-336, 1992) all produced a polar effect in at least one gene, a homologue of the uvrC gene of E. coli immediately downstream of gacA (Rahme et al., Science 268: 1899-1902, 1995; Laville et al., Proc. Na ti. Acad. Sci. USA 89: 1562-1566, 1992; Reimmann et al., Mol. Microbiol., 24: 309-319, 1997). To provide definitive evidence that the loss of the pathogenicity phenotypes of the gacA mutants described herein, was due to the rupture of the gacA open reading frame per se, rather than due to a polar effect on a gene downstream of gacA, we constructed a non-polar gacA mutation in UCBPP-PA14 using a DNA cartridge that encodes a gene that confers resistance to kanamycin. Importantly, the non-polar gacA mutant exhibited the same decreased level of pathogenicity in the mice assay (50 percent mortality) and in the Arabidopsis assay (culture at 3 x 105 cfu / cm2 after four days) as the gacA:; TnphoA mutant (1D7), but did not exhibit the extreme UV sensitivity of the polar gacA mutants. Like the 1D7, the non-polar gacA mutant also excreted lower levels of pyocyanin (50 percent) compared to the wild type. The pho34Bl2 mutant was selected for further analysis for the following reasons. First, the insertion in pho34B12 was located directly downstream of the phnA phnB biosynthetic genes of pyocyanin from P. aeruginosa (Essar et al, J. Bact 172: 884-900, 1990), in a region previously without genome characterization of P. aeruginosa. Second, the pho34Bl2 insertion induced a pleiotropic phenotype that included reduced elastase and hemolytic activities, suggesting that the gene in which the pho34Bl2 TnphoA insertion was located could encode a regulator of various pathogenicity factors. To rule out the possibility that a secondary mutation in pho34B12 was responsible for the loss of the pathogenicity phenotype, rather than the TnphoA insertion, we replaced the pho34B12:.-TnphoA mutation by homologous recombination with the corresponding wild-type gene. This resulted in the restoration of the pathogenicity defect in both plants and animals, as well as the restoration of hemolytic and elastolytic activity and the production of pyocyanin at wild type levels (Table 2, below). Table 2a a See Table 1 for an explanation of the entries in the table. These results in Table 2 show that the TnphoA insert in pho34B12 was the cause of the pleiotropic phenotype of this strain, including the loss of the pathogenicity phenotype. The fact that there were no putative ORFs present in the next 500 bp downstream of the stop codon after insertion of pho34B12:.-TnphoA (see below), made it unlikely that TnphoA would produce a polar effect on a current gene down which was responsible for the phenotype of the mutant pho34B12. The genetic complementation analysis of pho34Bl2 with a plasmid (pLGRE34B12) containing a 3.7 bp insert which included pho34B12 and part of the phnAB region, resulted in the restoration of elastase and hemolytic activities to the levels of the type wild and more than one overproduction of ten times more pyocyanin (Table 2). However, pLGRE34B12 did not contemplate the phenotype of impaired pathogenicity of pho34B12 in both Arabidopsis and mice (Table 2), most likely due to the presence of multiple copies of the wild-type gene corresponding to pho34B12. Additional DNA analysis showed that the region containing the pho34B12 mutation encoded two almost completely overlapping open reading frames (ORFs) (ORF1 and ORF2) that were transcribed in opposite directions. In addition, the ORFl had two potential methionine initiation codons (which are designated ORFl-S and ORFl-L). The predicted proteins encoded by ORF1-S and ORF1-L, which were transcribed in the same direction as the phnA, phnB, and phoA genes, contained a consensus motif that corresponded to a lipid adhesion site found in a variety of prokaryotic membrane lipoproteins (Hayashi and Wu, J. Bioenerg, Biomembr, 22: 451-471, 1990). These membrane lipoproteins are synthesized with a precursor signal peptide, which provides an explanation for the Pho + phenotype of the pho34B12 insert (Hayashi and Wu, J. Bioenerg, Biomembr, 22: 451-471, 1990). The predicted protein encoding ORF2 contained a N-terminal 'helix-turn-helix' DNA binding motif, similar to the 'helix-turn-helix' motif found in the LysR family of transcriptional regulators (Henikoff and collaborators, Proc. Na ti, Acad. Sci. USA 85: 6602-6606, 1988; Viale et al., J. Bacteriol., 173: 5224-5229, 1991). This class of proteins includes regulators that are included in the pathogenesis of both plants and mammals (Finlay and Falkow, Microbiol., And Mol. Biol. Rev. 61: 136-169, 1997). The existence of two functional ORFs, almost completely overlapping is unusual in bacterial genomes. To determine which of the ORFs encoded in the pho34B12 region was functional, an additional complementation analysis was performed using the plasmids containing the polymerase chain reaction products corresponding to ORF1-S, ORF1-L, and ORF2 ( Figure 7). The production of both pyocyanin and elastolytic activity was restored at 20-40 percent of wild-type levels by plasmid synthesizing the protein encoding ORF2 (pRRLE2). Likewise, the hemolytic capacity of this complemented strain was partially restored. Complementation of pho34B12 with the plasmids pRRLEl and pRRLE15, which correspond to ORF1-S and ORF1-L, respectively, also restored the hemolytic activities of pyocyanin and elastolytic. Interestingly, however, the presence of the plasmids pRRLEl and pRRLEl5 resulted in a ten-fold higher production of pyocyanin and a twice greater level of elastase activity. Neither the pRRLEl, the pRRLE15, or the pRRLE2 contemplated the loss of the pathogenicity phenotypes of the mutant pho34B12 neither in the plants nor in the animals (Table 2). The additional characterization of this region, which includes site-directed mutagenesis will further elucidate which of the three ORFs is required (n) for pathogenicity in plants and animals. The data presented above showed that the virulence factors (genes) of P. aeruginosa previously unknown that play a significant role in the pathogenesis of mammals can be easily identified by randomly selecting the mutants of P. aeruginosa for some that show attenuated pathogenic symptoms in plants. This is consistent with our previous study in which we demonstrated that at least three P. aeruginosa genes encode virulence factors involved in the pathogenesis of both plants and animals (Ausubel et al., Methods of Screening Compounds Useful for Prevention of Infection or Pa thogenicity, USSNs 08 / 411,560, 08 / 852,927, and 08 / 962,750, filed on March 25, 1995; May 7, 1997; and November 3, 1997, respectively; Rahme et al., Science 268: 1899-1902, 1995). On the other hand, we did not expect to find that the nine of the nine mutants that we isolated that were less virulent in the plants were also less virulent in the mice. The simplest interpretation of this result is that the pathogenesis of P. aeruginosa in plants and animals uses a substantially superimposed set of genes that can be considered as basic virulence genes. Another possible interpretation is that some of the genes that were identified can encode regulatory proteins (ie, pho34B12, which control different effector molecules, a subset of which can be specific to either plants or animals. mutants that would be identified in this study (7 out of 9), would correspond with genes that were previously unknown, using the Poisson distribution, a genome size for P. aeruginosa of 5.9 Mb and an average gene size of 1.1 kb, we calculated that the 2,500 mutants that were tested represent 25 percent of the total number that needs to be tested to give approximately a 95 percent chance to test each gene in the assay, therefore, because our selection for the virulent mutants of P. aeruginosa is not saturated, it is likely that there are many additional genes of P. aeruginosa with important roles in pathogenicity, it is Perando being discovered. Importantly, at least two of the previously known virulence factors (genes) that were identified in our model as being important in the pathogenesis of plants are not only important virulence factors for P. aeruginosa in a model of mouse burn, but have also been described as important virulence factors in other gram-negative pathogens. These latter factors (genes) of pathogenicity include dsbA, and gacA (Schevchik et al., Mol Microbiol., 16: 745-753, 1995; Peek and Taylor, Proc. Nati, Acad. Sci. USA 89: 6210-6214. , 1992; Watarai et al, Proc. Na ti, Acad. Sci. USA 92: 4927-4931, 1995; Johnston et al., Mol. Microbiol. 22: 715, 1996). This makes it likely that many of the previously unknown factors that were identified in P. aeruginosa will generally be relevant for gram-negative pathogenesis.

Another important conclusion from this study is that the high production in vivo selection method that we have developed can lead to the identification of pathogenicity factors that do not correlate with evident biochemical defects. The mutants 33C7, 33a 9,. 34? 4, 25F1, and 1 6G12 did not exhibit detectable defects in different pathogenicity factors of P. aeruginosa known and, importantly, mutants 33C7 and 33A9 were among the most weakened in the mouse model. Furthermore, although the pho34B12 and 25A12 mutants did exhibit a decreased production of known virulence factors, the genes corresponding to these mutants had not previously been identified, most likely because the biochemical defects in these mutants can not be easily identified efficiently in a simple selection of high production. This testifies to the sensitivity of our selection for the loss of pathogenicity phenotypes. In recent years, other high-production classifications have been described to identify bacterial pathogenicity factors. IVET (in vivo expression technology) identifies the promoters that are specifically activated during pathogenesis (Wang et al, Proc.Nat.Acid.Sci.USA 93: 10434-10439, 1996; Mahan et al., Science 259 : 686-688, 1993), the STM (signature-tagged transposon method) identifies the genes that are required for survival in a host (Hensel, Science 268: 400-403, 1995) and DFI (differential fluorescence induction) ) uses the green fluorescent protein and the selection of fluorescence-activated cells to identify genes that are activated under specific conditions or in specific host cell types (Valdivia and Falkow, Mol.Microbiol., 22: 367-378, 1996). These approaches are complementary to the one we have described in this application and each approach has advantages and disadvantages. An advantage of our selection procedure in an invertebrate host is that it directly measures pathogenicity while the IVET and DFI methods measure the expression of the gene associated with pathogenicity. Unlike the STM procedure, which identifies genes whose function can not be complemented in trans by the mixed population of the bacterial mutants that were used for the inoculum, the present selection in an invertebrate includes the test of each mutant clone separately . Other Virulence Targets The nucleic acid sequence 33A9 (Figures 5 and 6A-B) was also identified in a cosmid clone that was designated BI48 (Figure 1). This cosmid was sequenced in its entirety and in Figure 2 its nucleic acid sequence is shown. Using the standard database analysis, the nucleotide sequences and deduced amino acid sequences of several additional open reading frames were identified (Figures 3 and 4). A summary of this analysis is presented in Table 3. As with the sequences described above, any of the sequences found in Figures 3 and 4 can be used to select the compounds (e.g., using the methods described herein), which reduce the virulence of a pathogen The sequence obtained from the cosmid pBI48 of strain PA14 revealed that 33A9 is located approximately 5 kb upstream of a cluster of pili genes (Figure 1, Table 3). This cluster contains the pilS / pilR genes, which are known to be involved in the regulation of pili formation. In addition, analysis of the sequence upstream of 33A9 did not show any homology with previously identified sequences, suggesting the possibility that the entire region surrounding 33A9 might define an island of pathogenicity. Figure 3 (orf 19544), Figure 4 (orf 19544), 5, 6A, and 6B show the nucleotide sequence 33A9, as well as the ORFs that were identified. In addition, analysis of the sequence obtained from the cosmid gene pBI48 indicated the presence of a sequence "located approximately 2 kb downstream of 33A9, which showed strong homology with the tRNA sequences (ORF 22626, Figure 1 Because the analysis of the region that is located upstream of the tRNA sequence showed no homology with the sequences present in the database, and because the ARTNt sequences represent "hot spots" for the insertions of DNA, we arrive at the hypothesis that the sequence of the tRNA represents the correct limit for the insertion of an island of pathogenicity present in PA14. As seen in Figure 1, the size of the region that could represent the piece of foreign DNA which was inserted, is approximately 25 kb.The identification of the limit that is located upstream of the probable pathogenicity island, will help to establish the exact size of the p DNA that was inserted. In addition, analysis of the 33A9 region also indicated the presence of more than one sequence with protein-level homology to the integrases and transposases (ORF21421, ORF8109, respectively). Finally, our data showed that the 33A9 site was present in different clinical isolates of highly pathogenic P. aeruginosa, and absent in the PAO1, a less pathogenic strain of P. aeruginosa. Analysis of the sequencing data that was obtained from the cosmid pBI48 also indicated the presence of two sequences flanking the 33A9 gene, which contained the recognition motifs involved in the adhesion of the cells. Sequence analyzes of ORF11738 (2436 bp) and ORF23228 (2565 bp), upstream and downstream of 33A9 respectively (Figure 1), indicated the presence of the RGD motifs in these two open reading frames. The RGD tripeptide sequences are a characteristic eukaryotic recognition motif that binds cell surface integrins and has been found to be included in bacterial adherence. By mimicking the host molecules, bacterial adhesins containing these RGD motifs can effect host responses that are required to promote cell-cell adhesion. The expression of these two ORFs containing RGD was evaluated in both 33A9 and wild-type PA14 strain. Transcript levels were determined by hybridization with a radiolabeled DNA probe that corresponded to an internal region of ORF11738 and ORF23228. The data obtained for the two ORFs in the 33A9 mutant, showed reduced transcript levels compared to the wild-type PA14, indicating that both genes encoded by ORF11738 and ORF23228 are regulated by 33A9. These data indicated that 33A9 plays a role as a regulator of multiple genes responsible for regulating the expression of genes involved in bacterial adherence to the surfaces of host cells.

Table 3 15 fifteen In addition, using the plant and nematode selection assays (slow or rapid elimination assays) described by Ausubel et al. (Methods of Screening Compounds Useful for Prevention of Infection or Pathogenicity, USSNs 08 / 411,560. 08 / 852,927, and 08 / 962,750 , filed on March 25, 1995, May 7, 1997, and November 3, 1997, respectively), many other strains of the mutant Pseudomonas aeruginosa were identified as having decreased virulence. The slow and rapid elimination assays that were used for these studies are described below. Slow elimination test. For the slow clearance assay, 10 μl of a bacterial culture from the previous night was spread on an NG plate (modified from the NGM agar that was described in Sulston and Hodgkin (in: The Nematode Caenorhabditis elegans, WB Wood, ed. , Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 188, pp. 587-606): (0.35 percent peptone was used instead of 0.25 percent) and incubated at 37 ° C for 24 hours. 8-24 hours at room temperature (25-25 ° C) each plate (3.5 centimeters in diameter) was seeded with 40-50 C. elegans L4 hermaphrodites of the Bristol strain, for statistical purposes, 3-4 replications were made by test The plates were incubated at 25 ° C, and the number of dead worms was recorded every 4-6 hours.Worm is considered dead when it no longer moves when it is touched with an eyelash and no longer displays any action of pharyngeal pumping: For each batch of mutants that were tested, PA14 and to OP50 from E. coli as positive and negative controls. Any worm that had died as a result of having been immobilized against the wall of the plate was excluded. In order to determine the LT50s, the data were plotted on a graph (percentage of worms removed vs. time after exposure to the test strains (hour)). A curve of the form was adjusted: percentage removed = A + (1-A) / (1 + exp (B - G x log (hours after exposure))) to the data, using the computer program SYSTAT 5.2. 1, where A represents the fraction of worms that died in an OP50 control experiment, and B and G are the parameters that were varied to adjust the curve. Once determined B and G, LT50 is calculated by the formula: LT50 = exp (B / G) x (1 - 2 x A) "(l / G) In developing the selection, we take advantage of two observations. First, the longer the worms die, the more progeny are produced, and second, the early larval stages are apparently more resistant to elimination by P. aeruginosa, which provides us with a very sensitive convenient test for the identification of TnphoA mutants that are only slightly impaired in their pathogenic potential These attenuated mutants would be less efficient in eliminating the worms, and the progeny production by the survivors effectively "amplifies" up to a weak defect in an observable phenotype. In this way, hundreds of worms were obtained in the plates containing the attenuated PA14 :: TnphoA mutants, from the hermaphrodites that were initially sown. they were planted with a non-pathogenic mutant, thousands of worms were seen by day five and bacterial grass completely consumed, while none or very few live worms were found on the plates planted with the wild-type strain. The nonpathogenic or attenuated putative mutants that were identified in the preliminary selection were retested and subjected to a virulence assay to determine the kinetics of C. elegans elimination. Rapid elimination test. The rapid elimination test, such as the slow elimination test, is useful for identifying the microbial virulence factors that cause disease. further, the assay is useful to identify the therapeutic that can, either inhibit the pathogenicity or increase the resistance capacities of an organism to a pathogen. In preferred embodiments, the rapid elimination test is performed using a nematode strain having increased permeability for a compound, for example, a toxin such as colchicine. Examples of nematodes having this increased permeability include, without limitation, animals that have a mutation in a P glycoprotein, eg, PGP-1, PGP-3, or MRP-1. These mutant nematodes are useful in the rapid elimination test due to their increased sensitivity to toxins that is due to the increased permeability of the membrane. This characteristic results in an assay with an increased differential between complete susceptibility and complete resistance to toxic compounds. The rapid elimination test can also be performed by increasing the osmolarity of the culture medium, as described below. The conditions of the rapid elimination assay that were used herein are as follows: 5 μl of a culture of PA14 that was cultured overnight in Kings B on plates (3.5 centimeters in diameter) containing peptone medium was extended. -glucose (PG), (Bacto-Peptone at 1 percent, NaCl at 1 percent, glucose at 1 percent, Bacto-Agar at 1.7 percent). Because the effectiveness of rapid elimination was found to be dependent on osmolarity, the peptone-glucose medium was modified by the addition of 0.15M sorbitol. After spreading the bacterial culture, the plates were incubated at 37 ° C for 24 hours and then placed at room temperature for 8-12 hours. Fifteen to twenty worms were placed on the test plate, which was then incubated at 25 ° C. Each independent trial consisted of 3-4 replicates. Mortality of the worms was recorded over time, and a worm was considered dead when it did not respond to touch, as described above. E. coli strain DH5a was used as a control for the rapid elimination assays. An analysis of these strains, together with those identified above, indicated that they fell into many different classes, including the following: some mutants were less pathogenic in both plants and nematodes, while others were reduced in either the plants or animals, but not both. The less pathogenic bacterial mutants were defined in plants as those which, four days after infiltration (DPI), had an average maximum titer (from 5 leaf samples) of two standard deviations lower than the wild type. within the same set of experiments. Wild type control was necessary because the maximum level reached by the wild type in the fourth IPD could vary as much as an order of magnitude between the experiments, due to the effects of minor variations in growth conditions on the responses of defense of the plant. Similarly, a mutant was characterized as reduced in pathogenicity in the worms, if the average time required to eliminate 50 percent of the worms feeding on it (LT50 from 3 replicates) was two standard deviations less than T50 of wild-type PA14 in the same experiment.

In general, those mutant strains that had reduced pathogenicity in plants included 1 6G12, 25A12, 33A9 and 33C7; those that had reduced pathogenicity in nematodes included 33A9, 44B1, 1G2, 8C12, and 2A8, and those that had reduced pathogenicity in plants and nematodes included 25F1, 41A5, 50E12, phol d, 12A1, pho23, 34B12 , 34H4, 3E8, 23A2, and 36A4. Tables 4 and 5 (below) summarize the pathogenicity phenotypes of these mutant strains. Sequence analysis was performed for each of these strains that had reduced virulence due to insertion mutagenesis. DNA sequence analysis, summarized in Tables 4 and 5, showed that both novel and known genes were identified in our selection trials. Each of the sequences from 50E12 and 41C1 showed strong similarity to the open reading frames (ORFs) that were previously described for the unknown function in E. coli. The 35A9 mutant identified a mtrR homolog of the N. gonorrhoeae (SwissProt P39897). The 25F1 mutant identified an operon that encoded 3 proteins that had identity with the orfT of C. tepidium, MPK, and DjlAEc. Sequences from 48d9, 35H7, and 12A1 corresponded with the lemA, gacA, and R genes, respectively. The sequences that were interrupted in mutants 41A5 and 44B1 did not have a significant similarity with any of the sequences in GenBank. (The label of sequence 44B1 is only 148 bp because no sequence corresponding to the insertion of 44B1 in the PAOl database was identified). In accordance with the above, these sequences identify additional virulence factors. In Figures 10, 11, 12, 13, 14A, 14B, 15, 16, 16A, 16B, 17, 18A, 18B, 18C, 18D, and 18E and Figures 22, 23, 24a, 24B, 24C, 24D, 24E, 25, 26, 27 and 28, the nucleotide and amino acid sequences that were obtained from these experiments are shown. We also performed a series of standard biochemical tests on the mutants of TnphoA, 41A5, 50E12, 41C1, 48D9, 12A1, 44B1, and 35H7, to determine if any contained a lesion in the known virulence factors of P. aeruginosa important for the pathogenicity of mammals. These tests included: a standard plaque assay for sensitivity to H202, as well as the standard quantitative analysis of extracellular protease, elastase, phospholipase C and pyocyanin. Except for the following, most of the PA14 Tnphoa mutants were indistinguishable biochemically from their parent PA14 strain. Mutant 12A1 exhibited decreased elastolytic and proteolytic activities but an overproduction of pyocyanin. The 50E12 mutant produced 3 times larger levels of pyocyanin than PA14. Mutant 41A5 only had approximately 70 percent of the wild-type levels of proteolytic activity. Now, in the following sections, a detailed description of DNA sequence analysis and biochemical analysis is presented for each of these mutants that were identified using the slow elimination assay (described above). Mutant 12A1. The TnphoA insert was inserted into 12A1 within codon 154 of the lasR gene of P. aeruginosa PAO1 that was previously described. The 12A1 phenotype, like other known LasR mutants, is pleiotropic, and includes decreased production of elastase and protease. In addition, 12A1 produced 2-3 times more pyocyanin than the parent PA14 strain in the stationary phase. Additionally, an lasR mutant expressing GFP (PA141asi ?; GFP19-1) could not be established in the bowels of the worm, since very little fluorescence was detected in the intestines of C. elegans after 48 hours of feeding. Figure 34A shows that the slow elimination phenotype of the defective 12A1 nematode was completely restored when the lasR gene of P. aeruginosa PAO1 was expressed in trans under the control of the constitutive lacZ promoter in strain 12A1 (pKDT17). It was also found that the elastase production was restored to wild type levels in 12A1 (pKDT17), but not the overproduction of pyocyanin. Because the overproduction phenotype of pyocyanin was not expected, we constructed a new lasR mutant, the lasR:; Gm, by means of swapping a laR gene interrupted by a gentamicin cartridge within the PA14 genome. The mutant lasR:; Gm was as nonpathogenic as 12A1 (Figure 34A), but produced normal levels of pyocyanin, suggesting that 12 Al could harbor a second mutation that resulted in superior regulation of pyocyanin production. The result also indicated that the higher regulation of pyocyanin production during the stationary phase is not related to the attenuated pathogenicity phenotype. Mutant yhol d. It was found that the cleavage of the dsbA gene in the phold was responsible for the non-pathogenic phenotypes. Figure 24B shows the nucleotide sequence (SEQ ID NO: 166) and predicted the amino acid sequence (SEQ ID NO: 167) of PAl Aphol d. It was also found that the defective pathogenicity phenotype of phol d in C. elegans was completely restored by constitutive expression of the dsbAEc gene of E. coli or the PA14dsh gene > APa in trans in the background of the phold (Figure 34B). For these experiments, the dsbAEc gene of E. coli was cloned into the Kpnl and Kbal sites of pBADld to form pCH3. This placed E. coli dsbA under the arabinose promoter of E. coli. A 700 bp Kpnl / Sphl fragment containing the dsbA of E. coli was cloned under the lacZ promoter of the constitutive E. coli. Subsequently, pEcdsbA was used to transform PA14 and phol d to construct strains PA14 (pEcdsba) and phold (pEcdsbA), respectively. The PA14dsjAPa was built as follows. and TMW9 (5'-TGACGTAGCCGGAACGCAGGCTGC-3 '; SEQ:; based on sequences of paol dsbA (GenBank accession number U84726 from) the TMW8 primers (SEQ ID NO 177 5' -GCACTGATCGCTGCGTAGC ACGGC-3 ') were used ID NO: 178) to amplify an 1126 bp fragment containing the dsbA gene plus 176 bp upstream of the translational start of the dsbA gene from the PA14 genomic DNA. This fragment was cloned, using the TA cloning kit (Invitrogen), into the vector pCR2.1, to generate the pCRdsh > A. The dsbA containing the Sacl / Xbal fragment was cloned into the pUCPld digested by Sacl / Xbal to construct the pPAdsbA, placing the transcription of dsbA under the constitutive lacZ promoter. The phol d strain (PAdsbA) was constructed by transforming the phold with the pPAds¿ > APa. Mutant 23 Fl. In 25F1, it was found that TnphoA was inserted into codon 100 of a putative gene (orf 338) that encodes a protein of 338 amino acids, the first gene of an operon of 3 putative genes. The predicted downstream genes (orf 224 and orf2d2) encode the 224 and 252 amino acid proteins, respectively. The GAP analysis showed that the 338 orf is 28.5 percent identical (37.7 percent similar) to the orfT of the C. tepudim (GenBank accession number U58313). The BLASTP ORF224 identified mannose-1-fosfatoguanililtrans-ferasa (MPG; EC 2.7.7.13) from eukaryotes, arqueabacterias, cyanobacteria and Mycobacteria but not proteobacteria, close relatives of P. aeruginosa. It is not clear if 0RF224 is a functional MPG since all known MPGs consist of residues of 359-388 amino acids, while ORF224 consists of residues of only 224 amino acids. ORE252 is homologous to Dj 1AEC of E. coli. It is believed that the DjlAEc plays a role in the correct assembly, activity and / or maintenance of a number of membrane proteins, including the signal transduction systems of the two-component histidine kinase. To test whether orf338 is the gene responsible for the reduced worms pathogenicity compared the kinetics of a strain carrying the orf338 alone, 2dFl (pORF338), with PA14 wild type and 25F1 wearing a vector alone. The 2dFl (pORF338) was constructed as follows. a fragment of the polymerase chain reaction of 1.8 kb containing the sequence of promoter upstream 482 bp, the complete orf 338 and 224 truncated (System High Fidelity Expand ™, Boehringer Mannheim) from DNA orf was amplified genomic analysis of PA14, using primers F2327 (5 '-CGAGGAATCCAGTCGAGGTG-3'; SEQ ID NO: 179) and R4180 (5'-GCAAGATGCAGCCGAGAGTAG-3 '; SEQ ID NO.180). The product was cloned into the vector pCR2.1 (TA Cloning, Invitrogen) to construct the plasmid pMT403C-R. The Sacl / Xbal fragment of pMT403C-R, which contained the product of the polymerase chain reaction, was cloned into the Sacl / Xbal of pUCP18 to construct pORF338, placing the 338 orf to make the 2dFl strain (pORF338) .

In addition, a chain containing the complete operon (orf 338, orf 224 and dj lAPa) was constructed as follows. A polymerase chain reaction strategy was used to amplify a 3.6 kb genomic fragment containing the 338 orf, orf 224, and dj lAPa and their upstream transcriptional sequences using primers RIF3115 (5'-GTCAGAATTCTCAGCTTGACGTTGTTGCCC-3 '; SEQ ID NO: 181) and RIR6757 (5'-GTCAGAATTCGACTTCTATTACCGCGACGCC-3 '; SEQ ID NO: 182). EcoRI sites (underlined) are present in the primers, but absent in the genomic sequence. The two strains of the polymerase chain reaction product were sequenced to determine the sequence of orf 338, orf 224, and djlAPa in strain PA14. The EcoRI digestion product of the polymerase chain reaction was cloned into the EcoRI site of pUCP18, and the orientation of the insert was determined by restriction digestion. The plasmid p3-ORFs was then used, where orf 338, orf224, and dj lAPa are under control by their native promoter to transform 25F1 to make the strain 2dFl (p3-ORFs). As shown in Figure 34C, strain 2dFl (pORF338) could not fully complement the slow elimination phenotype. Strain 2dFl (pORF338), which contained the complete operon (orf 338, orf 224, and djlAPa), also showed only partial complementation of the mutant phenotype. This result indicated that Tnphoa is responsible for the pathogenicity phenotype; Partial complementation may be a consequence of the dose of genes. The highest mortality achieved by strain 25Fl (0RFs) compared to strain 2dFl (pORF338) further suggested that genes downstream, ORF224 and / or DjlAPa may also play a role in the virulence of PA14. Figure 24A shows the nucleotide sequence (SEQ ID NO: 173) of the PA14 25F1 encoding the ORFT (SEQ ID NO: 174), ORFU (SEQ ID NO: 175), and DjlAPa (SEQ ID N0.176). Mutant d0E12. The Tnphoa insert was inserted into 50E12 within codon 39 of a predicted protein of 759 amino acids that 43 percent identical (54 percent similar) to the PtsPEc protein, a 738 amino acid protein containing a terminal Nif-A domain N and an Enzyme I domain with C terminal; the latter works in the phosphoenolpyruvate-dependent phosphotransferase system. It is believed that the Nif-A domain serves as a signal transduction function, either by directly sensing small molecule signals or receiving signals from a protein similar to NifL. Any of these mechanisms can modulate the catalytic activity of the Enzyme I domain; which in turn suggests the NPr of phosphorylate (HPr related to nitrogen) and regulates by the same the transcription of the operons dependent on RpoN. Immediately upstream of the PA14 homologue ptsPPa is the open reading frame (orfld9) that was predicted to encode a 159 amino acid protein that apparently is transcribed together with the ptsPPa. Figure 24C shows the nucleotide sequence (SEQ ID NO: 168) of PA14 50E12 encoding YgdPPa (SEQ ID NO: 169) and PtsPPa (SEQ ID NO: 170). The ORF159 is 62.3-64.8 percent identical to the YgdP proteins of unknown function that were found in H. influenzae (accession number of GenBank Q57045) and E. coli (accession number of GenBank Q46930). These proteins are closely related to invasion protein A in Helicobacter pylori and Bartonella Bacilliformis. The invasion protein a from B. bacilliformis (accession number of SwissProt P35640) is encoded by ailA, which, when presented together with an adjacent but independently transcribed gene, ailB, confers to E. coli the ability to invade human erythrocytes. For the complementation of dOEl2, two strains were tested: d0E12 (pMT206-lac) and dOE12 (pMT206-nat). The dOEl2 strain (pMT206-lac) carried the plasmid pMT206-lac, where the transcription of orfl59 and ptsPPa is under the control of the constitutive lacZ promoter. For the d0El2 strain (pMT206-nat), the transcription of orfld9 and ptsPPa is controlled only by its native promoter. Each of these strains was constructed as follows. A 4.3 kb fragment, containing the EcoRI site at both ends, was amplified from the genomic DNA of PA14 from P. aeruginosa, using these primers: RIF1698 (5'-GTCAGAAT TCGATGTTCFCAGTCCCAGATCCC-3 '; SEQIDNO: 183) and RIR6002 (5'-GTCAGAA TTCCAGTAGACCACCGCCGAGAG-3 '; SEQ ID NO: 184). This fragment was cloned into the EcoRI site of pUCP18 to make pMT206-lac and pMT206-nat; his identity was confirmed by restriction digestion. In pMT206-lac, the transcription of orfld9 and ptsPPa is under the control of both the constitutive lacZ promoter and its native promoter. Only its native promoter controls the transcription of orfld9 and ptsPPa in the pMT206-nat. As shown in Figure 34D, the two strains partially supplemented the phenotype mutants, with the time required by these strains being supplemented to eliminate 100 percent of the worms, longer than that of the wild-type strain. Partial complementation was observed in the mouse-burn test: the mortality of the mice after infection by 5 x 105 bacteria from the 50E12 strain (pMT206-nat) was 39 percent compared to 100 percent and 0 percent morality when they were infected with the wild type strain and 50E12, respectively. These results indicated that the putative operon ofrld9-ptsPPa is involved in the pathogenesis of P. aeruginosa in nematodes and mice. Mutant 33 A9. The TnphoA insertion in 33A9 is located in a putative 210 amino acid protein (encoded by orf210) that is more closely related (31.5 percent identity) to the MtrRNg protein of N. gonorrhoeae, which belongs to the TetR family of helix-turn-helix that contains proteins regulating bacterial transcription. The ORF210 is adjacent to, and transcribed in a divergent manner from, three genes that are homologous to the components of the energy-dependent emanation system (EDE) in P. aeruginosa. The analyzes of the sequences from PAOl showed that together, these four genes defined a novel energy-dependent emanation system (EDE) in P. aeruginosa. The other EDE systems in P. aeruginosa described above are the mexR system, mexA-mexB-oprK, the nfxB system, mexC-mexD-oprJ, and the nfxC system, mexE-mexF-oprN. Figure 24D shows the nucleotide sequence (SEQ ID? O: 171) of the PA14 35A9 encoding the mtrRPa (SEQ ID? O: 172). Mutants 37H7 and 1D7. Analysis of the product of the reverse polymerase chain reaction from the 37H7 mutant showed that there is a TnphoA insert within codon 188 of the GacA protein of 214 amino acids. The spotting analysis of AD? showed that 1D7 also contained an insertion in the gacA gene. Mutant 48D9. TnphoA was inserted between codon 491 and 492 of the 925 amino acid homologue LemA, a detector kinase belonging to a family of two-component bacterial regulators. The cognate response regulator of LemA in P. syringae is GacA and GacA + LemA have been shown to affect the expression of a varied number of virulence factors.

Mutant 41C1. The TnphoA was inserted into the AefA homologue of the integral membrane protein of the putative E. coli (SwissProt P77338) in the 41C1 mutant - This is a member of the UPF0003 protein family of 30-40 kD (PROSITE PDOC00959). In addition to E. coli, strain PCC 6803 from Synechocystis and Methanococcus jannaschii is also present. In addition, strains pho34B12, 3E8, 8C12, 1G2, 33A9, and 23A2 were also found to have a phenotypic mutant phenotype. In addition, it was found that the mutants pho34B12, 3E8, 8C12, and 1G2 were reduced in pigment production. An additional mutant, 6A6, was also identified as having reduced pigment. The color characteristics of P. aeruginosa strains have been attributed to a group of tricyclic secondary metabolites that are collectively known as phenacenes, the most widely characterized of which is the blue-green pigment, pyocyanin (l-hydroxy). -5-methylphenacin). In order to test whether the reduction of pigmentation in the bacterial mutants was due at least in part to the reduction in pyocyanin, the levels of this pigment were quantified in the wild-type PA14, as well as in all the mutants that were obtained using the rapid elimination test. The results of this analysis showed that pho34Bl2, 3E8, 8C12, 1G2, and 6A6 that had a reduced pigment phenotype, were also reduced in pyocyanin production, with levels ranging from 10 to 50 percent of the type strain. wild. The other mutants, 13C9, 23A2, and 36A4, had pyocyanin levels comparable to the wild-type strain. In addition, it was found that the sequence that interrupted the TnphoA mutation in 3E8, predicted a protein with homology with the phzB gene from Pseudomonas fluorescens, which is part of an operon involved in the production of the secondary metabolite, phenazine (number of GenBank accession L48616). The phzB gene also has a homologue in Pseudomonas aureo faciens, which is referred to as phzY. (GenBank accession number AF007801). Using the tag sequence, a cosmid (1G2503) containing this region was identified in the P. aeruginosa database, which contains the phzA and phzB genes, as well as other genes that are thought to play a role in the biosynthesis of phenazine, pcnC and D genes (accession number of GenBank AF0054054). Four of these strains, 34B12, 3E8, 23A12, and 3dA9, were examined for pathogenicity in the mouse burn test. Surprisingly, these experiments showed that defective phenazine strains had reduced pathogenesis, indicating that the genes that disrupted TnphoA inserts are virulence factors in mammals. In Figures 7-9, 13, 14A, 14B, 15, 16A, 16B, 17, 18A, 18B, 18C, 18D, 18E, 22, 24A, 24B, 24C, 24D, 24E, and 33, the sequences are shown of nucleotides and deduced amino acids, which include sequence tags, for these strains. In addition, Figures 25 and 26 show the nucleotide sequence of the phnA and phnB genes of Pseudomonas aeruginosa and the deduced amino acid sequence of PHNA, respectively. Now, in the following sections, a detailed description of the DNA sequence and biochemical analyzes of each of the mutants that were identified using the rapid elimination assay (described above) is presented. Mutants 36A4, 23A2, and 13C9. The DNA sequence tags that were obtained from the three mutants that produced the wild-type pyocyanin levels had homologies with the genes known in the Pseudomonads. Mutant 36A4 contained the TnphoA inserted into a gene homolog with hrpM, which was previously identified as a site that controls the pathogenicity in the plant pathogen Pseudomonas syringae (Mills and Mukhopadhyay, in: Pseudomonas: Biotransformations, Pathogenesis, and Evolving Technology, S. Silver, AM Chakrabarty, B. Iglewiski, and S. Kaplan, eds., American Society for Microbiology, 1990, pp. 47-57, Mukhopadhyay et al, J. Bacteriol 170: 5479-5488, 1988) (accession number of GenBank 140793). This site also has homology with the mdoH gene of E. coli, which encodes an enzyme that is involved in the biosynthesis of periplasmic glucans (Loubens et al., Mol.Microbiol.10: 329-340, 1993, accession number of GenBank X64197). The TnphoA insert was inserted into the 23A2 mutant into a gene that was previously identified in the PAO1 strain of P. aeruginosa as mexA (Poole et al., Mol.Microbiol.10: 529-544, 1993, GenBank accession number. L11616). The product of mexA, which was predicted to be a lipoprotein associated with the cytoplasmic membrane, probably functions together with the products of the other two genes contained in the same operon, mexB and oprM, as a non-ATPase emanation pump. with broad substrate specificity (Li et al., Antimicrob, Agents, Chemother, 39: 1948-1953, 1995). The sequence analysis of the DNA flanking the third mutant that was wild-type for the production of pigment, 13C9, showed that it corresponds to another gene previously known in the PAO1 strain of P. aeruginosa, the orp (number of GenBank access U54794). The orp, or osmoprotector-dependent regulator of phospholipase C, was identified as a factor controlling the expression of the pathogenicity PlcH, one of two isoforms of phospholipase C produced by P. aeruginosa (Sage et al., Mol. Microbiol. 23: 43-56, 1997). Mutants 1G2 and 8C12. Molecular analysis of the non-pigmented mutants 1G2 and 8C12 showed that they contained insertions within novel genes, although the DNA flanking the 1G2 insert contained a characteristic motif of histidine-sensing kinases. This gene was not present in the PAO1 genome database. Although the 8C12 sequence tag identified a homologous gene in the PAOl database, no significant motifs were found within this gene. Mutants 3E8 and 6A6. Two mutants, 3E8 and 6A6, contained TnphoA inserts within the same gene, which was homologous with the phzB gene that was previously identified in strain 2-79 of P. fluorescens (accession number of GenBank AF007801) and the phzY in P. aureofaciens, strain 30-84 (access number of GenBank L48616). These two mutants contained the TnphoA insert in exactly the same position, however, they were isolated independently since they were obtained from two libraries of different mutants. Although the phzA and the phzY did not contain identifiable sequence motifs, they were present in the operons that are known to regulate the production of phenacyl-1-carboxylate (PCA) in both P. fluorescens and P. auerofaciens (Mavrodi and collaborators, J, Bacteriol 180: 2541-2548, 1998). Mutant pho34Al2. The DNA flanking the TnphoA insert in the final non-pigmented pho34B12 mutant was previously cloned and shown to be a novel site as described infra. Interestingly, this insertion is immediately downstream of the biosynthetic genes of phenazine, phnA and phnB, as identified in the PAO1 strain of P. aeruginosa (Essar et al., J, Bacteriol., 172: 884-900, 1990). Phenacines are required for the rapid elimination of C. elesans The isolation of both pigmented and non-pigmented mutants in the rapid elimination selection indicated that the rapid elimination process included more than one factor. However, molecular analysis of 3E8 and 6A6 mutants (which contained insertions in an operon known to regulate phenazine production) strongly suggested that phenacins represent a class of toxins involved in rapid elimination. In order to directly test this hypothesis, an additional mutation, the? PhnA phnB, was generated and studied as follows. The biosynthetic genes of phenazine phnA and phnB (Essar et al, J. Bacteriol 172: 884-900, 1990) lie upstream of the TnphoA insert of pho34B12 in PA14; (access number of GenBank AF031571). An EcoRI fragment of 3.7 kb corresponding to the wild-type sequence of this region (from plasmid pLGR34) was subcloned into pBluescript SK / + to produce Bs34B12. This plasmid contained 944 bp of phnA (full length of 1591 bp), the complete phnB gene (600 bp) and 1.7 kb of downstream sequences. The remaining 605 bp of the phnA and the 405 bp upstream were amplified, using the polymerase chain reaction from the PA14 genomic DNA with the oligonucleotide primers PHNA3 (5'-GGTCTAGA CGAACTGAGCGAGGAG-3 '; SEQ ID NO: 185) and PHNA2 (5 '-GCCTGCAGGCG TTCTACCTG-3'; SEQ ID NO: 186). The primers were based on the sequence of the phnA and PhnB genes that were previously cloned from the PAO1 strain of P. aeruginosa (Essar et al, J. Bacteriol 172: 884-900, 1990, GenBank accession number M33811 ). The 1010 bp amplified sequence was subcloned into the PstI sites of pBs34B12 to give the construct, pBs34Bl2phnA. A deletion was generated in the frame within phnA, phnB, by replacing 2.6 kb of the wild type sequence of the genes with 1 kb fragment (Figure 35) that was amplified by the polymerase chain reaction, using the primers PHNDEL1 (5 '-GGCTGCAGTGATTGACTGAGCGTCTGCTGGAGAACG-3'; SEQ ID NO: 187) and PHNDEL2 (5'-GGGAAGCTTCGTCTAGAATCACGTGAACA TGTTCC-3 '; SEQ ID NO: 188), to produce the plasmid pBs34bl2phndel. A 1.8 kb XbaI fragment containing the in-frame deletion of phnAphnB was subcloned into the suicide positive sucrose selection vector pCVD442 (Donnenberg and Kaper, Infect.Immun.59: 4310-4317, 1991). The resulting construct, pBs34bl2phndel, was used to introduce the phnA, phnB genes interrupted into the wild-type PA14 genome by homologous recombination resulting in the mutant PA14 AphnAphnB. DNA restriction and DNA staining analyzes were undertaken using the paternal PA14 DNA and the AphnAphnB strains of the derivative PA14 in order to verify that the mutant contained the desired suppression. Although little is known about the nature of the enzymes that catalyze the formation of phenazines in P. aeruginosa and related Pseudomonads, it is thought that the conversion of crosimate to anthranilate is a key step in the trajectory (Figure 35A). In the PAOl strain of P. aeruginosa, this step is most likely catalyzed by the anthranilate synthase encoded by the phnA and PhnB genes, because mutations in these genes result in a decreased production of phenazine pyocyanin (Essar et al, J. Bacteriol, 172: 884-900, 1990). The phnA and phnB genes were cloned from PA14 (Figure 35B). Importantly, this mutation was designed to be non-polar and therefore, it will not affect the two ORFs that are shown directly upstream of phnA and phnB (infra). Measurement of pyocyanin in the mutant? PhnAphnB showed that it generated only 10 percent of the wild-type levels, confirming that phnA and phnB are involved in the production of pyocyanin in strain PA14, such as in the PAOl. The trials that were conducted using the? PhnAphnB, revealed that this strain was severely reduced in rapid elimination. As seen in Figure 35C, less than 5 percent of the worms were dead three hours after exposure to? PhnAphnB, in contrast to the 100 percent case that they were exposed to the wild-type strain. The? PhnAphnB strain behaved in a manner similar to the other phenazine mutant, 3E8, which served as the control for an attenuated mutant in this experiment. These results demonstrated that phenacines are required for the rapid elimination of C. elegans. To discover if the bacterial factors involved in rapid elimination are relevant for pathogenesis in other hosts, the rapid elimination mutants were tested for virulence in the Arabidopsis leaf infiltration model, as well as the skin burn model. of full thickness mouse (infra). Five rapid elimination mutants were tested for the culture over the course of four days in Arabidopsis leaves as a quantitative measure of their pathogenicity and also in the full-thickness mouse skin burn model. As shown in Tables 4 and 5, the maximum growth level in Arabidopsis leaves on the fourth day after infection was significantly lower for 2 of the phenazine mutants, 3E8 and 8C12. In the mouse model, these two mutants caused significantly less mortality than the wild-type strain with a P < 0.05 when a 5 X 105 cell inoculum was used. The third phenazine mutant, 1G2, was not significantly different from the wild type strain in any of the plant or mouse models. Both the hrpM mutant, 36A4, and the mexA mutant, 23A2, were severely weakened in Arabidopsis leaf culture, indicating a strong pathogenicity defect in this model. In the mouse model, mutant 36A4 had a dramatic effect that did not cause mortality at the dose that was tested. In contrast, the mutant mexA, 23A2 was only marginally affected. These results demonstrated that the rapid elimination selection is extremely effective in identifying the genes required for pathogenesis in both plants and mice, and also provides the first in vivo demonstration that phenacenes are required for pathogenesis in these two hosts. We also noticed that we have identified a regulator, the phzR of the phz operon. Figure 18E shows the nucleotide sequence (SEQ ID NO: 164) and predicted the partial amino acid sequence (SEQ ID NO: 165) of the phzR of PA14. Fenacinas and pathogenesis. The PA14 mutants reduced in the rapid elimination also affected the synthesis of the pigment. Our molecular analysis revealed that the association between pigment production and pathogenesis was not simply due to the coordinated regulation of pigmentation and the production of toxins by regulatory factors. Instead, we found that the mutations in the biosynthetic genes of phenazine were reduced in virulence, strongly implicating the phenacins as toxins in the rapid elimination process. Phenacenes, pigmented tricyclic compounds that give Pseudomonads their characteristic colors (Turner and Messenger, Adv. Microb Physiol.27: 211-273, 1986), are secondary metabolites that are thought to increase the survival of organisms under competitive factors (Maplestone et al., Gene 115: 151-157, 1992). Although the repertoire of phenacines produced by PA14 is unknown, the PAO1 strain of P. aeruginosa produces at least six different phenacines, including the well-characterized blue-green pigment pyocyanin. Phenaceins, including pyocyanin, have been shown to have antibiotic action against different species of bacteria, fungi, and protozoa, a quality that is attributed to their active redox. In its highly reactive reduced state, it has been reported that phenacins undergo a redox cycle in the presence of different molecular oxygen or reduction agents resulting in the formation of superoxide and hydrogen peroxide (Hassan and Fridovich, J. Bacteriol. 141: 1556-163, 1980). In vitro, these moderately cytotoxic oxygen radicals can be converted by an iron catalyst to the highly cytotoxic hydroxyl radical (Britigan et al., J. Clin., Invest 90: 2187-2196, 1992). It is also believed that the formation of the reactive oxygen species by the phenacins contributes to their cytotoxic effects that are observed in eukaryotic cells in vitro. These effects include inhibition of cellular respiration in mammals, interruption of the ciliary beat, and immunomodulatory effects such as stimulation of the inflammatory response, inhibition of lymphocyte proliferation and alteration of the response of the T lymphocyte to antigens. .

The bisosynthetic pathways that lead to the production of phenacins in P. aeruginosa have been poorly defined, making it difficult to identify steps in the pathway that block PA14 mutants defective in the production of phenazine. However, transposon insertion in two mutants, 3E8 and 6A6, broke a gene with homology to phzB, which had previously been characterized as being included in the production of phenazine in the related Pseudomonads, P. fluorescens and the P. aureo faciens. In P. fluorescens, it was shown that phzB was part of an operon of seven genes (phzA-G) included in the production of phenacene-1-carboxylic acid. Comparison of this operon in P. fluorescens and P. aureofaciens showed that both were highly homologous, suggesting that the trajectories leading to the production of phenazine are conserved in the fluorescent Pseudomonads (Mavrodi et al., J. Bacteriol 180: 2541-2548, 1998). Although the DNA flanking the phzA and phzB genes has been sequenced only partially in the PA14 strain of P. aeruginosa, our analysis suggests that the region shares a conserved structure with the phzA-F operon of P. fluorescens. The predicted products that were transformed from the phzA and phzB genes from PA14 and P. fluorescens, share 68 and 74 percent identity, respectively. In addition, a region containing the genes similar to phzA-F is present in the PAOl strain of P. aeruginosa, and the predicted products that were transformed from these genes, exhibited between 69 and 85 percent identity with their counterparts of P. fluorescens (accession number of GenBank AF005404). Extrapolation from the role of the phz operon in P. fluorescens and P. aureo faciens, the isolation of the phzB mutants of PA14 that are defective in rapid elimination strongly suggests that phenazines are involved in this process. The hypothesis was further tested that phenazines, including pyocyanin, are one of the mediators of rapid elimination by non-polar disruption of the genes, phnA and phnB, which encode the two subunits of an anthranilate synthase, which is previously showed that it was specifically included in the synthesis of phenazine in the PAO1 strain of P. aeruginosa (Essar et al, J. Bacteriol 172: 884-990, 1990). Consistent with a role in the biosynthesis of phenazine, the deletion of the phnA and phnB genes in PA14, severely reduced the production of phenazine. Additionally, the mutant? PhnaphnB was defective in rapid elimination, demonstrating the critical role of the phenacins in this process. The role of phenacenes in pathogenesis was also examined in Arabidopsis and in mice. The two functional mutants that contained the inserts within the phzB gene, 3E8 and 6A6, were dramatically reduced in pathogenicity in both the Arabidopsis leaf infiltration model and in the full-thickness mouse skin burn model (Tables). 4 and 5), suggesting that phenacins are pathogenicity factors of multiple hosts. It is interesting to note that many of the other pathogenicity factors of multiple hosts that were identified in this and our previous studies are likely to be involved in the production of several other virulence factors and are not effectors, or molecules that interact directly with the host (described below). In this way, phenacins represent the only known class of multi-host pathogenicity effectors that we have identified. These findings are also significant since, regardless of the in vitro intensive analyzes of the phenacins, the physiological significance of their production and their role in P. aeruginosa infections remains controversial, and prior to this study, there has not been a demonstration of his role in vivo. The rapid elimination is multifactorial The analysis of the rapid elimination mutants that generated levels of wild-type pigments, showed that although phenacins were essential mediators of rapid elimination, there were other factors involved in this process. Molecular analysis of one of these mutants, 23A2, revealed that the transposon was inserted into a gene that had previously been identified in the PAO1 strain of P. aeruginosa as the MexA, which is part of the operon of 3 MexA genes. , B, OprM (Poole et al., Mol.Microbiol.10: 529-544, 1993). The products of these genes are located in the cytoplasmic (MexA, MexB) and in the outer membranes (OprM) where it is proposed that they function as a broad specific emission pump of non-ATPase (Li et al., Antimicrob. Agents Chemother 39: 1948-1953, 1995). Originally identified due to its contribution to the multi-drug resistance process in P. aeruginosa, it is believed that this pump plays a general role in the export of secondary metabolites, although its natural substrates remain unknown (Poole, Antimicrob Agents Chemother. : 453-456, 1994). The defect of the mexA mutant in rapid elimination, a process mediated by diffusible toxins, is most likely due to the lack of export of one or more factors involved in the process. Because the mutant mexA was pigmented, it is unlikely that the phenacins are a substrate for the pump. In addition to its defect in rapid elimination, the mexA mutant was marginally reduced in pathogenicity in the mouse model and severely weakened in the Arabidopsis leaf infiltration model. Although the lack of export of specific virulence factors could explain these defects, an additional model is that bacteria of the mutant mexA can not protect themselves against the host defense factors that are generated in response to bacterial infection. This protective function has been demonstrated for the sap genes, which encode proteins related to the ATP binding cartridge (ABC) transporters and mediate resistance to the antimicrobial peptides of the host in the human pathogen, Salmonella typhimurium, as well as in the pathogen Erwinia chrysanthemi (Taylor, Plant Cell 10: 873-875, 1998). A second mutant that was identified in the selection, 36A4, contained a transposon insert within a gene with homology to the E. coli MdoH, which is part of the mdoGH operon. In E. coli, the products of this operon are included in the synthesis of membrane-derived oligosaccharides (MDO) or linear, periplasmic glucans (Loubens et al., Mol.Microbiol.10: 329- 340, 1993). A similar site, named hrpM, is present in the pathogen of plants Pseudomonas syringae pv. syringae (Mukhopadhyay et al, J. Bacteriol 170: 5479-5488, 1988), which was originally identified because the mutations in place abolished both the development of disease symptoms in the host plants, as well as the hypersensitive response in non-host plants (Anderson and Mills, Phytopath, 75: 104-108, 1985). Periplasmic glucans have also been found in a wide range of gram-negative bacteria, where diverse, but poorly understood functions have been assigned. In addition to being essential virulence factors in P. syringae, other functions include adaptation to hypoosmotic environments, and cell signaling leading to the recognition of eukaryotic hosts by Rhizobium and Agrobacterium species (Kennedy, in: Escherichia and Salmonella, FC Neidardt, ed., American Society for Microbiology Press, Washington, DC, pp. 1064-1071, 1996). However, despite being present in the periplasm of different animal pathogens such as Salmonella and Klebsiella, up to this study, which shows that P. aeruginosa carries a mutation in a place similar to mdoH is severely reduced in pathogenicity in a mouse model, periplasmic glucans have not been shown to play a role in the infection of animal hosts.

Table 4 Summary for the Pathogenicity of the UCBPP-PA14 mutants of the P. aeruginosa strain on different hosts Pathogenicity Phenotypes Number Leaf Culture Name Capacity for% Mortality Gene Identity Isolation of the Arabidopsis strain eliminate the C. of mice the strain elegans0 5X105"PA14 PA14 5.5 X 107 100 rep (reduced pathogenicity in plants) 16G12 repl 2.3 x 105 + 100 without correspondence 49H2 rep2 1.2 x lO6 + 63 not sequenced 25A12 rep3 1.7 x lO6 75 without correspondence 33A9 rep4 5.1 x 106 + 0 without correspondence 33C7 rep5 8.4 105 + 0 without correspondence ren (reduced patosenicity in nem? All) 35A9e renl 5.7 x 107 55 mtrR 44B1 ren2 5.4 x 107 56 without correspondence lG2f'8-h NT NT without correspondence 8C12f * h NT NT without correspondence 2A8f-h NT NT without correspondence rpn (reduced ptosinicity in plants and nem? All) 25F1 rpnl 1.5 x lO4 20 orfT 35H7C rpn2 1.2 x lO4 NTC gacA 41A5 rpn3 1.3 x lO4 100 without correspondence 41C1 rpn4 2.4 x 105 85 aefA 50E12 rpn5 2.0 x 105 0 ptsP phol5 rpnó 3.9 x lO4 62 dsbA 12A1 rpnJ 1.7 x lO6 50 lasR pho23 rpn8 6.4 x 10"5 without correspondence 34B12 &hrpnll 4.0 x 104 50 dst * from phnB 34H4 rpnl2 3.8 x lO6 50 without correspondence 3E8g-h rpnl 3 lx lO6 12.5 phzB 23A2h rpnl4 1.7 x lO5 71 mexA 36A4h rpnl 5 4 x 10"- O hrpN b Leaf area CFU / cm2 of bacterial counts at four days of inoculation of bacteria of 103: media from four to five samples.Mutants are defined as less pathogenic when the CFU / cm2 intermediate leaf area of the bacterial counts is 2 standard deviations lower than the wild type within the same set of experiments c A mutant is considered attenuated in pathogenicity of the nematode (-) if the average time required to eliminate 50 percent of the worms that feed on it (LT50 of 3 replicates) is two standard deviations less than the LT50 of the maternal UCBPP-PA14 in the same experiment; LT50 calculations, see Materials and Methods. d Six-week-old male ARK / J inbred strain mice (from Jackson Laboratories) weighing between 20 and 30 grams with 5 x 105 cells were injected as described by Stevens et al., J. of Burn Care and Rehabil. 15: 232-235, 1994. The number of animals that died of sepsis daily for ten days was monitored. c Two other gacA mutants isolated independently are ID7 (rpn9) and 33D11 (rpnl O). The rpn9 mutant has been tested in mice and showed a 50 percent mortality. f tested only in nematodes g mutants defective in phenazine mutants defective in rapid elimination, unaffected in the slow elimination dst * = downstream Table 5 Pathogenicity in Rapid Elimination Mutants PA14 in Plants and Mice Cepa Cultivation in Hojab% Mortality (n) Identity of the Arabidopsis Gene of Mice 5X10sd PA14 7 X lO8 100 (> 16) 1G2 3 x l07 100 (8) without correspondence , contains the histidine kinase motif i-ES, 6A6 3 x 105 18 (16) phzB 8C12 5 x l05 63 (8) without correspondence 23A2 1.2 x lO4 85 (16) mexA 36A4 2 x 10"0 (16) hrpM Leaf area CFU / crtr of bacterial counts at five days of inoculation of bacteria of 103. Values represent average terms of four to five samples.Mutants are defined as less pathogenic when the mean value of bacterial counts is two standard deviations lower than the wild type within the same experimental set b Six-week old ARK / J inbred male mice (from Jackson Laboratories) weighing between 20 and 30 grams with 5 x 105 bacterial cells were injected. (n) is the total number of mice that were injected. The number of mice that died of sepsis daily for seven days was monitored. ° 3E8 and 6A6 are independently generated mutants that contain the TnphoA inserted exactly in the same place. The numbers that are reported are those that are obtained using the 3E8. Similar results were obtained with 6A6 (data not shown). Isolation of Additional Virulence Genes Based on the nucleotide and amino acid sequences described herein, it becomes possible to isolate additional coding sequences from virulence factors, using the standard strategies and techniques that are well known in the art. technique. Any pathogenic cell can serve as the source of nucleic acid for the molecular cloning of this virulence gene, and these sequences are identified as those that encode a protein that exhibits structures, properties, or activities associated with pathogenicity. In a particular example of this isolation technique, any of the nucleotide sequences described herein can be used, along with conventional selection methods of nucleic acid hybridization screening. These hybridization techniques and selection procedures are well known to those skilled in the art and are described, for example, by Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Nati, Acad. Sci., USA 72: 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 1997); Bergery Kimmel (supra); and Sambrook et al., Molecular Cloning: A Labora tory Manual, Cold Spring Harbor Laboratory Press, New York. In a particular example, all or part of the sequence of 33A9 (which is described herein) can be used as a probe to screen a DNA library of recombinant plants for genes that have a sequence identity with the 33A9 gene ( Figures 5 and 6A-B). Hybridization sequences are detected by hybridization of the plate or colony, in accordance with standard methods. Alternatively, using all or a portion of the amino acid sequence of the 33A9 polypeptide, one can readily design the oligonucleotide probes of 33A9, which include the degenerate oligonucleotide probes (i.e., a mixture of all possible coding sequences). for a given amino acid sequence). These oligonucleotides can be based on the sequence of any DNA strand and on any appropriate portion of the 33A9 sequence (Figures 5 and 6A-B; SEQ ID NOs: 102 and 103, respectively) of the 33A9 protein. General methods for designing and preparing these probes are provided, for example, in Ausubel et al. (Supra), and in Berger and Kimmel, Guide to Molecular Cloning Techniques, 1987, Academic Press, New York. These oligonucleotides are useful for the isolation of the 33A9 gene, either through its use as probes that can hybridize to the complementary sequences of 33A9 or as primers for different amplification techniques, for example, cloning strategies of the polymerase chain reaction (PCR). If desired, a combination of detectably labeled oligonucleotide probes may be used, different for the selection of a recombinant DNA library. These libraries are prepared according to methods well known in the art, for example, as described by Ausubel et al. (supra), or can be obtained from commercial sources. As discussed above, sequence specific oligonucleotides can also be used as primers in amplification cloning strategies, for example, using the polymerase chain reaction. Polymerase chain reaction methods are well known in the art and are described, for example, in PCR Technology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, Innis et al., Eds., Academic Press, Inc., New York, 1990; and Ausubel et al. (supra). The primers are optionally designed to allow cloning of the amplified product into a suitable vector, for example, by including the appropriate restriction sites at the 5 'and 3' ends of the amplified fragment. (as described herein). If desired, the nucleotide sequences can be isolated using the "RACE" technique of the polymerase chain reaction, or the Rapid Amplification of the cDNA ends (see, eg, Innis et al. (supra)). By this method, oligonucleotide primers that are based on a desired sequence are oriented in the 3 'and 5' directions and are used to generate fragments of the overlapping polymerase chain reaction. These 3'- and 5'-end-terminated RACE products are combined to produce an intact full-length cDNA. This method is described in Innis et al. (Supra); and in Frohman et al., Proc. Nati Acad. Sci. , USA 85: 8998, 1988. Partial virulence sequences, eg, sequence tags, are also useful as hybridization probes to identify sequences of full lengths, as well as to select databases to identify genes for related virulence that had not previously been identified. For example, the sequences of 36A4, 25A12, and 33C7 were expanded for those encompassed by contigs 2507, 1126, and 1344, respectively. Confirmation of a sequence affinity with a pathogenicity polypeptide can be achieved by a variety of conventional methods including, but not limited to, functional complementation assays and sequence comparison of the gene and its expressed product. In addition, the activity of the gene product can be evaluated in accordance with any of the techniques described herein, for example, the functional or immunological properties of its encoded product. Once an appropriate sequence is identified, it is cloned in accordance with standard methods and can be used, for example, to select compounds that reduce the virulence of a pathogen. Expression of the Polypeptide In general, the polypeptides of the invention can be produced by transforming a suitable host cell with all or part of a nucleic acid molecule or fragment thereof encoding the polypeptide in a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to provide the recombinant protein. The host cell requires that it is used, it is not critical to the invention. A polypeptide of the invention can be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insert cells, e.g., Sf21 cells, or mammalian cells, for example, NIH 3T3, HeLa, or preferably COS cells). These cells are available from a wide range of sources (for example, the American Type Culture Collection, Rockland, MD; also, see, for example, Ausubel et al., Supra). The transformation or transfection method and the selection of the expression vehicle will depend on the host system that was selected. Transformation and transfection methods are described, for example, Ausubel et al. (Supra); Expression vehicles can be selected from those provided, for example, in Cloning Vectors: A Labora tory Manual (P.H. Pouweis et al., 1985, Supp. 1987). A particular bacterial expression system for the production of polypeptides is the expression system of E. coli (Novagen, Inc., Madison, Wl). In accordance with this expression system, the DNA encoding a polypeptide is inserted into a pET vector in an orientation that is designed to allow expression. Because the gene encoding this polypeptide is under the control of T7 regulatory signals, expression of the polypeptide is achieved by inducing expression of the T7 RNA polymerase in the host cells. This is typically achieved by using host strains that express the T7 RNA polymerase in response to the induction of IPTG. Once produced, the recombinant polypeptide is isolated according to standard methods known in the art, for example, those described herein. Another bacterial expression system for the production of polypeptides is the expression system pGEX (Pharmacia). This system employs a fusion system of the GST gene, which is designed for the high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of the functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S protein transferase from Schistosoma japonicum and is easily purified from the bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion proteins can be recovered under mild conditions by levigation with glutathione. Dissociation of the S-transferase domain of glutathione from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins that are expressed in plasmids pGEX-2T can be dissociated with thrombin; those that are expressed in pGEX-3X can be dissociated with factor Xa. Once the recombinant polypeptide of the invention has been expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., which occurs as described herein) that was raised against a polypeptide of the invention can be bound to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of the cells harboring the polypeptide before affinity chromatography can be performed by standard methods (see, for example, Ausubel et al., Supra). Once isolated, the recombinant protein can, if desired, be further purified, for example, by high performance liquid chromatography (see, for example, Fisher, Labora tory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980). The polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Con., Rockford, IL. ). These general techniques of expression and purification of the peptide can also be used to produce and isolate useful peptide fragments or analogues (described herein). Antibodies To generate antibodies, a coding sequence for a polypeptide of the invention can be expressed, such as a C-terminal fusion with glutathione S transferase (GST) (Smith et al., Gene 67: 31-40, 1988). The fusion protein is purified on glutathione-Sepharose pellets. levigated with glutathione, dissociated with thrombin (at the designed dissociation site), and purified to the extent necessary for the immunization of rabbits. Primary immunizations were performed with Freund's complete adjuvant and subsequent immunizations with incomplete Freund's adjuvant. Antibody titers are monitored by Western blot and immunoprecipitation assays, using the thrombin dissociated protein fragment of the GST fusion protein. The immune serum is purified by affinity using the coupled protein of CNBr-Sepharose. The specificity of the antiserum is determined using a panel of unrelated GST proteins. As an alternative immunogen or adjunct to the GST fusion proteins, peptides corresponding to relatively unique immunogenic regions of a polypeptide of the invention can be generated and coupled with orifice limpet hemocyanin (KLH). through a terminal C lysine that was introduced. The antiserum of each of these peptides is also purified by affinity in the peptides that are conjugated with bovine serum albumin, and the specificity is tested in the enzyme-linked immunosorbent assay and Western blots using the peptide conjugates, and by Western blotting and immunoprecipitation using the polypeptide that was expressed as a GST fusion protein. Alternatively, monoclonal antibodies that specifically bind any of the polypeptides of the invention, were prepared in accordance with standard hybridoma technology (see, for example, Kohler et al., Na ture 256: 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; Ausubel et al., Supra). Once they were produced, the monoclonal antibodies are also tested for specific recognition by Western blotting or immunoprecipitation analysis (by the methods described in Ausubel et al., Supra). Antibodies that specifically recognize the polypeptide of the invention are considered useful in the invention; these antibodies can be prepared using the polypeptide of the invention described above and a phage display library (Vaughan et al., Na ture Biotech 14: 309-314, 1996). Preferably, the antibodies of the invention are produced using fragments of the polypeptide of the invention, which lie outside generally conserved regions and which appear to be probably antigenic, by criteria such as the high frequency of charged residues. In a specific example, these fragments are generated by standard techniques of polymerase chain reaction and are cloned into the pGEX expression vector (Ausubel et al., Supra). The fusion proteins are expressed in E. coli and purified using an affinity matrix of glutathione agarose, as described by Ausubel et al. (Supra). To try to minimize the potential problems of the low affinity or specificity of the antisera, two or three fusions are generated for each protein, and each fusion is injected into at least two rabbits. The antisera are collected by injections in a series, preferably including at least three booster injections. Antibodies against the polypeptides described herein can be used to treat bacterial infections. Selection Trials As discussed above, we have identified a number of virulence factors of P. aeruginosa that are involved in pathogenicity and that can therefore be used to select compounds that reduce the virulence of that organism, as well as other microbial pathogens. For example, the invention provides methods for selecting compounds to identify those that improve (agonists) or block (antagonists) the action of a polypeptide or the expression of a gene of a nucleic acid sequence of the invention. The method to select may include high performance techniques. Any number of methods are available to perform these selection trials. In accordance with one approach, the candidate compounds are added in varying concentrations to the culture medium of the pathogenic cells expressing one of the nucleic acid sequences of the invention. The expression of the gene is then measured, for example, by standard Northern blot analysis (Ausubel et al., Supra), using any appropriate fragment that has been prepared from the nucleic acid molecule as a hybridization probe. The level of expression of the gene in the presence of the candidate compound is compared to the level that was measured in a control culture medium lacking the candidate molecule. A compound that promotes a decrease in the expression of the pathogenicity factor is considered useful in the invention; this molecule can be used, for example, as a therapeutic to combat the pathogenicity of an infectious organism. If desired, the effect of candidate compounds can be measured in the alternative, at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for a pathogenicity factor. For example, immunoassays can be used to detect or monitor the expression of at least one of the polypeptides of the invention in a pathogenic organism. Polyclonal or monoclonal antibodies (which are produced as described above) can be used which can bind to that polypeptide in any standard immunoassay format (e.g., the enzyme linked immunosorbent assay, Western blotting, or the RIA assay), to measure the level of the pathogenicity polypeptide. A compound that promotes a decrease in expression of the pathogenicity polypeptide is considered particularly useful. Again, this molecule can be used, for example, as a therapeutic to combat the pathogenicity of an infectious organism. Alternatively, or in addition, candidate compounds can be selected by those that specifically bind to, and inhibit a pathogenicity polypeptide of the invention. The efficacy of this candidate compound depends on its ability to interact with the pathogenicity polypeptide. This interaction can easily be tested using any number of standard fixation techniques and functional assays (for example, those described by Ausubel et al., Supra). For example, a candidate compound can be tested in vivo for interaction and binding with a polypeptide of the invention and its ability to modulate pathogenicity can be tested by any standard assays (eg, those described in FIG. I presented) . In a particular example, a candidate compound that binds to a pathogenicity polypeptide can be identified using a technique based on chromatography. For example, a recombinant polypeptide of the invention can be purified by standard techniques from cells designed to express the polypeptide (e.g., those described above) and can be immobilized on a column. A solution of the candidate compounds is then passed through the column, and a compound specific for the pathogenicity polypeptide is identified on the basis of its ability to bind to the pathogenicity polypeptide and immobilized on the column. To isolate the compound, the column is washed to remove the fixed molecules in a non-specific manner, and then the compound of interest is released from the column and harvested. The compounds isolated by this method (or any other suitable method) can, if desired, be further purified (for example, by high performance liquid chromatography). In addition, these candidate compounds can be tested for their ability to produce a less virulent pathogen (e.g., as described herein). Compounds that are isolated by this approach can also be used, for example, as therapeutics to treat or prevent the establishment of a pathogenic infection, disease, or both. Compounds that are identified as binding to the pathogenicity polypeptides with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. In yet another approach, candidate compounds are selected for the ability to inhibit the virulence of a Pseudomonas cell by monitoring the effect of the compound on the production of a phenazine (e.g., pyocyanin). In accordance with one approach, the candidate compounds are added in varying concentrations to a culture medium of pathogenic cells. The pyocyanin is then measured according to any standard method, for example, by monitoring its absorbance at 520 nm in acidic solution (Essar et al, J. Bacteriol 172: 884, 1990). To maximize the production of pyocyanin in the liquid culture for quantification, the cells can be cultured in a modified KA broth (King et al., J. Lab. Clin. Med. 44: 301, 1954), by adding 100 μM. of FeCl3. The level of pyocyanin production in the presence of the candidate compound is compared to the level that was measured in the control culture medium lacking the candidate molecule. A compound that promotes a decrease in the expression of a pyocyanin, is considered useful in the invention; this molecule can be used, for example, as a therapeutic to combat the pathogenicity of an infectious organism. Similar techniques can also be used to select other suitable phenacines including, without limitation, piorubine, aeruginosine A, myxin, and tubermycin A. Other phenazines are described in Turner and Messenger (Advances In Microbial Physiology 27: 211-1275, 1986), Sorensen and Joseph (in: Pseudomonas aeruginosa as an Opportunistic Pa thogen, Campa, M. ed., Plenum Press, NY, 1993), Ingram and Blackwood (Advances in Applied Microbiology 13: 267, 1970), and Gerber ( in: CRC Handbook of Microbiology, Laskin, AI, and Lechevalier, eds., 2nd edition, volume 5, Chemical Rubber Con., Cleveland, Ohio, 1984, pp. 573-576). Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind to a nucleic acid or polypeptide sequence of the invention and thereby inhibit or extinguish their activity. Potential antagonists also include small molecules that bind to, and occupy, the binding site of the polypeptide, thereby avoiding binding to cellular binding molecules, so that normal biological activity is avoided. Other potential antagonists include antisense molecules. Each of the DNA sequences provided herein can also be used in the discovery and development of antipathogenic compounds (e.g., antibiotics). The encoded protein, after expression, can be used as a target for the selection of antibacterial drugs. Additionally, DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation that facilitates the sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest. The invention also provides the use of the polypeptide, polynucleotide, or inhibitor of the invention, to interfere with the initial physical interaction between a pathogen and a mammalian host responsible for the infection. In particular, the molecules of the invention can be used: in the prevention or adhesion and colonization of bacteria to mammalian extracellular matrix proteins; to extracellular matrix proteins in wounds; to block the invasion of mammalian cells; or to block the normal progress of pathogenesis. The antagonists and agonists of the invention can be used, for example, to inhibit and treat a variety of bacterial infections. Optionally, compounds that were identified in any of the assays described above can be confirmed as being useful for conferring protection against the development of a pathogenic infection in any standard animal model (e.g. mouse described herein) and, if successful, can be used as an anti-pathogen therapeutic (eg, antibiotics). Test Compounds and Extracts In general, compounds that can reduce pathogenic virulence are identified from large libraries of extracts from both the natural or synthetic (or semi-synthetic) product or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of the test extracts is not critical to the selection procedure (s) of the invention. In accordance with the above, virtually any number of chemical extracts or compounds can be selected using the methods described herein. Examples of these extracts or compounds include, but are not limited to, extracts based on plants, fungi, prokaryotes or animals, fermentation broths, and synthetic compounds, as well as modification of existing compounds. There are also numerous methods available to generate randomized or directed syntheses (for example, semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, compounds based on saccharides, lipids, peptides, and nucleic acids. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenove (Slough, UK), Harbor Branch Oceanographics Institute (Ft. Pierce, FL), and PharmaMar, USA (Cambridge, MA). In addition, libraries that are produced naturally or synthetically are produced, if desired, in accordance with methods known in the art, for example, by standard extraction and fractionation methods. Additionally, if desired, any library or compound can be easily modified using standard chemical, physical, or biochemical methods. In addition, those skilled in the art of drug discovery and development will readily understand that methods for de-reproduction (e.g., taxonomic de-production, biological de-reproduction, and chemical de-reproduction, or any combination thereof) or the elimination of replicas or repetitions of materials that are already known for their anti-pathogenic activity, should be used whenever possible. When it is found that a crude extract has an anti-pathogenic or anti-virulence activity, or a binding activity, additional fractionation of the positive guide extract is necessary to isolate the chemical constituents responsible for the observed effect. In this way, the goal of the processes of extraction, fractionation, and purification, is the characterization and careful identification of a chemical entity within the crude extract that has the antipathogenic activity. The methods of fractionation and purification of these heterogeneous extracts are known in the art. If desired, the compounds shown to be useful agents for the treatment of pathogenicity are chemically modified in accordance with methods known in the art. Pharmaceutical Therapeutics and Plant Protectors The invention provides a simple means to identify compounds (including peptides, small molecule inhibitors, and mimetics) that can inhibit the pathogenicity or virulence of a pathogen. In accordance with the foregoing, a chemical entity that was found to have medicinal or agricultural value using the methods described herein, are useful either as drugs, plant protectants, or as information for the structural modification of the existing anti-pathogenic compounds, for example, by rational drug design. These methods are useful for selecting compounds that have an effect on a variety of pathogens including, but not limited to, bacteria, viruses, fungi, annelids, nematodes, flatworms, and protozoa. Examples of the pathogenic bacteria include, without limitation, Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bortella, Brucella, Calymma tobacterium, Campylobacter, Citrobacter, Clostridium, Cornyebacterium, Enterobacter, Escherichia, Francisella, Haemophilus, Hafnia. , Helicobacter, Klebsiella, Legionella, Listeria, Morganella, Moraxella, Proteus, Providence, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio, and Yersinia. For therapeutic uses, the compositions or agents that were identified using the methods described herein, can be administered in a systematic manner, for example, formulated in a pharmaceutically acceptable pH regulator, such as physiological saline. The treatment can be achieved directly, for example, by treating the animal with the antagonists that interrupt, suppress, attenuate, or neutralize the biological events associated with a pathogenicity polypeptide. Preferred routes of administration include, for example, subcutaneous, intravenous, interperitoneal, intramuscular, or intradermal injections, which provide sustained, sustained levels of the drug in the patient. The treatment of human patients or other animals will be carried out using a therapeutically effective amount of an anti-pathogenic agent in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin, the amount of the anti-pathogenic agent (e.g., an antibiotic) to be administered, varies depending on the manner of administration, the age and body weight of the patient, and the type of disease and extent of the disease. In general, the amounts will be in the range of those that are used for other agents that are used in the treatment of other microbial diseases, although in some cases smaller amounts will be needed due to the increased specificity of the compound. A compound is administered at a dose that inhibits microbial proliferation. For example, for routine administration, a compound is typically administered in the range of 0.1 ng-10 g / kg body weight. For agricultural uses, the compositions or agents that were identified using the methods described herein can be used as chemicals that are applied as sprays or powders on the foliage of the plants. Typically, these agents should be administered on the surface of the plant before the pathogen, to avoid infection. The seeds, bulbs, roots, tubers, and bulbous stems are also treated to avoid the pathogenic attack after planting, by controlling the pathogens that carry them or that exist in the soil at the site where they were planted. The land on which plants, ornamental plants, shrubs, or trees are to be planted can also be treated with chemical fumigants to control a variety of microbial pathogens. The treatment is preferably done several days or weeks before they are planted. Chemical products can be applied either by a mechanized route, for example, a tractor or with manual applications. In addition, chemicals that were identified using the assay methods, such as disinfectants, can be used. Other Modalities In general, the invention includes any nucleic acid sequence that can be isolated as described herein or which are readily isolated by homology selection or amplification of the polymerase chain reaction, using the sequences of nucleic acids of the invention. Also included in the invention are polypeptides that are modified in ways that do not negate their pathogenic activity (which are tested, for example, as described herein). These changes may include certain mutations, deletions, insertions, or post-translational modifications, or may involve the inclusion of any of the polypeptides of the invention as a component of a larger fusion protein. Also, polypeptides that have lost their pathogenicity are included in the invention. Thus, in other embodiments, the invention includes any protein that is substantially identical to a polypeptide of the invention. These homologs include other polypeptides that occur substantially naturally, as well as allelic variants; natural mutants, induced mutants; proteins encoded by the DNA that hybridizes to any of the nucleic acid sequences of the invention under conditions of high stringency or, less preferably, under conditions of low stringency (e.g., washing at 2X SSC at 40 ° C with a length of probe of at least 40 nucleotides); and proteins that are specifically bound by the antisera of the invention. The invention further includes analogues of any naturally occurring polypeptide of the invention. The analogs may differ from the naturally occurring polypeptide of the invention by differences in amino acid sequences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85 percent, more preferably 90 percent, and most preferably 95 percent or up to 99 percent identity with all or part of a naturally occurring amino acid sequence. of the invention. The length of the sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably residues of more than 35 amino acids. Again, in any exemplary approach to determining the degree of identity, a BLAST program can be used, with a probability of between e "3 and e" 100 indicating a closely related sequence. Modifications include chemical derivatization in vivo and in vitro of the polypeptides, for example, acetylation, carboxylation, phosphorylation, or glycosylation; these modifications may occur during the synthesis or processing of the polypeptide or after treatment with the isolated modified enzymes. The analogs may also differ from the naturally occurring polypeptides of the invention by alterations in the primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis through irradiation or exposure to ethamethylsulfate or through site-specific mutagenesis, as described by Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Pres, 1989, or Ausubel et al., Supra). Also included are cyclic peptides, molecules, and analogs that contain residues other than L amino acids, for example, D amino acids, or naturally occurring or synthetic amino acids, e.g., β or y amino acids. In addition to the full-length polypeptides, the invention also includes fragments of any of the polypeptides of the invention. As used herein, the term "fragment" means at least 5, preferably 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 amino acids. contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or can be the result of normal processing of the protein (e.g., removal of the amino acids from the incipient polypeptide that is not required for the activity biological or the removal of amino acids by splicing alternative mRNA or processing events of the alternative protein). Additionally, the invention includes nucleotide sequences that facilitate the specific detection of any of the nucleic acid sequences of the invention. Thus, for example, the nucleic acid sequences described herein or the fragments thereof can be used as probes to hybridize the nucleotide sequences by standard hybridization techniques under conventional conditions. The sequences that hybridize to a coding sequence of the nucleic acid sequence or its complement, are considered useful in the invention. Sequences that hybridize to a coding sequence of a nucleic acid sequence of the invention or its complement and that encode a polypeptide of the invention are also considered useful in the invention. As used herein, the term "fragment" as applied to nucleic acid sequences, means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and more preferably at least 40 to 80 contiguous nucleotides. Fragments of nucleic acid sequences can be generated by methods known to those skilled in the art. The invention further provides a method for inducing an immune response in an individual, particularly a human being, which includes inoculating the individual with, for example, any of the polypeptides (or a fragment or analog thereof or fusion protein) of the invention, to produce an antibody and / or an immune response of the T cell, to protect the individual from infection, especially bacterial infection (e.g., an infection by Pseudomonas aeruginosa). The invention further includes a method for inducing an immune response in an individual that includes supplying the individual with a nucleic acid vector to direct the expression of a polypeptide as described herein (or a fragment or fusion thereof), with the goal of inducing an immune response. The invention also includes vaccine compositions that include the polypeptides or nucleic acid sequences of the invention. For example, the polypeptides of the invention can be used as an antigen for vaccination of a host, to produce specific antibodies which protect against the invasion of bacteria, for example, by blocking the production of the phenacins. The invention therefore includes a vaccine formula that includes an immunogenic recombinant polypeptide of the invention, together with a suitable carrier. The invention further provides compositions (e.g., probes of nucleotide sequences), polypeptides, antibodies, and methods for diagnosis of a pathogenic condition. All publications and patent applications referred to in this specification are hereby incorporated by reference to the same extent as if it were specifically and individually indicated that each publication or independent patent application is incorporated by reference. Other embodiments are within the scope of the claims.

Claims

CLAIMS 1. An isolated nucleic acid molecule, comprising a sequence substantially identical to SEQ ID No: 252.
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises the sequence shown in SEQ ID. NO: 252.
3. An isolated nucleic acid molecule, comprising a sequence substantially identical to SEQ ID NO: 105.
4. The isolated nucleic acid molecule of claim 3, wherein said nucleic acid molecule comprises the sequence shown in SEQ ID NO: 105.
5. An isolated nucleic acid molecule, comprising a sequence substantially identical to SEQ ID NO: 106.
6. The isolated nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises the sequence shown in SEQ ID NO: 106.
7. A substantially pure polypeptide, comprising an amino acid sequence that is substantially identical to the amino acid sequence. os of SEQ ID NO: 253.
8. The substantially pure polypeptide of claim 7, wherein said amino acid sequence comprises the sequence shown in SEQ ID NO: 253.
9. A substantially pure polypeptide, comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 107.
10. The substantially pure polypeptide of claim 9, wherein said amino acid sequence comprises the sequence shown in SEQ. ID NO: 107.
11. A substantially pure polypeptide, comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 108.
12. The substantially pure polypeptide of claim 11, wherein said sequence of amino acids comprises the sequence shown in SEQ ID NO: 108.
13. A method for identifying a compound that is capable of reducing the expression of a pathogenic virulence factor, said method comprising the steps of: (a) providing a pathogenic cell expressing a nucleic acid molecule of claim 1; and (b) contacting said pathogenic cell with a candidate compound, a reduction in the expression of said nucleic acid molecule following contact with said candidate compound by identifying a compound that reduces the expression of a pathogenic virulence factor.
The method of claim 13, wherein said pathogenic cell infects a mammal.
15. The method of claim 13, wherein said pathogenic cell infects a plant.
16. A method for identifying a compound that is capable of reducing the expression of a pathogenic virulence factor, said method comprising the steps of: (a) providing a pathogenic cell expressing a nucleic acid molecule of claim 3; and (b) contacting said pathogenic cell with a candidate compound, a reduction in the expression of said nucleic acid molecule following contact with said candidate compound by identifying a compound that reduces the expression of a pathogenic virulence factor.
17. The method of claim 16, wherein said pathogenic cell infects a mammal.
18. The method of claim 16, wherein said pathogenic cell infects a plant.
19. A method for identifying a compound that is capable of reducing the expression of a pathogenic virulence factor, said method comprising the steps of: (a) providing a pathogenic cell expressing a nucleic acid molecule of claim 5; and (b) contacting said pathogenic cell with a candidate compound, a reduction in the expression of said nucleic acid molecule following contact with said candidate compound by identifying a compound that reduces the expression of a pathogenic virulence factor.
20. The method of claim 19, wherein said pathogenic cell infects a mammal.
The method of claim 19, wherein said pathogenic cell infects a plant.
22. A method for identifying a compound that binds to a polypeptide, said method comprising the steps of: (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of claim 7 under conditions that allow ligature; and (b) detecting ligation of the candidate compound to the polypeptide.
23. A method for identifying a compound that binds to a polypeptide, said method comprising the steps of: (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of claim 9 under conditions that allow ligature; and (b) detecting ligation of the candidate compound to the polypeptide.
24. A method for identifying a compound that binds to a polypeptide, said method comprising the steps of: (a) contacting a candidate compound with a substantially pure polypeptide comprising an amino acid sequence of claim 11 under conditions that allow ligature; and (b) detecting ligation of the candidate compound to the polypeptide.
25. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of claim 1 in said pathogen.
26. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; . and (b) administering to said mammal a therapeutically effective amount of a composition that inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of claim 3 in said pathogen.
27. The method of claim 26, wherein said pathogen is Pseudomonas aeruginosa.
28. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that inhibits the expression or activity of a polypeptide encoded by a nucleic acid molecule of claim 5 in said pathogen.
29. The method of claim 28, wherein said pathogen is Pseudomonas aeruginosa.
30. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that binds and inhibits a polypeptide encoded by an amino acid sequence of claim 5.
31. The method of claim 30, wherein said pathogen is Pseudomonas aeruginosa.
32. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that binds and inhibits a polypeptide encoded by an amino acid sequence of claim 7.
33. The method of claim 32, wherein said pathogen is -Psudomonas aeruginosa. 3 .
A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that binds and inhibits a polypeptide encoded by an amino acid sequence of claim 9.
35. The method of claim 34, wherein said pathogen is Pseudomonas aeruginosa.
36. A method of treating a pathogenic infection in a mammal, said method comprising the steps of: (a) identifying a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that binds and inhibits a polypeptide encoded by an amino acid sequence of claim 11.
37. The method of claim 36, wherein said pathogen is Pseudomonas aeruginosa.
38. A method of identifying a compound that inhibits the virulence of a Pseudomonas cell, said method comprising the steps of: (a) providing a Pseudomonas cell; (b) contacting said cell with a candidate compound; and (c) detecting the presence of a phenazine, wherein a reduction in said phenazine relative to an untreated control cell is an indication that the compound inhibits the virulence of said Pseudomonas cell.
39. The method of claim 38, wherein said cell is Pseudomonas aeruginosa.
40. The method of claim 38, wherein said phenazine is detected by spectroscopy.
41. The method of claim 38, wherein said phenazine is a pyocyanin.
42. The method of claim 41, wherein said piocyanipa is detected by measuring the absorbance at 520 nm.
43. The method of claim 38, wherein said cell is present in a cell culture.