CZ20001858A3 - Molecule of isolated nucleic acid, polypeptide, method of determining substances and treatment method - Google Patents

Abstract

Bacterial virulent polypeptides and nucleic acid sequences acids (e.g., DNA) that encode such polypeptides and methods for producing such polypeptides by recombinant means way. Methods of using such polypeptides for testing of antibacterial or becteriostatic agents methods of substance identification and treatment modalities identified antiviral agents.

Description

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Molecule isolated identification of substances and method nuclear treatment. acids, polypeptide, method Field of the invention The invention relates of molecules nuclear acids, genes and

polypeptides that are associated with microbial pathogenicity. Pathogens use a variety of genetic strategies to infect their hosts with infection or possibly disease. The expression of microbial pathogenicity depends on complex genetic regulatory circles. Knowledge of microbial pathogenicity is essential for understanding the mechanisms of virulence of the pathogen and for developing new anti-virulent or anti-pathogenic agents that are necessary to induce infection and disease.

BACKGROUND OF THE INVENTION

Holt, 1984).

In one particular example, the opportunistic human pathogen Pseudomonas aeruginosa is a gram-negative bacteria isolated from soil, water and plants everywhere (Palleroni, JN in the Bergey's Manual of Systematic Bacteriology, ed., JG Williams and Wilkins, Baltimore, MD, pp. 141-172 ,

Various virulent factors of P. aeruginosa are described and most of them, such as exotoxin A, elastase and phospholipase C, were first detected biochemically based on their cytotoxic activity. (Fink, R. B., Pseudomonas aeruginosa the Opportunist: Pathogenesis and Desease, Boca Raton, CRC Press Inc., 1993). Subsequently, genes corresponding to these factors or genes that regulate the expression of said factors were identified. In general, most pathogenicity-associated genes in mammalian bacterial pathogens were first detected using a bioassay. In contrast to mammalian pathogens, simple systemic gene strategies have been used to identify genes associated with pathogenicity in plant pathogens. After random transposon-mediated mutagenesis, thousands of mutant phytopathogen clones were separately inoculated into individual plants to determine if they contained a mutation that caused pathogenic interaction with the host (Boucher et al., J. Bacteriol. 168: 5626-5623, 1987, Comai and Kosuge, J. Bacterial 149: 40-46, 1982; Lindgren et al., J. Bacteriol. 168: 512-522, 1986; Rahme et al., J. Bacteriol. 173: 575-586, 1991; Willis et al. al., Mol Plant-Microbe Interact. 3: 149-156, 1990). Comparable experiments using mammalian pathogenicity models on whole animals are not possible because large numbers of animals have to be exposed to the pathogen.

SUMMARY OF THE INVENTION

Numerous nucleic acid, polypeptide, and small molecules (e.g., phenazines) that are involved in the pathogenicity and virulence of pathogens have been identified and characterized. This discovery therefore forms the basis of assays for testing drugs useful for evaluating and identifying "antivirulent agents that are capable of blocking pathogenicity and virulence of a pathogen, for example, by selectively switching the expression of the pathogen gene on and off, or capable of inactivating or inhibiting the activity of the polypeptides involved. on the pathogenicity of a microbe. Drugs that act on said molecules can be used as antiviral agents.

The invention provides an isolated nucleic acid molecule comprising a sequence that is substantially identical to any of sequences B148 (SEQ ID NO: 1), ORF2 (SEQ ID NO: 2), ORF3 (SEQ ID NO: 4), ORF602c (SEQ ID NO). ORF214 (SEQ ID NO: 8), ORF1242C (SEQ ID NO: 10), ORF594 (SEQ ID NO: 12), ORF1040 (SEQ ID NO: 14), ORF1640c (SEQ ID NO: 16), ORF2228c ( 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.

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), ORE7180 (SEQ ID NO: 58), ORF7501 (SEQ ID NO: 60), ORF7584 (SEQ ID NO: 62), ORF8208c (SEQ ID NO: 64), ORF8109 (SEQ ID NO) 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) 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), ORE13156c (SEQ ID NO: 98), ORF12795 (SEQ ID NO: 100), ORF13755c (SEQ ID NO: 210), ORF13795C (SEQ ID NO: 212), ORF14727c (SEQ ID NO: 214), ORF13779 (SEQ ID NO: 216), ORF14293c (SEQ ID NO: 218), ORF14155 (SEQ ID NO: 220), ORF14360 i ISEQ ID NO: 222), ORF115342c (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), ORE19305 (SEQ ID NO: 248), ORF19519 (SEQ ID NO : 250), ORF19544 (SEQ ID NO: 252 ), ORF20008 (SEQ ID NO >: 254), < ORF20623B (SEQ ID NO: 256), ORF21210C (SEQ ID NO: 258 ), ORF21493c (SEQ ID NO: 260) ORF21333 (SEQ ID NO: 262), ORF22074C (SEQ ID ORF21421 (SEQ ID NO: 264) 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: 3% 4), 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), ORF34726 (SEQ ID NO: 384) , ORF34916 (SEQ ID NO: 387) ORF35464C (SEQ ID NO: 388); ORF35289 (SEQ ID NO: 390) ORF35410 (SEQ ID NO : 392), ORF35907c (SEQ ID NO: 394), ORF35534 (SEQ ¹ ID NO: 396), ORF35930 (SEQ ID NO: (398), ORF36246 (SEQ ID NO: 400), ORF26640c (SEQ ID NO: 402), ORF36739 (SEQ ID NO: 404), ORE37932c (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), ORF42234 (SEQ ID NO: 428), ORF41764c (SEQ ID NO: 430),

5 • · · · • · · • · • · · · • · · · • · • · • · • · ORF41284 (SEQ ID NO: 432), ORF 411598 (SEQ ID NO: 434), ORF42172C (SEQ ID NO: 436), ORF42233c (SEQ ID NO NO: 151), 33A9 (SEQ ID NO : 102 , 190, 190, 191, 192, 193, 194, 195, 196, 197 a

from a bacterial pathogen, the invention includes a vector

198), 34B12 (SEQ ID NO: 104), 34B12-ORF1 (SEQ ID NO: 105),

34B12-ORF2 (SEQ ID NO. 106), 36A4 (SEQ ID NO: 109), 36A4 contig (SEQ ID NO: 111), 23A2 (SEQ ID NO: 112), 3E8phn (~) (SEQ ID NO: 114) , 3E8contigPAO1 (SEQ ID NO: 115), 34H4 (SEQ ID NO:

118), 33C7 (SEQ ID NO: 119), 25a2.3 (SEQ ID NO: 120), 8C12 (SEQ ID NO: 121), 2A8 (SEQ ID NO: 122), 41A5 (SEQ ID NO: 123),

50E12 (SEQ ID NO: 124), 35A9 (SEQ ID NO: 125), pho 23 (SEQ ID NO

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 NO: 136), ORFA (SEQ ID NO: 153), ORFB (SEQ ID NO: 154), ORFC (SEQ ID NO: 155), phzR (SEQ ID NO: 164) and 1G2 (SEQ ID NO). : 137). It is preferred that the isolated nucleic acid molecule comprise any of the sequences or fragments thereof described above and be derived from a pathogen (e.g., Pseudomonas aeruginosa). Additionally, and a cell, each comprising at least one of the isolated nucleic acid molecules of the invention and a method of producing a recombinant polypeptide that produces a cell transformed with a nucleic acid molecule of the invention that is present in the cell for expression, culturing the transformed cell under conditions expressing the nucleic acid molecule. and recovering the recombinant polypeptides. The invention further encompasses features of recombinant polypeptides produced by expressing an isolated nucleic acid molecule of the invention and substantially pure antibodies that specifically recognize and bind such recombinant polypeptides.

The invention further provides a substantially pure polypeptide comprising an amino acid sequence that is substantially identical to an amino acid sequence selected from the group consisting of ORF2 (SEQ ID NO: 3), ORF3 (SEQ ID NO: 5), ORF602c (SEQ ID NO: 7), ORF214 (SEQ ID NO: 9), ORF1242c (SEQ ID NO: 11), ORF594 (SEQ ID NO:

13), ORF 1040 (SEQ ID NO.

(SEQ ID NO: 17)

ORF3965C ORF3568 (SEQ ID NO: 33), ORF4271 (SEQ ID NO: 31), ORF3973

15), ORF1640c

ORF2228C (SEQ ID NO: 19), ORF2068c (SEQ ID NO: 21), ORF1997 (SEQ ID NO: 23), ORF2558c (SEQ ID NO: 25), ORF2929c (SEQ ID NO: 29), ORF3218

ORF4506c (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) ORF87755c (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), ORF10765C (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.

NO: 97) ORF13755c ORF13156c (SEQ NO: ID NO: 211), 99), ORF12795 (SEQ ID NO i NO: NO: 101), 213), (SEQ ID ORF13795c (SEQ ID ORF14727C (SEQ ID NO: 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), 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 ID NO: 243), ORF18479 (SEQ ID NO: 245), ORF19027c (SEQ ID NO: 247), ORF19305 (SEQ ID NO: 249), ORF19519 (SEQ ID i NO : 251 ORF19544 (SEQ ID NO: 253) , QRF20008

(SEQ ID NO: 255), ORF20623c

ORF21493c (SEQ 263), ORF22074c (SEQ ID NO: 259)

(SEQ

ID NO 265)

ID NO: 257), ORF21210c (SEQ

261), ORF21333 (SEQ ID NO: 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 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 NO: 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) 1, 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 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) ORF38604C (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 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: 431) ORF41284 (SEQ ID NO: 433), ORF41598 (SEQ ID NO: 435) ORF42172c (SEQ ID NO: 437), ORF42233C (SEQ ID NO: 156), ORF42233c (SEQ ID NO NO: 152) , 33A9 (SEQ ID NO : 103 , 200, 200, . 202, 203, 204, 205, 206, 207 a 208), 34B12-ORF1 (SEQ ID NO. 107) 34B12-ORF2 SEQ ID NO: 108), 36A4 (SEQ SEQ ID NO: 110), 3E8phzA (SEQ ID NO: 116) 3E8phz (SEQ ID NO: 117), PhzR (SEQ ID NO: 165) , ORFA (SEQ ID NO: 156), ORFB (SEQ ORFC (SEQ ID NO: 157); NO: 158) and PA14 degP

(SEQ ID NO: 132). It is preferred that the substantially pure polypeptide comprise any of the above-described sequences of fragments thereof and obtain a pathogen (for example from a bacterial pathogen such as Pseudomonas aeruginosa).

The invention further provides a method for identifying a substance that is capable of attenuating the expression of a pathogenic virulence factor (e.g., at or after transcription level), comprising (a) preparing a pathogenic cell expressing any of the isolated nucleic acid molecules; and down-regulating the expression of the nucleic acid molecule following contact with said agent that identifies a agent that depresses the expression of a pathogenic virulence factor. In a preferred embodiment of the invention, the pathogenic cell infects a mammal (e.g., a human) or a plant.

The invention provides a method of identifying a substance that binds to a polypeptide comprising (a) contacting the substance with a substantially pure polypeptide comprising any of the amino acid sequences of the invention under conditions that allow binding and (b) detecting binding of the substance to the polypeptide.

In addition, the invention provides a method of treating a pathogenic infection in a mammal comprising (a) identifying a mammal having a pathogenic infection and (b) administering to the mammal a therapeutically effective amount of a substance that inhibits expression or activity of a polypeptide encoded by any of the nucleic acid molecules of the invention. In a preferred embodiment of the invention, the pathogen is Pseudomonas aeruginosa.

The invention further provides a method of treating a pathogenic infection in a mammal which comprises (a) identifying a mammal having a pathogenic infection and (b) administering a therapeutically effective amount of the agent to the mammal, said agent binding and inhibiting a polypeptide encoded by any of the amino acid sequences of the invention. In a preferred embodiment of the invention, the pathogenic infection is caused by Pseudomonas aeruginosa.

Further, the invention provides a method for identifying a substance that inhibits virulence of a Pseudomonas cell comprising (a) obtaining a Pseudomonas cell, (b) contacting the cell with a candidate agent, and (c) detecting the presence of phenazine wherein reducing phenazine to an untreated control culture inhibits virulence of a Pseudomonas cell. In a preferred embodiment of the invention, the cell is a Pseudomonas aeruginosa cell. The cell is present in cell culture and phenazine is detected spectroscopically (for example, pyocyanine is detected by absorbance at 520 nm). Pyocyanine is generally detected according to any standard method, for example the method described herein.

The term "isolated nucleic acid molecule" means a nucleic acid (for example DNA) that does not contain genes that flank the gene in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. The term therefore encompasses, for example, recombinant DNA which has been incorporated into a vector, into an autonomously replicating plasmid or virus, or into genomic DNA of prokaryotes and eukaryotes, or which exist as separate molecules (e.g., cDNA produced by PCR)

DNA or restriction endonuclease cDNA fragment) or genomic or by cleavage independently of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule as well as recombinant DNA that is part of a hybrid gene encoding another polypeptide sequence.

The term "polypeptide" refers to any chain of amino acids regardless of length and post-translational processing (e.g., glycosylation or phosphorylation).

The term "substantially pure polypeptide" refers to a polypeptide of the invention that has been separated from the components that naturally accompany it. Typically, a polypeptide is substantially pure when at least 60% (w / w) is free of proteins and naturally occurring organic molecules with which it naturally associates. It is preferred that the composition comprises at least 75%, more preferably at least 90% and most preferably 99% (w / w) of the polypeptides of the invention. A substantially pure polypeptide of the invention may be obtained, for example, by extraction from a natural source (e.g., a pathogen), expression of a recombinant nucleic acid encoding such a polypeptide, or chemically synthesizing protein. Purity is measured by any suitable method such as column chromatography, polyacrylic gel electrophoresis, or HPLC analysis.

The term "substantially identical" means a polypeptide or nucleic acid molecule having at least 25% identity with respect to a reference amino acid sequence (e.g., any of the amino acid sequences described herein or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). 30%, 40%, 50%, 60%, more preferably 80% and most preferably 90% or even 95% identical at the amino acid or nucleic acid level to the sequence used for comparison.

University Avenue, BESTFIT, GAP or%, more with at least 99 'of the invention

Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710).

Madison, WI 53705, BLAST programs,

PILEUP / PRETTYBOX). Such software matches identical or similar sequences by estimating the degree of homology with various substitutions, deletions and / or other modifications. Conservative substitutions typically include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine arginine; and phenylalanine, thyrosine. In an exemplary identity determination approach, a BLAST program with a probability score of e ^-3 and e ~ ¹⁰⁰ may be used, indicating a closely related sequence.

The term "transformed cell" means a cell into which (into its ancestor) a DNA molecule encoding a polypeptide of the invention has been introduced by a recombinant DNA method.

The term "location suitable for expression" means that the DNA molecule is placed close to the DNA sequence that directs the transcription or translation of the sequence (that is, allows the production of, for example, a recombinant polypeptide of the invention or RNA molecule). 60% (w / w) without proteins and naturally occurring organic molecules with which they naturally associate. It is preferred that the composition comprises at least 75, preferably 90%, and most preferably (w / w) antibodies. Purified antibodies of the invention were obtained, for example, by affinity chromatography using the recombinantly produced polypeptide of the invention and standard methods.

The term "specifically binds" refers to a substance or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind the polypeptide of the invention. other molecules in the sample, for example, a biological sample that naturally comprises a polypeptide of the invention.

The term "derived from" means that it has been isolated from or exhibited a naturally occurring sequence (e.g., cDNA, genomic DNA, synthetic DNA, or a combination thereof).

The term "pathogen inhibiting" means the ability of a candidate agent to reduce, suppress, attenuate, reduce or block the development or progression of a pathogen-mediated disease or infection in a eukaryotic host organism. It is preferred that such inhibition reduces pathogenicity by at least 5%, more preferably by at least 25%, and most preferably by at least 50%, as compared to symptoms in the absence of the candidate agent in any suitable pathogenicity assay (e.g., the assays described herein). In one particular example, inhibition can be measured by monitoring the pathogenic symptoms in a host organism that has been exposed to a candidate or extract substance. A decrease in the level of symptoms relative to the amount of pathogenic symptoms in the non-exposed host organism indicates the agent-mediated inhibition of the pathogen.

"Pathogenic virulent factor" means a cellular component (for example, a protein such as a transcription factor as well as a gene that encodes such a protein) without the pathogen being able to induce a disease or infection of a eukaryotic host organism.

The invention describes a number of targets that can be used in the development of drugs that specifically block the pathogenicity of a microbe. In addition, the methods of the invention make it possible to identify substances that are suitable for use in eukaryotic host organisms (i.e., substances that do not disrupt the development or physiology of the organism) and counteract pathogenic microbes (i.e., suppress the virulence of the pathogen). The methods of the invention make it possible to analyze for any number of anti-9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 «99 · 9 99 virulent effect with high permeability, high sensitivity and low complexity. The methods of the invention are also relatively inexpensive, since they allow the analysis of small amounts of active substances which have been detected either in the form of purified extract or in the form of crude extract.

Identification and characterization of virulence factor

As disclosed herein, plants were used as an in vivo pathogenesis model to identify virulent factors of the human opportunistic pathogen Nine of the nine TnphoA mutant P.aeruginosa strain UCBPP-PA14 identified in a plant leaf assay that is less pathogenic mutants also showing significantly reduced pathogenicity in the burned mouse assay, suggesting that P. aeruginosa microorgasnimus used a number of common strategies to infect both hosts. Seven of the nine mutants contained TnphoA insertions in previously unknown genes. These results demonstrated that an alternative non-vertebrate host of a human bacterial pathogen could be used in high throughput in vivo testing to identify novel bacterial virulence factors involved in mammalian pathogenesis.

These experiments were carried out using the following methods.

Pseudomonas aeruginosa. derivatives of the microorganism

Strains, growth conditions and plasmids.

P. aeruginosa strain UCBPPPA14, a human clinical isolate used in experiments suitable for the identification of new virulence-associated genes, has been studied in many laboratories (Ausubel et al., Methods of Screening Compounds). Pathogenicity, USSN 08 / 411,560, 08 / 852,927;

No. 08 / 962,750, filed March 25, 1995, May 7, 1997;

November 1997 and Rahme et al., Science 268: 1899-1902, 1995), P. aeruginosa PAK strains (Ishimoto and Lory, Proc. Natl. Acad. Sci. USA 86: 1954-1957, 1989) and PAO1 (Holloway et al., Microbiol. Rev. 43: 73-102, 1979). Luria Bertaini broth and agar were used to cultivate P. aeruginosa and E. coli strains and cultured at 37 ° C. Minimal medium (M9) was also used to cultivate P. aeruginosa.

Transposon mutagenesis

Transposon-mediated mutagenesis of UCBPP-PA14 was performed using TnphoA, which is found on plasmid pRT731 in the E. coli SM1OXpir strain (Taylor et al., J. Bacteriol. 171: 1870-1878, 1989). Donor and recipient cells cultured on the medium were plated on agar plates and Luria Bertani broth and incubated at 37 ° C for 8-10 hours, and then plated on Luria Bertani broth plates containing rifampicin (100 qg / ml). (for the selection of E. coli donor cells) and kanamycin (200 µg / ml) (for the selection of P. aeruginosa cells containing TnphoA). Colonies that are cultured on rifampicin and kanamycin media were replicated on Luria Bertani broth containing ampicillin (300 µg / ml). Ampicillin resistant colonies indicated the incorporation of pRT731 into the UCBPP-PA14 genome and were discarded.

Alkaline phosphatase activity

500 phototrophic mutants of UCBPP-PA14 TnphoA were tested on peptonglucose agar plates (Ostroff et al., J. Bacteriol. 172: 5915-5923, 1990) containing 40 µg / ml 5bromo-4-chloro-3-indolyphosphate (XP) ). Peptone medium was chosen because it suppressed the production of the endogenous blue-green pyocyanine pigment and the fluorescent yellow pyoverdine pigment, which allows the visibility of the blue color resulting from XP dephosphorylation by periplasmic alkaline phosphotase generated by PhoA ⁺ mutants.

Growth conditions and mutant isolation strategy.

P. aeruginosa strains that were grown to saturation in L-soil at 37 ° C were washed with 10 mM MgSO ₄ and resuspended to an optical density of 0.2 (OD600 0.2) in 10 mM. MgSO ₄ and diluted 1: 100 and 1: 1000 (corresponding to a bacterial density of approximately 10 ⁶ and 10 ⁵ colony forming units). Approximately 10 ml of the diluted cells were inoculated by pipette into the stem of salad plants, which are approximately 12 weeks old (variety Romain or Graet lake) and cultivated in a cauliflower in MetroMix soil in a greenhouse at 26 ° C. The stems were washed with 0.1% bleach and placed on 15 cm diameter petri dishes containing a Whatman filter (Whatman # 1) which was impregnated with 10 mM MgSO ₄ . The middle rib of each lettuce leaf was inoculated with three different P. aeruginosa mutants produced by TnphoA for testing, and the UCBPP-PA14 strain was used as a control. The plates were stored in a growth chamber at 28-30 ° C and a relative humidity of 90-100% during the experiment. Symptoms were monitored daily for 5 days.

In a leaf infiltration model of Arabidopsis, P. aeruginosa strains were cultured and washed as described above and diluted with 10 mM MgSO ₄ 1: 100 (corresponding to a bacterial density of 10 ³ / cm ² leaf disk area) and injected into leaves of Arabidopsis plants that are six weeks old, as described for infiltration of Pseudomonas syringae (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / Nos. 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, Rahme et al., Science 268: 18991902, 1995; Dong et al., Plant Cell 3: 61-72, 1991). Conditions Incubation and symptom monitoring were performed in the same way as in salad experiments. The intracellular leaf fluid containing bacteria was collected and the number of bacteria determined (Rahme et al., Science 268: 1899-1902, 1995, Donge et al., Plant Cell 3: 61-72, 1991). Four different samples were taken using two leaf discs for each sample. Control plants inoculated with 10 mM MgSO ₄ showed no symptom development.

A mouse mortality study% of the total surface burn area was generated on the stretched abdominal skin of AKR / J mice. Six-week-old (Jackson Laboratories) females weighing between 25 and 30 grams are used (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997,

Rahme et al., Science 268: 1899-1902, 1995; Stevens, J.Burn

Care Rehabil. 15: 232-235,1994). Immediately after burn, mice were treated with 5 x 10 ³ or 5 x 10 ⁵ cells of P. aeruginosa and the number of animals that died from sepsis was recorded every day for ten days. Animal study protocols were approved by the Animal Studies of the Massachusetts General Hospital. Statistical significance of the mortality data was determined using the χ2 Yates correction test or Fisher's exact test. The differences between groups are considered statistically significant when P is less than or equal to 0.05.

DNA manipulation, molecular cloning and TnphoA mutant sequence.

Chromosomal DNA of P. aeruginosa with phenol extraction (Strom and Lory, J. Bacteriol., 372, 1986) and DNA blotting and hybridization analysis isolated

165: 367 studies were performed as described by Ausubel et al., Current Protocols in Molecular Biology, Wiley, New York, 1996).

The oligonucleotides 5'-AATATCGCCCTGAGCAGA-3 '(LGR1) (SEQ ID NO: 138) and 5'-AATACACTCACTATGCGCTG-3' (LGR2) (SEQ ID NO: 139) corresponded to sequences on opposite strands at the 5'-end of TnphoA. The oligonucleotides 5'-CCATCTCATCAGAGGGTA-3 '(LGR3) (SEQ ID NO: 140) and 5'-CGTTACCATGTTAGGAGGTC-3' (LGR4) (SEQ ID NO: 141) corresponded to sequences on opposite strands at the 3'-end of TnphoA. LGR1 + LGR2 or LGR3 + LGR4 were used to amplify DNA sequences by inverse PCR (IPCR) led TnphoA insertion sites, as described in Ochman et al., 1993, A Guide to Methods and Applications, eds., Innis, MA, States (DJ, 1990). Amplified DNA fragments ranging in size from 350 to 650 base pairs were cloned into pBlueScript SK +/-. Before subcloning into the EcoRV pBlueScript SK +/- restriction site, the IPCR products were filled with ends. Double stranded DNA sequencing was performed using Sequenase 2.0 kits (U.S. Biochemical, Inc) to determine the sequence of IPCR amplified products. The sequences obtained were compared to non-redundant peptide sequence databases at the National Center for Biotechnology Information (NCBI) using the BLASTX program (Gish and States, Nat. Gent. 3: 266-272, 1993).

Isolation and DNA manipulation of a wild-type clone containing a gene corresponding to the pho34B12 mutation from the UCBPP-PA14 genomic library. The IPCR product was generated from the mutant UCBPP-PA14 TnphoA. The pho34B12 mutant was labeled using a random DNA insertion kit (Boehringer Mannheim, Indianapolis, IN) and used chromosomal DNA in pJSR1 (Rahme et al., Science 268: 18991902, 1995) to test the genome library UCBPP-PA14 (1995) as probes. to find a clone containing a gene corresponding to pho34B12 mutations. The 3.7 kb EcoRI fragment found in cosmid clone pLGR34B12, which corresponds to the pho34B12 mutation, was subcloned into the EcoRI restriction site of pRB54 (Roberts et al., J. Bacteriol. 172: 6204-6216).

1990) after filling the ends of the vector and fragment to construct pLGRE34B12. The same fragment (which was blunt-ended) was subcloned into the SmaI restriction site of pCVD (Donnenberg and Kaper, Infect. Immun. 59: 4310-4317,

1991) to form pLGR34. pLG34 was used to replace the mutated pho34B12 gene with wild-type copies as described in Donnenberg and Kaper, Infect Immun. 59: 4310-4317

1991). The 3.7 kb EcoRI fragment was also subcloned into the EcoRI restriction site of pBlueScript SK +/- to construct pBSR34B12 and used for DNA sequence analysis.

A sequence of 1,659 pairs in size corresponds to the incorporation of pho34B12 containing two overlapping open reading frames (ORF1 and ORF2) on opposite strands was inserted into GenBank and designated AF031571. ORF1 is 1148 bp

509) and ORF2 is 1022 bp large

The overlap of the two ORFs is from nucleotide 436 to 1458. ORF1 contains, at nucleotide 751, a second putative translation initiation site corresponding to a coding region of 758 bp. Oligonucleotide primers 5'-CGCATCGTCGAAACGCTGGCGGCC-3 '(SEQ ID NO: 142) were used to amplify the fragment from pBSR34B12 containing ORF1 of 100 bp in size.

5'-GCCGATGGCGAGATCATGGCGATG-3 '(SEQ ID NO: 143).

Due to the two putative initiation sites present in ORF1, oligonucleotide primers were used to amplify 1,659 pairs of pBSR34B12 that contain ORF1.

5'-TGCGCAACGATACGCCGTTGCCGACGATC-3 '(SEQ ID NO: 144) and 5'-GATTCCACCTTCGCAGCGCAGCCC-3 ^of (Reg3) (SEQ ID NO: 145).

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 pBSR34B12 containing ORF2. All primer combinations were large (nucleotides 361 to (nucleotides 1458 to 436)).

· · Designed to contain putative upstream regulatory elements of each ORF. The obtained PCR products (1100, 1659 and 1302 bp) were subcloned into pCR2.1 (Invitrogen Inc) to produce pLE15, pLE1 and PLE2 constructs. All three PCR products were subcloned into pRR54 to construct pRRLE15, pRRLE1 and pRRLE2.

Enzymatic activities of TnphoA mutants

P. aeruginosa strains were cultured for 18 hours in LB medium and used for testing enzymatic activities. Proteolytic and elastolytic activities were determined as previously described (Toder et al., Mol. Microbiol. 5: 20032010, 1991). The amount of pyocyanine was determined as described by Essar et al., J. Bact. 172: 884-900

1990). Hemolytic activity was determined after incubation of plates containing trypticase soy agar (BBL) supplemented with 5% sheep red blood cells (Ostroff and Vasil, J. Bacteriol. 169: 4957-4601, 1987).

Generation of non-polar GacA mutation

A non-polar gacA mutation in UC BPP-PA14 was constructed by cloning a 3.5 kb PstI fragment containing the gacA gene from cosmid pLGR43 (Rahme et al., Science 268: 18991902, 1995) into a single BamHI restriction site of the pEGBR vector (described (Akerley et al., Call 80: 611-620, 1995) using BamHI linkers. The 950 bp EcoRI-HincII fragment with Klenow-filled ends containing the kanamycin resistance cassette of pUC18K (Menard et al., J. Bacteriol. 175: 5899-5906, 1993) was then cloned into a single fragment. a BamHI restriction site (and blunt ends prepared) in gacA such that transcription was maintained and translation of a portion of gacA downstream of the gene was reinitialized at the 3 'end of the kanamycin cassette. The resulting SW 7-4 construct containing the kanamycin gene cassette in the gacA gene and in orientation with its transcription was used to indicate a change by homologous recombination of the damaged gacA gene into the wild-type UCBPP-PA14 genome.

Isolation and characterization of virulent factors of P.aeruginosa organism.

Using the procedures described above, the UCBPP-PA14 genome of P. aeruginosa was mutagenized with the TnphoA transposon and 2,500 phototrophic mutants were tested for pathogenicity using a lettuce stem assay. This assay allows testing of several mutants on a single lettuce stem. Interestingly, the salad was found to be not only susceptible to UCBPP-PA14 infection, but also susceptible to well characterized PAK strains of P. aeruginosa (Ishimoto and Lory, Proc. Natl. Acad. Sci USA 86: 1954-1957, 1989) and PAO1 (Holloway et al., Microbiol. Rev. 43: 73, 1979). Both of these strains multiplied on lettuce leaves and caused disease symptoms similar to those accompanying UCBPP-PA14 infection, which characterizes water saturation, followed by mild rot, four to five days after infection. At later stages of infection, all three strains of P.aeruginosa infected the entire central rib of the lettuce leaf, resulting in complete maceration and tissue collapse.

As summarized in Table 1, nine Tnphol generated UCBPP-PA14 mutants were identified among 2500 phototrophs and tested for the ability to induce zero, mild, or moderate lethal stem rot symptoms. These results are compared to the wild-type strain.

Table 1 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Strain Growth in the leaves grew. Arabidops is ^a The symptoms induced in grew. Arabidops is ^b % mortality of mice ^c Identity gene 5 x 10 ³ 5 x 10 ⁵ PA14 5.5 x 10 ⁷ Heavy 53 100 ALIGN! 33C7 8.3 x 10 ⁴ None 0 0 Unknown ³ 1D7 7.5 x 10 ⁵ Faintly 0 50 (Network 3 cA 25A12 1.7 x 10 ⁶ Faintly 11 87 Unknown 33A9 5.1 x 10 ⁶ Medium 0 0 Unknown 25F1 1.5 x 10 ⁴ Medium 0 20 May Unknown 34H4 3.8 x 10 ⁶ Medium 0 33 Unknown Pho34B12 4.0 x 10 ⁶ Medium 0 56 Unknown Phol5 3.9 x 10 ⁴ Medium 0 62 DsbA 16G12 2.3 x 10 ⁵ medium 20 May 100 ALIGN! Unknown

^and Four different samples were taken using two leaf discs / sample. Control plants inoculated with 10 mM MgSO ₄ showed no symptoms during the course of the test. Three independent experiments showed the same results.

^b Symptoms were observed four to five days after infection. None means no symptoms, chlorosis, chlorosis delimiting the site of inoculation; weak means localized water intake and tissue chlorosis delimiting the site of inoculation; medium means moderate water soaking and chlorosis of most tissues around the inoculation site; heavy means severe rotting of the whole leaf characterized by a water-absorbed reaction zone and chlorosis around the site of inoculation two to three days after infection.

^c All animal experiments were performed at least twice using 8 to 10 animals per experiment. Independent experiments show the same percentage of deaths. Mice were injected with approximately 5 x 10 or 5 x 10 cells.

^d BLAST analysis showed uncoded proteins with substantial homology.

No strong maceration was observed in any mutant. DNA blot analysis showed that each of the nine mutants contained a single insertion of TnphoA. A 1,542 base pair BglI-BamHI fragment containing the kanamycin TnphoA resistance gene was used as a probe (Taylor et al., J.

Bacteriol. 171: 1870-1878 (1989). Two of the nine UCBPPPA14 mutants TnphoA, pho34B1 and phol5, expressed alkaline phosphatase activity, suggesting that genes containing these TnphoA insertions encoded membrane spanning or secreted proteins (Taylor et al., J. Bact. 171: 18701878, 1989) Manoil and Beckwith, Proc. Natl. Acad. Sci USA 82: 5117, 1985).

Nine additional TnphoA mutants were further tested by measuring their growth rate in Arabidopsis leaves over four days, a quantitative measurement 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 differences in their growth rate, which is compared to a wild-type strain in both rich and minimal medium. The growth rate over time of all nine mutants in the leaves of Arabidopsis was lower than that of the wild-type strain. Table 1 shows the maximum rate achieved by each mutant on the fourth day after infection. For all nine mutants, less severe symptoms reflected the number of bacteria in the leaves. All mutants except 33C7 induce either mild or moderate putrefaction and water soaking with varying degrees of chlorosis (yellowing) (see Table 1). As shown in Table 1, the potency of proliferation of individual mutants does not show a direct dependence on the strength of the symptoms they induce. For example, even the 25A12 mutant (Figure 21) grows to the same height as the 33A9 mutants (Figure 5 and 6A to B), pho34B12 (Figures 7, 8 and 9) and

34H4 (Figure 19) and only occasionally less than wild-type UCBPP-P14. The 25A12 mutant causes very weak symptoms. Similarly, mutants 33C7 (Figure 20), phol5 (Figure 24B), and 25F1 (Figure 24A) reach maximum growth levels (approximately 10 ³ times less than wild type growth). Only the 33C7 mutant causes no symptoms (Table 1). The differences caused by the degree of symptoms and the level of proliferation among the ten mutants suggest that these mutants are likely to carry insertion genes that are involved at various stages of the plant infection process.

The pathogenicity of each of the nine generated TnphoA mutants, which appear less pathogenic in the plant leaf assay, was measured in a model of mice burned with full skin thickness (Rahme et al., Science 268: 1899-1902), 1995; Stevens et al., J. of Burn Care and Rehabil. 15: 232-235,1994). As shown in Table 1, all nine mutants were significantly different from the wild type, with a P value of less than or equal to 0.05 at both doses except for the 25A12 and 16G12 mutants (Figure 24A), which did not differ significantly from the wild type at a higher cell dose of 5 x 10 ⁵ . In addition to the data shown in Table 1, the 33A9 mutant also causes no death, even at higher doses containing 5 x 10 ⁶ cells.

We used DNA sequence blot analysis to determine if TnphoA in the nine less pathogenic mutants carried the less pathogenic genes incorporated. DNA blot analysis showed that the 1D7 mutant contained the incorporation of TnphoA in the gacA gene (Laville et al., Proc. Natl. Acad. Sci. USA 89: 1562-1566, 1992; Gaffney et al., Mol. Plant-Microbe Interact. 7 : 455-463, 1994), which have previously been shown to be important pathogenic factors of P. aeruginosa in plants and animals (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560). , 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, and Rahme et al.

Science 268: 1899-1902 (1995)). For the other eight mutants, an inverse polymerase chain reaction (IPCRR) was used to generate amplified products that correspond to DNA sequences adjacent to the TnphoA insertion sites (Ochman et al., A Guide to Methods and Applications, eds. Innis, MA, DJ 1990). IPCR products were cloned and then DNA sequenced. The phol5 mutant contained TnphoA inserted into the P. aeruginosa gene (from strain PA01), which had previously been introduced into GenBank (U84726), which showed a high degree of similarity to the dsbA gene of Azotobacter vinlandia, which encodes the periplasmic disulfide bond of the forming enzyme (Berdwell et. al., Cell 67: 581-589 (1991). DsA homologues in the bacterial phytopathogen Erwinia chrysanthemi and in human pathogens Shigella flexneri and Vibrio cholera are required for pathogenesis (Shevchik et al., Mol. Microbiol. 16: 745-753, 1995; Peek and Taulor, Proc. Natl. Acad. Sci. USA 89: 6210-6214, 1992; Watarai et al., Proc. Natl. Acad. Sci. USA 92: 4927-4931, 1995).

Computer analysis using BLASTX showed that when the DNA sequences correspond to the remaining seven InphoA insertions, they translated into all possible reading frames. No significant similarity was found to any of the known genes (Table 1).

We conducted a number of biochemical tests to categorize the nine less pathogenic mutants of UCBPP-PA14 based on whether the defects carried previously described the primary virulence factor and / or metabolic pathways. All mutants were tested for protease, elastase, and phospholipase activity and their ability to secrete the secondary metabolite pyocyanine (Toder et al., Mol. Microbiol. 5: 20032010, 1991; Essar et al., J. Bact. 172: 884 -900, 1990; Ostroff and Vasil, J. Bacteriol. 169: 45974601, 1987). Pyocyanine is a redox active phenazine secreted by most clinical strains of P. aeruginosa, which disrupts the elastase activity of pyocyanine production.

mammalian and bacterial cells through the formation of reactive oxygen intermediates and which is implicated as a virulence factor 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. 64: 182, 1996). Mutants 33C7, 33A9

34H4, 25F1 and 16G12 show no defects in the biochemical assays used. The pho34B12 mutant exhibits decreased hemolytic activity on blood agar plates, decreased (approximately 50%) and did not detect

The pho15 mutant shows only traces of elastase activity and a decrease in proteolytic activity (60-70%) compared to the wild type. The 25A12 mutant shows a 50% reduction in elastolytic activity. Finally, the 1D7 mutant that contained the insertion into the gacA gene showed a reduced amount of pyocyanine (approximately 50%) compared to the wild type. In plant testing, a second independent gacA: 3nphoA mutant designated 33D11 was identified in addition to the 1D7 mutant. This latter mutant also shows a similar reduction in pyocyanine production and reduced virulence in both plants and mice.

Based on DNA sequence analysis and mutant biochemical assays, genes targeted by TnphoA insertions in AD7 and pho34B12 mutants were selected for further analysis. As discussed above, 1D7 contained an insertion in the gacA gene that encodes the virulence factor of P. aeruginosa (Rahme et al., Science

268: 1899-1902 (1995)].

important virulent

The gacA-like gene also appears to be a factor in the Salmonella typhimurium microorganism (Johnston et al., Mol. Microbiol. 22: 715, 1996). Mutants with two gacA inserts were constructed: TnphoA (1D7 and 33D11) and a gacA insert mutant (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927). and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, and

Rahme et al., Science 268: 1899-1902, 1995) and independently constructed gacA mutation of P. aeruginosa, which affects the production of several known virulent factors (Hasset et al., Infect. Immun. 60: 328-336 , 1992). Both mutants produce a polar effect on at least one gene that is a homolog of the E. coli uvrC gene immediately downstream of the gacA gene (Rahme et al., Science 268: 1899-1902, 1995; Laville et al., Proc. Natl. Acad. Sci., USA 89: 1562-1566, 1992; Reimman et al., Mol. Microbiol. 24: 309-319 (1997). To obtain definitive evidence that the loss of the pathogenic phenotype of the gacA mutants is due to a gacA open reading frame violation rather than a polar effect on the gene downstream of the gacA gene, a non-polar gacA mutation in UCBPP-PA14 was constructed using a DNA cassette encoding carries kanamycin resistance. The non-polar gacA mutant showed the same attenuated pathogenicity in the mouse test (50% mortality) and in the Arabidopsis plant test (growth after 4 days is 3 x 10 ⁵ colony forming units per square centimeter) as well as the gacA :: TnphoA mutant (1D7), but does not exhibit extreme UV sensitivity to polar gacA mutants. Like the 1D7 mutant, the non-polar gacA mutant also secreted a small amount of pyocyanine (50%) compared to the wild type.

The mutant pho34B12 was selected for further analysis for the following reasons. First, the incorporation of pho34B12 was located directly downstream of the pynocyanine phnA and phnB biosynthesis genes in P. aeruginosa (Essar et al., J. Bact. 172: 884-900, 1990) into the previously characterized region of the microorganism genome. P. aeruginosa. Second, the incorporation of pho34B12 caused a pleitropic phenotype that includes reduced elastase activity and hemolytic activity, suggesting that the gene in which the insertion of pho34B12 ^r FnphoA is located may encode a regulator of diverse pathogenic factors.

• ·

To exclude that the secondary mutation in pho34B12 is responsible for the loss of the pathogenic phenotype rather than the incorporation of Tnphoh, the pho34B12 :: TnphoA mutation was replaced by homologous recombination corresponding to the wild-type gene. This leads to the restoration of the pathogenic defect in plants and animals as well as the restoration of hemolytic and elastolytic activity and the production of pyocyanine in amounts as in the wild type (described in Table 2 below).

Table 2 ^a

strain Grow in sheets grew. Arabidopsís Symptoms induced v rošt. Arabidopsís % mortality of 5 x 10 ⁵ mice 0. 0 pyocyanine PA14 5,5 x 10 ⁵ hard 100 ALIGN! 100 ALIGN! pho34B12 4,0 x To ⁵ medium 56 <1 pho34B12 reconstructed called on wild type 3,9 x 10 ⁷ hard 100 ALIGN! 120 pho34B12 + pLGRE34B12 6,1 x 10 ⁵ medium 0 600 pho34B12 + pRRLE2 7,0 x 10 ⁵ medium 13 40 pho34B12 + pRRLEl 5,0 x 10 ⁵ medium 13 1 400 pho34B12 + pRRLE15 1,0 x 10 ^p medium 22nd 1 360

^and in Table no

These results, shown in Table 2, show that the insertion of TnphoA in pho34B12 caused the pleitope phenotype of this strain, which involved the loss of the pathogenic phenotype. The fact that the putative ORF is located at an additional 500 bp downstream of the stop codon following insertion of pho34B12 :: TnphoA (described below) makes it unlikely that TnphoA induces the polar effect of the gene responsible for the phenotype of the pho34B12 mutant . Genetic complement analysis of pho34B12 with a plasmid (pLGRE34B12) containing a 3.7 kb insert that included pho34B12 and a portion of the phnAB region resulted in restoration of elastase and hemolytic activity to a value equal to that of wild-type and more than ten times the excess pyocyanine production. No. 2). However, the disrupted pathogenic phenotype of pho34B12 was not complemented with pLGRE34B12 (Table 12) in mice or Arabidopsis plants (Table 12), possibly due to the presence of multiple copies of the wild-type gene corresponding to pho34B12.

DNA sequence analysis showed that the region containing the pho34B12 mutation encodes two nearly completely overlapping open reading frames (ORFs) (ORF2 and ORF2), which are transcribed in opposite directions. ORF1 had two potential methionine start codons (designated ORF1-S and ORF1-L). The predicted proteins encoded by ORF1-S and ORF1-L, which transcribed in the same direction as the phnA, phnB and phoA genes, contain a consensus motif that corresponds to the lipid attachment site found in many prokaryotic membrane lipoproteins (Hayashi and Wu) (J. Bioenerg. Biomembr. 22: 451471, 1990). These membrane lipoproteins were synthesized by a precursor signal peptide that provides an explanation of the Pho ⁺ phenotype of pho34B12 insertion (Hayashi and Wu, J. Bioenerg. Biomembr. 22: 451-471, 1990). The predicted protein encoded by ORF2 contained an N-terminal helix-turn-helix DNA-binding motif similar to the helixturn-helix motif found in the family of LysR transcriptional regulators (Henikoff et al., Proc. Natl. Acad. Sci.). USA 85: 6602-6606 (1988; Viale et al., J. Bacteriol., 173: 5224-5229, 1991). This class of proteins includes regulators involved in mammalian and plant pathogenesis (Finlay and Falkow, Microbiol. And Mol. Biol. Rev. 61: 136-169, 1997). The existence of two functional nearly completely overlapping ORFs is unusual in the bacterial genome.

In order to determine whether the ORF encoded in the pho34B12 region is functional, additional complement analysis was performed using plasmids containing the PCR products corresponding to ORF1-S and ORF1-L (Figure 7). Production of pyocyanine and elastolytic activity was restored to 20-40% wild-type level by plasmids synthesizing the ORF2 encoded protein (pRRLE2). Similarly, the hemolytic ability of this complemented strain was also partially restored. Complementation of pho34B12 plasmids pRRLE and PRRLE15 corresponding to ORF1-S and ORF1-L also restored hemolytic, pyacyanine and elastolytic activity. The presence of plasmids pRRLE1 and pRRLE10 results in a 10-fold pyocyanine production and twice as high elastase activity. Neither pRRLE1, pRRLE15 nor pRRLE2 supplemented the loss of the pathogenic phenotypes of the pho34B12 mutant in plants or animals (Table 2). Further characterization of this region, including site-directed mutagenesis, further elucidates which of the three ORFs is required for pathogenicity in plants and animals. The data presented above have shown that unknown virulence factors (genes) of P. aeruginosa, which play an important role in mammalian pathogenicity, can often be identified by testing random mutants of P. aeruginosa. In this way, it is possible to find a mutant that exhibits attenuated pathogenic symptoms in plants. This is consistent with a previous study in which at least three genes of P. aeruginosa have been shown to encode virulent factors that are involved in plant and animal pathogenicity (Ausubel et al., USSN. Methods of Screening Compounds). Nos. 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997 and Rahme et al., Science 268: 1899-1902, 1995). On the other hand ·

• * «·

* * It was not expected that nine of the nine mutants that were isolated and less virulent in plants would also be less virulent in mice. The simplest interpretation of this result is that the pathogenesis of P. aeruginosa in plants and animals utilizes a set of substantially overlapping genes, which can be considered as essential virulence genes. Another possible interpretation is that some of the genes identified may encode regulatory proteins (i.e., pho34B12) that drive different effector molecules, the nature of which may be either plant or animal specific. Also, most of the mutants identified in this study (7 out of 9) were not expected to correspond to previously unknown genes. Using the Poisson distribution, the gene size for P. aeruginosa is 5.9 Mb and the average gene size is 1.1 kb. The 2,500 mutants tested were calculated to represent 25% of the total number to be tested to determine the approximately 95% probability of testing each gene in the assay. At least two of the previously known virulence factors (genes) identified in our model that are important in plant pathogenesis are not only important virulence factors in the case of P. aeruginosa in a burned mouse model, but also described as important virulence factors in other grams -negative pathogens. These later pathogenic factors (genes) include dsbA and gacA (Shevchik et al., Mol. Microbiol. 16: 745-753, 1995; Peek and Taylor, Proc. Natl. Acad. Sci. USA 89: 6210-). 6214, 1992, Watarai et al., Proc Natl Acad Sci USA

92: 4927-4931 (1995); Johnston, et al., Mol. Microbiol. 22: 715 (1996). Thus, it is likely that a number of previously known factors identified in P. aeruginosa will generally be relevant for gram-negative pathogenesis. Another important conclusion from this study is that developed high throughput in vivo test methods can lead to the identification of pathogenic factors that do not correlate with common biochemical defects. Mutants 33C7, 33A9, 34H4, 25F1 and 16G12 showed undetectable defects in several known pathogenic factors of P. aeruginosaa, and mutants 33C7 and 33A9 were among the most attenuated in the mouse model. Even pho34B12 and 25A12 mutants show reduced production of known virulence factors, genes corresponding to these mutants have not been previously identified, therefore it is probably not possible to effectively identify these mutants by high-throughput assays. This confirms the sensitivity of our test to the loss of the pathogenic phenotype.

In the past few years, other high-throughput tests identifying bacterial pathogenic factors have been described. IVET (in vivo expression technology) has identified promoters that are specifically activated during pathogenesis (Wang et al., Proc. Natl. Acad. Sci. USA. 93: 10434-10439, 1996; Mahan et al., Science 259: 686-688, 1993), the STM method identified genes that are required for host survival (Hensel, Science 268: 400403, 1995) and DFI (differential fluorescence induction) utilizes green fluorescent protein and fluorescence activated distribution cells to identify genes that activate under specific conditions or specific types of host cells (Valdivia and Falkow, Mol. Microbiol. 22: 367-378, 1996). These approaches are complementary to one approach described in the application and each such approach exhibits certain advantages and disadvantages. One advantage of our non-vertebrate host test procedure is that it directly measures pathogenicity, while the IVET and DFI methods measure the pathogenicity associated with gene expression. Unlike the STM procedure, which identifies genes whose function cannot be complemented by an in-mixed mixed population of bacterial mutants used as inoculum, current invertebrate assays involve testing each mutant clone separately.

• 0 »

Other virulent targets

The 33A9 nucleic acid sequence (Figures 5 and 6A to B) was also identified in the cosmid clone designated BI48 (Figure 1). This cosmid was sequenced over its entire length and its nucleic acid sequence is shown in Figure 2. Nucleotide sequences and deduced amino acid sequences of several other open reading frames were identified using standard database analysis (Figures 3 and 4). A summary of this analysis is given in Table 3. As a sequence described above, any of the sequences shown in Figures 3 and 4 can be used to test substances (for example using the methods described herein) that reduce virulence of a pathogen.

The sequence obtained from cosmid pBI48 strain PA14 showed that 33A9 is approximately 5 kb upstream of the pili gene cluster expression (Figure 1, Table 3). This cluster contains the pilS / pilR genes that are known to be involved in regulating pili formation. Sequence analysis of the upstream gene expression suggests the possibility that the entire region surrounding 33A9 may have defined pathogenicity. Figure 3 (orf 19544), Figure 4 (orf 19544), 5, 6A and 6B show the nucleotide sequence of 33A9 as well as the determined ORFs.

In addition, sequence analysis obtained from cosmid pBI48 also points to the presence of two sequences flanking the 33A9 gene, which contain recognized motifs that are involved in cell capture. Sequence analysis of ORF11738 (2436 bp) and ORF23228 (2565 bp) upstream and downstream of the 33A9 gene (Figure 1) indicates the presence of RGD motifs in the two open reading frames. The RGD tripeptide sequences are characteristic of a eukaryotic recognition motif that binds to the surface integrins of the host cell and have been found to be involved in bacterial adherence. By mimicking host molecules with bacterial adhesins that contain these RGD motifs, they can effectively affect host responses that are required to induce cell-cell adhesion.

Expression of the two ORFs containing RGD was evaluated in both 33A9 and wild type PA14. Transcriptional strength was determined by hybridization with a radiolabeled DNA probe that corresponded to the inner regions of ORF11738 and ORF23228. The data obtained for the two ORFs in mutant 33A9 show a reduced amount of transcript compared to wild type PA14 strain, indicating that the genes encoded by ORF11738 and ORF23228 are regulated by 33A9. These data have shown that 33A9 has the role of a multiple gene regulator responsible for regulating the expression of genes involved in the capture of bacteria on the surface of host cells.

• · · co · · · · ··· · · ·· co

Table

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In addition, using plant and nematode assays (slow or rapid killing assays (Ausubel et al., Methods of Screening Compounds), USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750 filed 25 March 1995, May 7

1997 and November 3, 1997), several other mutant strains of Pseudomonas aeruginosa have been identified that have reduced virulence. The slow and rapid killing tests used for these studies are described below.

Slow-killing test.

For the slow-killing test, 10 μΐ overnight culture was plated on NG (modified NGM agar described in Sulston and Hodgkin, The Nematode Caenorhabditis elegans, WB Wood, ed., Cold Spring Harbor, NY). , 188, pp. 587-606): (using 0.35% peptone instead of 0.25%) and incubated at 37 ° C for 24 hours, after 8 to 24 hours incubation at 23 to 25 ° C. 40 to 50 hermaphrodite L4 C. elegans strain Bristol was applied to each plate (3.5 cm diameter), 3 to 4 replicates were performed for statistical processing, and the plates were incubated at 25 ° C and the number of dead worms was monitored each time. 4 to 6 hours The worm is considered dead when it does not move with tweezers and shows no pharyngeal action For each set of mutants tested, strains PA14 and E. coli OP50 were used as positive and negative controls Any worm that dies as result of immobilization on the wall plates are eliminated from the analysis. In order to determine the value of LT _50, the data is plotted (the percentage of killed worms versus time after exposure to the test strains (h)). Form Curve: Percentage Dead = A + (1-A) / (1 + exp (BG x log (hours post-exposure))) matched data using the SYSTAT 5.2.1 computer program, where A represents the fraction of worms dying in a control experiment OP50 and B and G are parameters that change to fit the curve. When the values of B and G are determined, LT ₅₀ is calculated according to the formula LT ₅₀ = exp (B / G) x (1-2 x A) (1 / G).

In developing the tests we took advantage of two observations. First, the longer the worms die, the more offspring are produced. Second, the early stage larvae are apparently more resistant to killing by P. aeruginosa. This allows us to obtain a very sensitive assay to identify mutants of TnpnoAA that show only a mild disorder in their pathogenic potential. These attenuated mutants are less effective in killing worms, and the production of offspring by survivors effectively amplifies even a weak defect into a well observable phenotype. Thus, on plates containing attenuated PA14 :: TnphoA mutants, hundreds of worms were obtained from the initial deposited hermaphrodites. Thousands of worms are found on the patches where the non-pathogenic mutant is found on the fifth day and the layer of bacteria has been completely consumed. No or very few living worms were detected on wild-type plates. The putative non-pathogenic or attenuated mutants identified in the previous assay were retested. They were subjected to a virulence test to determine the kinetics of C. elegans killing.

Fast killing test

The rapid kill test as well as the slow kill test can be used to identify microbial virulence

In addition, an assay that is capable of being able to carry out disease-causing factors can be used to identify therapeutics, inhibit pathogenicity, or enhance pathogen resistance. In preferred embodiments, the rapid killing assay is performed using a nematode strain that exhibits enhanced permeability of substances such as colchicine toxin. Examples of nematodes that exhibit such increased permeability include, but are not limited to, animals showing mutations in P-glycoprotein, for example PGP-1, PGP-3

• or MRP-1. Such mutant nematodes, due to their increased sensitivity to toxins, can be used in the rapid kill assay. This increased sensitivity is caused by increased membrane permeability. These characteristics result in a test with an increased difference between complete susceptibility and complete resistance to toxic substances. The rapid kill assay is performed based on the increasing osmolarity of the culture medium as described below.

The rapid killing test conditions used here are as follows: 5 μΐ of a culture of overnight PA14 culture in Kings B was plated (3.5 cm diameter) containing peptone-glucose (PG) culture medium (1% bacto-peptone) , 1% NaCl, 1% glucose, 1.7% bacto-agar). Since the efficacy of the rapid kill assay was found to depend on osmolarity, the PG culture medium was adjusted by adding 0.15 M sorbitol. After loading the bacterial culture, the plates were incubated at 37 ° C for 24 hours and then left for 8 to 12 hours at room temperature. 15-29 worms were placed on the test plate ate and incubated at 25 ° C. Each independent assay includes 3-4 replicates. The mortality of the worms was monitored over time, and the worm was considered dead when it did not respond to touch as described above. The E. coli DH5α strain was used as a control strain.

Analysis of these strains together with those identified above shows that they fall into different classes, including the following: some mutants were less pathogenic in both nematodes and plants, while others were reduced in either plants or nematodes but not in both. Bacterial mutants with lower pathogenicity in plants are defined as those that, four days after infection (DPI), exhibit a maximum titer (obtained from 5 leaves) by a value corresponding to two standard deviations lower compared to wild type in the same set of experiments. Wild-type control was necessary because the maximum amount achieved by wild-type on the fourth day after infection may vary by order of magnitude between experiments, causing the effects of minor variations in plant growth conditions exhibiting defensive responses. Similarly, the mutant was characterized as having reduced pathogenicity in worms if the average time required to kill 50% of the worms (LT50 of three replicates) was less by a value that corresponds to two standard deviations compared to the LT50 of wild-type PA14 in the same experiment.

Generally, these mutant strains showing reduced pathogenicity in plants include 16G12, 25A12, 33A9 and 33C7. Those strains showing reduced pathogenicity in nematodes include 35A9, 44B1, 1G2, 8C12, and 2A8 and those showing reduced pathogenicity in plants and nematodes include 25F1, 41A5, 50E12, pholS, 12A1, pho23, 34B12, 34H4.3E8, 23A2 and 36A4. Tables 4 and 5 (below) summarize the pathogenic phenotypes of these mutant strains. Sequence analysis was performed for each strain showing reduced virulence due to established mutagenesis. DNA sequence analyzes summarized in Tables 4 and 5 show that both new and known genes were detected in our assays. The sequences from mutants 50E12 and 41C1 show strong similarity to the previously described open reading frames (ORFs), which are known to be functional in E. coli. The 35A9 mutant identified an mtrR homolog of N. gonorrhoeae (Swissprot P39897). The 25F1 mutant has identified an operon encoding 3 proteins that are consistent with the orfT of C. tepidium, MPK, and DjIA _Ec . The sequences from mutants 48D9, 35H7 and 12A1 corresponded to the lamA, gacA and lasR genes. The disrupted sequences in mutants 41A5 and 44B1 do not show substantial similarity to any sequence deposited with GenBank. (the 44B1 mutant tag sequence is only 149 bp in size, there is no sequence corresponding to 44B1 insertion in the PAO1 database). These sequences identify other virulence factors. The nucleotide and amino acid sequences obtained from these experiments are shown in Figure 10, 11, 12, 13, 14A, 14B, 15, 16, 16A.

16B, 17, 18A, 18B, 18C, 18D and 18E and in Figures 22, 23, 24A, 24B, 24C, 24D, 24E, 25, 26, 27.

We have also performed a number of standard biochemical tests with mutants 41A5, 50E12, 41C1, 35A9, 48D9, 12A1, 44B1 and 35H7 to assess whether any lesions involved in known virulent factors of P. aeruginosa are important for mammalian pathogenicity. These assays include: standard plate assays for sensitivity to hydrogen peroxide, as well as standard quantitative analysis of extracellular protease, elastase, phospholipase, and pyocyanine. Most of the PA14TnphoA mutants were not biochemically distinguishable from the parental strain PA14. The 12A1 mutant showed reduced elastolytic and proteolytic activity, but overproduced pyocyanine. The 50E12 mutant produced 3 times the amount of pyocyanine compared to PA14. The 41A5 mutant showed only about 70% of the amount of wild-type proteolytic activity.

A detailed description of DNA sequence analysis and biochemical analysis of each of these mutants identified using the slow kill assay (described above) is provided in the following section.

Mutant 12A1

The insertion of TnphoA into mutant 12A1 was inserted into codon 154 of the previously described P. aeruginosa PA01 lasR gene. Phenotype 12A1, like other known lasR mutants, is pleitopic and involves increased production of elastase and protease. Furthermore, the 12A1 mutant produces 2-3 times more pyocyanine compared to the stationary phase strain PA14. Further, the GFP-expressing lasR mutant (PA141asR :: GFP19-1) did not self-capture in the worm gut. After 48 hours of feeding, very weak fluorescence was shown in the intestines of C. elegans.

Figure 34A shows that the 12A1 mutant slow killing defective nematode was completely restored when the P. aeruginosa PAO1 lasR gene was expressed in trans under the control of the constitutive lacZ promoter in strain 12A1 ( pKDT17). It was found that elastase production was also restored to the wild-type value of mutant 12A1 (pKDT17), but overproduction of pyocyanine was not achieved. Since the phenotype with overproduction of pyocyanin was not expected, a new lasR mutant (lasR :: Gm) was constructed, replacing the lasR gene marker with a gantamicin cassette in the PA14 genome. The lasR: Gm mutant was as non-pathogenic as 12A1 (Figure 34A), but produced a normal amount of pyocyanine, suggesting that the 12A1 mutant may carry a second mutation that results in increased pyocyanine production. The result also shows that the enhancement of pyocyanine production during the stationary phase is not associated with an attenuated pathogenic phenotype.

Mutant phol5

Disruption of the dsbA gene in the mutant phol5 has been found to be responsible for non-pathogenic phenotypes. Figure 24B shows the nucleotide sequence (SEQ ID NO: 166) and predicted amino acid sequence (SEQ ID NO: 167) of the mutant PA14phol5. It was also found that the pathogenicity of the defective phol5 phenotype in C. elegans was also completely restored by constitutively expressing the dsbA _Ec gene of E. coli or the PA14 dsbA _Pa gene in position in the original phol5 mutant (Figure 34B). For these experiments, the dsbA _Ec gene of E. coli was cloned into plasmid pUCP18 as follows. PCR amplified dsbA _{Ec of} E. coli was cloned into KpnI and XbaI restriction sites of pBAD18 to generate pCH3. This was introduced into dsbA of E. coli and expression was driven by the arabinose promoter of E. coli. Fragment of restriction enzymes

A 700 bp KpnI / SphI was cloned into KpnI / SphI sites of pUC18 to generate pEcdsbA. E. coli dsbA is under the control of the constitutive lacZ promoter of E. coli. PScdsbA is then • · • ·

4i used to transform PA14 and the mutant pho15 to construct the strain PA14 (pEcdsbA) and pho15 (pEcdsbA).

PA14dsbA _Pa was constructed as follows. Based on the dsbA sequences of strain PA14 (GenBank deposit number U84726), primer TMW8 (5'-GCACTGATCGCTGCGTAGCACGGC-3 '; SEQ ID NO: 177) and primer TMW9 (5'-TGACGTAGCCGGAACGCAGGCTGC-3SEQ ID NO: 178) were used for amplification. a 1126 bp fragment containing the dsbA gene plus 176 bp from the translation start site upstream of the dsbA gene from the PA14 genomic DNA. This fragment was cloned using the TA cloning kit (Invitrogen) into the pCR2.1 vector to generate pCRdsbA. A restriction enzyme fragment of Sacl / XbaI containing dsbA was cloned into pUCP18 digested with Sacl / XbaI to give pPAdsbA, wherein transcription of the dsbA gene is driven by the constitutive lacZ promoter. The phol5 strain (PadsbA) was constructed by transforming pho15 pRAdsbA _Pa .

Mutant 25F1

It was found that in the 25F1 mutant, TnphoA was inserted into codon 100 of a putative gene (orf338) which encodes a protein of 338 amino acids, the first putative operon gene of three genes. The predicted downstream genes (orf224 and orf252) encode proteins of 224 and 252 amino acids, respectively. GAP analysis showed that the orf338 gene was 28.5% identical (37.7% similarity) to the C. tepidum orfT gene (deposited with GenBank U58313). BLASTP ORF224 identified mannose-1-phosphateguanylyltransferase (MPG; EC 2.7.7.13) from eukaryotes, archebacteria, cyanobacteria and mycobacteria, not from proteobacteria that are closely related to P.aeruginosa. It is unclear whether ORF224 in MPG, since all known MPGs contain 359 to 388 amino acid residues, while ORF224 contains only 224 amino acid residues. ORF252 is homologous to the DjlA _Ec gene of E. coli. The Dj1A _Ec gene is believed to play an important role in repairing

arranging, activating, and / or maintaining a number of membrane proteins that include a signal transduction of two component systems.

To test whether the orf338 gene is responsible for limited pathogenicity in worms, the kinetics of the killing strain carrying the wild-type orf338, 25F1 (pORF338) gene alone were compared.

PA14 and 25F1, which carries the vector itself. 25F1 (pORF338) was constructed as follows.

A 1.8 kb PCR fragment containing the 482 bp promoter sequence, the whole orf338 gene and the truncated orf224i gene was amplified (ExpandTm High Fidelity System, Boehringer Manheim) from genomic DNA of strain PA14 using primer F2327 (5'-CGAGGAATCCAGTCGAGGTG-3 SEQ ID NO: 179) a

R4180 (5'-GCAAGATGCAGCCGAGAGTAG-3 '; SEQ ID NO: 180). The product was cloned into pCR2.1 vector (TA Cloning, Invitrogen) to give plasmid pMT403C-R. The Sac I / Xba I fragment from pMT403C-R, which contains the PCR product, was cloned into Sac I / Xba I pUCP18 to generate pORF338. The orf338 gene is under the control of its natural promoter. The 25F1 mutant was transformed with pORF338 to form the 25F1 strain (pORF338).

The stem that contained the entire operon (orf338, orf224 and djIA _Pa ) was constructed as follows. PCR was used to amplify a 3.6 kb genomic fragment containing orf338, orf224, and dj 1A _Pa and their transcriptional sequences downstream of the gene. PCR was performed using primers RIF3115 (5'-GTCAGAATTCTCAGCTTGACGTTGTTGCCC-3 '; SEQ ID NO: 181) and RIR6757 (5'-GTCAGAATTCGACTTCTATTACCGCGACGCC-3'; SEQ ID NO:

182). The EcoRI site (underlined) is present in the primers but not present in the genomic sequence. Both strands of the PCR product were sequenced to determine the sequence of orf338, orf224, and djIA _Pa in the PA14 strain. The EcoRI restriction enzyme digested PCR product was cloned into the EcoRI site of pUCP18. The orientation of the insert was determined by restriction enzyme digestion. Plasmid p3-ORFs where genes orf338, orf224 and djlAp _and are controlled by their natural promoter. This plasmid was used to transform 25F1 to form the 25F1 strain (p3-ORF).

As shown in Figure 34C, strain 25F1 (pORF338) fails to completely complement the slow killing phenotype. Strain 25F1 (p3-ORF), which contains the entire operon (orf338, orf224, and djIA _Pa ) also shows only partial complementation of the mutant phenotype. This result indicates that TnphoA is responsible for the pathogenic phenotype. Partial complementation may be dose-dependent. Higher mortality was achieved with 25F1 (p3-ORF) compared to 25F1 (pORF338), suggesting that ORF224 and / or DjIA _Pa genes may also play an important role in the virulence of PA14.

Figure 24E depicts the nucleotide sequences (SEQ ID NO: 173) of PA14 25F1 encoding ORFT (SEQ ID NO: 174, ORFU (SEQ ID NO: 175) and Dj 1A _Pa (SEQ ID NO: 176)).

Mutant 50E12

The incorporation of FnphoA into the 50E12 mutant was carried out at codon 39 of a predicted 759 amino acid protein that showed 43% identity (54% similarity) to the E. coli PtsP _Ec protein. Based on sequence analysis, the ptsP _Ec gene was predicted to encode the enzyme INTr, which is a protein containing 738 amino acids and the N-terminal domain of Nif-A and the Cterminal domain of enzymes I. Later, it is used in a phosphoenolpyruvate-dependent phosphotransferase system. It is contemplated that the Nif-A domain serves in signal transduction, either directly sensing small molecule signals or receiving signals from a NifL-like protein. Any mechanism can modulate the catalytic region of enzyme I. This, in turn, is indicated by phosphorylation of Npr (nitrogen-related Hpr), thereby regulating the transcription of RpoN-dependent operons. Immediately upstream of the PA14ptsP _Pa homolog expression is an open reading frame (orf159) which is believed to encode a protein of 159 amino acids that appears to be transcribed together with ptsPPa. FIG. 24C shows the nucleotide sequence (SEQ ID NO: 168) of PA14 50E12 encoding mutant YgdPp _and (SEQ ID NO: 169) and PTSD _Fa (SEQ ID NO: 170). ORF159 is 62.3 to 64.8% identical to YgdP proteins found in H. influenzae (GenBank deposit number is Q57045) and E. coli (GenBank deposit number is Q46930). These proteins are also closely related to the invasive protein of Helicobacter pyroli and Bartonella bacilliformis. Invasive protein A of B. bacilliformis deposit number (SwissProt P35640) is encoded by the ailA gene, which, when present with the adjacent but independently transcribed ailB gene, confers the ability of E. coli to infect human erythrocytes.

Two strains were tested for complementarity of the 50E12 mutant: the 50E12 (pMT206-lac) and 50E12 (pMT206-nat) strains. Strain 50E12 (pMT206-lac) carries the plasmid pMT206-lac, where transcription of orf159 and ptsPPa is driven by the constitutive lacZ promoter. In the case of strain 50E12 (pMT206-nat), the transcription of orf159 and ptsPPa is driven only by their natural promoter. Each of these strains was constructed as follows.

A 4.3 kb PCR fragment containing an EcoRI restriction site at both ends was amplified from the P. aeruginosa genomic DNA strain PA14 using primers RIF1698 (5'-GTCAGAATTCGATGTTCCAAGTCCCAGATCCC-3 '; SEQ ID NO: 183) and RIR6002 (5') -GTCAGAATTCCAGTAGACCACCGCCGAGAG-3 '; SEQ ID NO: 184). This fragment was cloned into the EcoRI restriction site of pUCP18 to give pMT206-lac and pMT206-nat. Their identity was confirmed by restriction enzyme digestion. In pMT206lac, the transcription of orf159 and ptsPPa is driven by the constitutive lacZ promoter and their natural promoter. The transcription of the orf159 and ptsPPa genes in pMT206-nat is driven only by their native promoter.

As shown in Figure 34D, both strains partially complement the mutant phenotype. The time it takes for these supplemented strains to kill 100% of the worms is longer than the wild-type strain. Partial complementation was observed in the burned mice assay: mortality of mice after infection with 5 χ 10 ⁵ bacteria of the 50E12 strain (pMT206-nat) was 39% compared to 100% and 0% mortality when infection with wild-type strain These results indicated that respectively putative with strain 50E12. orf159-ptsP _Pa operon is involved in pathogenesis in nematodes and mice.

Mutant 35A9

The insertion of TnphoA into mutant 35A9 is found in a putative 210 amino acid protein (encoded by the orf210 gene), which is very closely related (31.5% identity) to the N. gonorrhoeae MtrRNg protein, which belongs to the family of proteins regulating bacterial transcription of TetR. ORF210 is adjacent, but transcribed separately, to three genes that are homologous to components of the energy-dependent runoff system (EDE) in P. aeruginosa. Other previously described EDE systems in P. aeruginosa 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 NO: 171) of PA14 35A9 encoding mtrR _Pa (SEQ ID NO: 172).

Mutants 37H7 and 1D7

Analysis of the IPCR product from human 37H7 shows that there is an insertion of inphoA at codon 188 of the GacA protein, which contains 214 amino acids. DNA blot analysis showed that the 1D7 mutant also contains an insertion in the gacA gene.

Mutant 48D9

TnphoA was inserted between codon 491 and 492 of a LemA homolog containing 925 amino acids, a sensor kinase belonging to the family of two components of bacterial regulators. It has been shown that a related LamA regulator in P. syringae microorganism 9 · ·

99 9

9 · ·

99 9 9 9

GacA and GacA + Lem A also affect the expression of different numbers of virulent factors.

Mutant 41C1

TnphoA was incorporated into the AefA homolog of the putative integrated membrane protein of E. coli (SwissProt P77338) in mutant 41C1. It is a member of the UPF0003 protein family with a protein molecular weight of 30,000 to 40,000 (PLEASE

E. coli is also present with PCC 6803 and Methanococcus

PDOC00959). In addition to the organism in the microorganism Synechocystis jannaschii.

In addition, strains pho34B12, 3E8, 8C12, 1G2, 35A9 and 23A2 were found to exhibit the phenazine mutant phenotype minus. In addition, mutants pho34B12, 3E8, 8C12, and 1G2 were found to exhibit reduced pigment production. The 6A6 mutant also exhibits reduced pigment production. The characteristic color of P. aeruginosa strains has been classified as a group of tricyclic secondary metabolites collectively known as phenazines, of which the blue-green pigment pyocyanine (1-hydroxy-5-methylphenazine) is the most characterized. In order to test whether the reduction in pigmentation in bacterial mutants was at least in part due to pyocyanine reduction, the amount of this pigment was determined in wild type PA14 as well as in mutants obtained using the rapid kill assay. The results of this analysis show that the mutants pho34B12, 3E8, 8C12, 1G2 and 6A6, which exhibit a reduced pigmentation phenotype, also exhibit limited pyocyanine production. The amount ranges from 10 to 50% of the production of the wild-type strain. Other mutants such as 13C9, 23A2 and 36A4 show an amount of pyocyanine comparable to the wild-type strain.

Furthermore, it was found that the sequence interrupted by the mutation of TnphoA in mutant 3E8 encodes a protein with homology to the phzB gene from P. fluorescence microorgasmic, which is part of the operon, which is A * AI

Four of the assays involved in the production of the secondary metabolite phenazine (GenBank deposit number is L48616). The phzB gene also has a homologous strain in P. aureofaciens, known as the phzY gene (GenBank deposit number AF007801). Using the tag sequence, a cosmid (1G2503) containing this region was identified in the Pseudomonas aeruginosa microorganism database that contains the phzA and phzB genes, as well as other genes likely to play an important role in the biosynthesis of pcnC and D phenazine genes (GenBank ref. Number AF005404;

of these strains 34B12, 3E8, 23A12 and 35A9 with burned mice tested for pathogenicity. Surprisingly, these experiments show that phenazine defective strains have reduced pathogenicity, indicating that genes disrupted by YnphoA insertions are mammalian virulent factors. The nucleotide and deduced amino acid sequences comprising the tag sequences of these strains are shown in Figure 7 to 9, 13, 14A, 14B, 15, 16A, 16B, 17, 18A, 18B, 18C, 18D, 18E, 22,

24A, 24B, 24C, 24D, 24E, and 33. 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.

A detailed description of the DNA sequence and biochemical analysis of each of the mutants identified using the rapid kill assay (described above) follows in the following sections.

Mutants 36A4, 23A2 and 13C9

DNA tag sequences obtained from all three mutants that produce a wild-type amount of pyocyanine showed homologies to the Pseudomonas micro-organism genes. The 36A4 mutant contained Ynpho inserted into the gene homologous to the hrpM gene, which, as previously determined, is a locus controlling the pathogenicity of the plant pathogen Pseudomonas syringaei (Mills and Mukhopadhyay, Pseuodmonas: biotransformations, pathogenesis and evolving technology, S. Silver, A.).

¹ 48 • » • t • »· · * _ · * «·· · * · · > * · E »· ··· Chakrabarty, B. Iglewiski, and S. Chaplain, eds., American Sociaty for Microbiology, 1990, s. 47-57, Mukhopadhyay et al., J. Bacteriol. 170: 5479-5488, (1988) (storage number

in GenBank is 140793). This locus also shows homology to the mdoH gene of the E. coli microorganism, which encodes an enzyme involved in the biosynthesis of periplasmic glucan (Loubens et al., Mol. Microbiol. 10: 329-340, 1993; GenBank deposit number). is X64197). The insertion of TnphoA in mutant 23A3 was made into a previously determined P. aeruginosa gene of strain PAO1 as mexA (Poole et al., Mol. Microbiol. 10: 529-544. 1993; GenBank deposit number is L11616). The mexA gene product, which encodes a cytoplasmic membrane-associated lipoprotein, is likely to work together with the products of the other two genes contained in the same mexB and oprM containing operon, as a non-ATPase drainage pump with broad substrate specificity (Li et al., Antimicrobiol. Agents Chemother 39: 1948-1953 (1995). Sequence analysis of the DNA flanking the third mutant, the wild-type for the production of 13C9 pigment, showed that it corresponded to another previously determined gene in the P. aeruginosa microorganism strain PAO1, orp (GenBank deposit number is U54794). Orp or phospholipase C-dependent regulator has been identified as a factor driving expression of the pathogenic factor PlcH, one of the 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 two non-pigment secreting mutants, 1G2 and 8C12, showed that they contained insertions of two new genes, although the DNA flanking the 1G2 incorporation contained a motif characteristic of the histidine sensor kinase. This gene is not present in the PAO1 genome database. Although the sequence of the mutant 8C13 tag identified the homologous gene in the PAO1 database, no substantial gene was found in this gene.

Mutants 3E8 and 6A6

Two mutants 3E8 and 6A6 contain insertion of TnphoA in the same gene that is homologous to the previously determined phzB gene of P. aeruginosa strain 2-79 (GenBank deposit number AF007801) and phzY of P. aeruginosa strain 30-84 (deposit number GenBank is L48616). These two mutants contained TnphoA incorporation at exactly the same site, but are independent isolates because they were obtained from different mutant libraries. Although the phzB gene and the phzY gene contained non-detectable sequence motifs, they are present in operons known to regulate the production of phenazine-1-carboxylate (PCA) in both P. fluorescens and P. aureofaciens (Mavrodi et al., J. Bacteriol., 180: 2541-2548 (1998).

Mutant pho34A12

The DNA flanking the insertion of TnphoA in the final mutant pho34B13 without pigment was previously cloned and turned out to be a new locus as previously described. This insertion is immediately downstream of the phenazine biosynthesis genes phnA and phnB, as identified in the P. aeruginosa microorganism strain PAO1 (Essar et al., J. bacteriol. 172: 884900, 1990).

Phenazines are required to quickly kill C. elegans

Isolation of pigmented unpigmented mutants in rapid killing assays indicates that the rapid killing process involves more than one factor. Molecular analysis of mutants 3E8 and 6A6 (containing operon insertions that regulate phenazine production) strongly suggests that phenazines represent one class of toxins that mediate rapid killing. For

in order to directly test this hypothesis, further mutations of AphnAphnB were generated and studied as follows.

The phnA and phnB biosynthesis genes (Essar et al., J. bacteriol. 172: 884-900, 1990) are upstream of the previously characterized pho34B12TAiphoA insertion in PA14 (GenBank deposit number AF031571). A 3.7 kb EcoRI fragment corresponding to the wild-type sequence of this region (from plasmid pLGR34) was subcloned into pBluescript Sk / + to produce Bs34B12. This plasmid contained the 944 bp phnA gene (full length 1,591 bp), the entire phnB gene (600 bp), and a 1.7 kb sequence downstream of the gene. The missing 405 bp phnA gene and the 405 bp downstream gene were amplified using PCR from PA14 genomic DNA using the PHNA3 (5'-GGTCTAGACGAACTGAGCGAGGAG-3 '; SEQ ID NO: 185) and PHNA2 (5'-GCCTGCAGGGGGTTCTCT oligonucleotide primers). SEQ ID NO: 186). The primers are based on the sequence previously cloned into the phnA and phnB genes from P. aerugonoas strain PAO1 (Essar et al., J, Bacteriol. 172: 884-900, 1990; GenBank deposit number is M33811). The 1,010 bp amplified sequence was subcloned into the Pst restriction site of pBs34B12 to generate the pBs34B12phmA construct. Within the phnA deletion, phnB was generated by replacing the 2.6 kb sequence of wild-type genes with a 1 kb fragment (Figure 35) amplified by PCR using primer PHNDEL1 (5'GGCTGCAGTGATTGACTGAGCGTCTGCTGGAGAACG-3 '; SEQ ID NO: 187) and PHNDEL2 (5'-GGGAAGCTTCGTCTAGAATCACGTGAACATGTTCC-3 '; SEQ ID NO: 188) to give plasmid pBs34bl2ohndel. The 1.8 kb XbaI fragment containing the deletion within phnAphnB was subcloned into vector pCVD442 (Donnernberg and Kapper, Infect. Immun. 59: 4310-4317, 1991).

The resulting pCVD34B12phndel construct was used to introduce the phnA, phnB genes into the wild-type PA14 genome by homologous recombination, which resulted in the PA14 mutant AphnAphnB. In order to verify that the mutant contains the desired deletion, restriction and blot analysis of the DNA using DNA from parental PA14 was performed. and derivative strains of PA14 AphnAphnB.

Although little is known about the nature of the enzymes that catalyze the formation of phenazines in P. aeruginosa and related Pseumonads, the conversion of chorismate to anthranilate is considered a key step in this pathway (Figure 35A) in P. aeruginosa strain PA01. This step is most likely catalyzed by anthranilate synthase encoded by the phnA and phnB genes. Since these mutations in these genes result in a decrease in the production of fensin pyocyanine (Essar et al., J. Bacteriol. 172: 884-900, 1990). The phnA and phnB genes were cloned from the PA14 strain and an AphnAphnB mutant was created containing a 1,602 bp deletion in these genes (Figure 35B). This mutation was designed to be non-polar and therefore does not affect two ORFs that are known to be located downstream of the phnA and phnB genes. Measurement of the amount of pyocyanine in the AphnAphnB mutant showed that only 10% of the wild-type amount was generated, confirming that the phnA and phnB genes are involved in pyocyanine production in the PA14 strain as well as in the PAO1 strain. Tests performed using AphnAphnB showed that it was greatly reduced by rapid killing. As can be seen in Figure 35C, less than 5% were dead three hours after the AphnAphnB challenge compared to the wild-type strain, where nearly 100% died. The AphnAphnB strain behaves in a manner similar to the other phenazine mutant 3E8, which serves as a control for the attenuated mutant in this experiment. These results showed that phenazines are required for rapid killing of C. elegans.

In order to determine whether bacterial factors mediate rapid killing are relevant to the pathogenicity of other hosts, the rapidly fatal mutants were tested for virulence in an infiltration model on leaves of Arabidopsis as well as in a skin model of burned mice.

described above. Five rapidly-fatal mutants were tested for their ability to grow over four days on Arabidopsis leaves as a quantitative measure of their pathogenicity. This was also tested in a burned mouse model. As shown in Tables 4 and 5, the maximum growth value on the leaves of Arabidopsis obtained four days after infection was significantly lower in the 2 phenazine mucins 3E8 and 8C12. In the murine model, the two mutants caused a significantly lower mortality compared to the wild-type strain where the P value is less than or equal to 0.05 when a 5 x 10 ⁵ inoculum was used. The third phenazine mutant 1G2 did not differ significantly from the wild-type strain in either plant or animal models.

Both hrpM mutants mexA and 35A4 mutated in growth on Arabidopsis leaves, indicating a strong pathogenic effect in this model. In the murine model, the 36A4 mutant has a dramatic effect that causes no death at the dose tested. In contrast, the mexA mutant 23A2 was only marginally affected. These results demonstrate that the rapid killing assay is extremely effective in identifying genes that are required for plant and mouse pathogenesis and further provides a first in vivo demonstration that phenazines are required for the pathogenesis of the two hosts.

It must also be noted that the phzR regulator of the phz operon has been identified. Figure 18E shows the nucleotide sequence (SEQ ID NO: 164) and the predicted partial amino acid sequence (SEQ ID NO: 165) of PA14phzR.

Phenazines and pathogenesis

PA14 mutants with limited rapid killing also affect pigment synthesis. Molecular analysis revealed that the association between pigment production and pathogenesis was not due solely to the regulation of pigmentation and toxin production by regulatory factors. Instead, mutations in phenazine biosynthesis genes were found to reduce virulence strongly implying rapid killing phenazine. Phenazines, which pigmented substances they provide as process toxins are tricyclic

Pseudomonads have their characteristic color (Turner and

Messenger, Adv. Microb, secondary metabolites,

Physiol which increases the number of surviving organisms under competitive conditions (Maplestone et al., Gene 115: 151-157, 1992). Although the composition of the phenazines produced by strain PA14 is known, the P. aeruginosa microorganism strain PA01 produces at least six different phenazines that include the well characterized blue-green pyocyanine pigment. Phenazines including pyocyanine have been shown to contain antibiotics against several species of bacteria, fungi and protozoa, and the quality depends on their redox activity. It is reported that phenazines in their highly reactive reduced stage undergo a redox cycle in the presence of various reducing agents or molecular oxygen, leading to the formation of hyperoxide and hydrogen peroxide (Hassen and Fridovich, J. Bacteriol. 141: 1556-163, 1980). In vitro, these moderately cytotoxic oxygen radicals can be converted by a ferric catalyst to highly cytotoxic hydroxyl radicals (Britigan et al., J. Clin. Invest. 9: 2187-2196, 1992). The formation of reactive oxygen species by phenazines is also thought to contribute to their cytotoxic effects observed in eukaryotic cells in vitro. These effects include inhibition of mammalian cell respiration, ciliary movement disruption, and immunomodulatory effects such as stimulation of inflammatory response, inhibition of lymphocyte proliferation and alteration of T-lymphocyte response to antigens. The biosynthetic pathways leading to phenazine production in P. aeruginosa microrganism are poorly defined, which makes it difficult to define pathway-blocked mutants of PA14 that are deficient in phenazine production. Transposon insertion into

27: 211-273, 1986) are likely involved in two mutants 3E8 and 6A6 disrupted a gene that exhibits homology to the phzB gene, which was previously characterized by being involved in the production of phenazine in related P. fluorescens pseudomonas and P. aurofaciencs. It turned out that in microorganism P.

The fluorescent gene phzB is part of the operon, which contains seven genes (phzA-G), which are involved in the production of phenazine-1-carboxylic acid. A comparison of this operon in P. fluorescens and P. aureofaciens shows that the two operons are highly homologous, suggesting that the pathway to production is conserved in fluorescent psedomonads (Mavrodi et al., J. Bacteriol. 180). : 2541-2548 (1998). Although the DNA flanking the phzA and phzB genes in the P. aeruginosa microorganism PA14 were only partially sequenced, our analysis suggests that the region shares a conserved structure with the P. fluorescens phzA-F operon. The predicted translated products of the phzA and phzB genes from the PA14 strain and from P. fluorescens share 68% and 74% identity, respectively. In addition, a region containing phzF-like genes is present in the P.aeruginosa microorganism strain PAO1 and exhibits approximately 60-85% identity with their P. fluorescens homologues in the predicted translated products of these genes (GenBank deposit number AF005404). Extrapolation from the role of the phz operon in P. fluorescence and P. aureofaciens, isolation of PA14phzB mutants that are deficient in rapid killing strongly suggests that phenazines are involved in this process. The hypothesis that pyazyanine-containing phenazines are one of the mediators of rapid killing was further tested by nonpolar disruption of the phnA and phnB genes, which encode two anthalinate synthase subunits, which, as previously shown, is involved in the phenazine synthesis of aeruginosa strain PAO1 (Essar et al. , J.

in microorganism P.

Bacteriol. 172: 884-990 (1990)

Consistent with the role in phenazine synthesis, deletion of the phnA and phnB genes in the PA14 strain reduces pyocyanine production. Further, the AphnAphnB mutant was deficient in rapid killing, indicating a critical role of phenazines in this process.

The role of phenazines in pathogenesis has also been tested in Arabidosis and mice. Two independent mutants containing insertions in the phzB gene 3E8 and 6A6 show dramatically reduced pathogenicity in the leaf infiltration model of Arabidopsis as well as in the burned mouse model (Tables 4 and 5), suggesting that phenazines are pathogenic factors for multiple hosts. It is interesting to note that many other multi-host pathogenic factors identified in this or previously performed studies are likely to be involved in the production of several other virulent factors and are not effectors or molecules that directly interact with the host (described above) . Thus, phenazines represent only a known class of identified pathogenic effectors suitable for multiple hosts. These findings are also essential because, despite intensive in vivo analyzes of phenazines, the physiological relevance of their production and their role in P. aeruginosa infection remains controversial. Prior to this study their role in vivo was not shown.

Multiple factors cause rapid killing

Analysis of the fast killing mutants that make up the amount of pigment corresponding to that of the wild-type strain shows that although phenazines are essential mediators of fast killing, other factors are involved in this process. Molecular analysis of such a single 23A2 mutant has shown that transposon has been incorporated into a gene previously identified in P. aeruginosa as a PAO1 strain as MexA, which is part of an operon containing 3 MexA, B, OprM genes (Poole et al. Mol. Microbiol. 10: 529-544, 1993). The products of these genes are found in the cytoplasmic (MexA, MexB) membrane and in the outer membrane (OprM), where they are thought to function as drain pumps or

Broad specificity ATPas (Li et al., Antimicrobiol. Agents Chemother. 39: 1948-1953, 1995). This pump appears to play a general role in the expert of secondary metabolites, although their natural substrates remain unknown (Poole, Antimicrobiol. Agents Chemother. 34: 453-456, 1994). The defect of the mexA mutant in rapid killing, a process mediated by diffuse toxins, is most likely due to the lack of export of one or more factors involved in this process. Since the mexA mutant is pigmented, it is unlikely that phenazines will form a substrate for the pump. In addition to the rapid killing defect, the mexA mutant exhibits markedly reduced pathogenicity in the mouse model and a strongly reduced pathogenicity in the leaf infiltration model of the Arabidopsis plant. Although the lack of specific virulence factor exports may explain this defect, another model is that mutant mexA bacteria are not able to protect themselves against host defense factors that have developed in response to bacterial infection. Such a protective function has been implicated in the sap gene, which encodes proteins related to ATP binding cassette transporters (ABC) and mediates resistance to host antimicrobial peptides in the human pathogen Salmonella typhimurium as well as in the phytopathogen Erwinia chrysanthemi (Taylor et al., Plant Cell). 10: 873875 (1998).

The second mutant 36A4 identified in this assay contained a transposon insertion into a gene that showed homology to the MdoH gene of E. coli, which is part of the mdoGH operon. In E. coli, the products of said operon are involved in the synthesis of membrane-derived oligosaccharide (MDO) or linear periplasmic glucan (described in

in the publication Loubens et al. , Mol. Microbiol. 10: 329-340, 1993). A similar locus, who is called hrpM Yippee present in plant pathogen Pseudomonas syringae pv. syringae

(Mukhopadhyay et al., J. Bacteriol. 170: 5479-5488, 1988), which was originally identified because mutations at this locus eliminate the development of disease symptoms in host plants as well as hypersensitivity responses in non-hostile plants (describes (Anderson and Mills, Phytopath. 75: 104-108, 1985). Periplasmic glucans have also been found in a wide range of bacteria attributed to various misunderstood functions. In addition to being essential virulence factors in P. syringea, other features include adaptation of the hypoosmotic environment and cellular signals leading to recognition of eukaryotic hosts of Rhizobium and Agrobacterium species (Kennedy, Escherichia and Salmonella, FC Neidardt, ed, American Society for Microbiology Press, Washington, DC, pp. 1064-1071, 1996). Despite the presence in the periplasm of several animal pathogens, such as Salmonella and Kleibsiella, in this study, it shows that P. aeruginosa carrying a mutation in a mdoH-like locus is a strongly reduced pathogenicity in a mouse model. Periplasmic glucans have not been shown to play any role in the infection of animal hosts.

• • •

Table 4

Summary of pathogenicity of P. aeruginosa mutant strain UCBPP-PA14 in various hosts.

Pathogenic phenotypes Number insulation strain Name strain Growth on leaves Arabidops is ^b Killing ability C. elegans ^c o mortality of 5x10 ^5d mice Identity gene PA14 PA14 5.5 x 10 ⁷ + 100 ALIGN! Rep (limited pathogenicity in plants) 16G12 rep 1 2.3 x 10 ^p + 100 ALIGN! None steam 4 9H2 rep2 1.2 x 10 ⁶ + 63 It is not sequential no 25A12 rep3 1.7 x 10 ⁶ + 75 None steam 33A9 rep 4 5.1 x 10 ⁶ + 0 None steam 33C7 rep5 8.4 x 10 ⁵ + 0 None steam Ren (limited pathogenicity in nematodes) 35A9 ⁹ renl 5.7 x 10 ⁷ - 55 mtrR 44B1 ren2 5.4 x 10 ⁷ 56 None steam lG2 ^{f, g, h} NT NT None Couples 8G12 ^f ' ^g NT NT None steam 2A7 ^{f, h} NT NT None steam Rpn (reduced pathogenicity in plants and nematodes)

25F1 rpnl 1.5 X 10 ⁴ - 20 May orfT 35H7 ^e rpn 2 1,2 X 10 ⁴ - NT ^e ga cA 41A5 rpn 3 1.3 X 10 ⁴ 100 ALIGN! None steam 41C1 rpn4 2.4 X 10 ^p - 85 aefA 50E12 rpn 5 2,0 X 10 ^p - 0 ptsP Phol5 rpn 6 3.9 X 10 ⁴ - 62 dsbA 12A1 rpn 7 1.7 X 10 ⁶ - 50 lasR Pho23 rpn 8 6.4 X 10 ⁴ 5 None steam 34B12 ^{g, h} rpn 11 4.0 X 10 ⁴ - 50 dst * phnB 34H4 rpn 12 3.8 X 10 ⁶ 50 None steam 3E8 ^g rpn 13 1 x 10 6 - 12.5 phzB 23A2 ^h rpnl 4 1.7 X 10 ⁵ - 71 MexA 36A4 ^h rpn 15 4 x 10 4 - 0 HrpN

^b colony-forming units / cm ² leaf area of bacterial count four days after inoculation with 10 ³ bacteria: average value obtained from four to five samples. Mutants are defined as less pathogenic based on an average of four to five samples. Mythants are defined as less pathogenic when the average colony forming unit / cm ² leaf area of the bacterial count is lower by two standard deviations relative to the wild-type strain in the same experiment.

^{c A} mutant is considered attenuated in the case of non-nematode pathogenicity (-) if the average time to kill 50% of the worms (LT ₅₀ of three replicates) is less than the LT _{50 of the} parent strain UCBPP-PA14 in the same experiment. The calculation of LT ₅₀ is described in the chapter Meterial and methods.

^d 6 week old female inbred strain of ARK / J mice (originating from Jackson Laboratories), weighing 20-30 g, were injected with 5 x 10 ⁵ cells as described in Stevens et al., J. of Burn Care and Rehabil. 15:

• ·

232-235, 1994. The number of animals that die from sepsis was malted every day for ten days.

^c Two other independently isolated gacA mutants are ID7 (rpm9) and 33D1 (rpn10). The rpn9 mutant was tested in mice and showed 50% mortality.

^f tested only on nematodes ⁹ mutants deficient in phenazine ^h mutants with insufficient fast killing, not affected by slow killing dst * means downstream of gene expression

Table 5:

The pathogenicity of PA14 rapidly killing mutants in plants and mice

Strain Growth in Arabidopsis ³ % mortality (n) 5x10 ^5b Gene identity PA14 7 x 10 ⁸ 100 (> 15) 1G2 3 x 10 ⁷ 100 (7) No pairs, contains theme histidine kinases 3E8 ^C , 6A6 3 x 10 ^p 18 (16) PhzB 8C12 5 x 10 ^p 63 (8) No couples 23A2 1.2 x 10 ⁴ 85 (16) mexA 36A4 2 x 10 ⁴ 0 (16) hrpM

^and colony-forming units / cm ² of leaf area of bacterial count ^three days after inoculation with 10 ³ bacteria. The values represent the average of four to five samples. Mutants were defined as less pathogenic when the average number of bacteria was two standard deviations less than that of the wild type in the same experiment. ^b six week old female inbred strain of ARK / J mice (originating from Jackson Laboratories), weighing 20-30 g, were injected with 5 x 10 ⁵ cells; (n) is the total number of mice injected with cells. The number of mice that die from sepsis was malted every day for ten days.

^c 3E8 and 6A6 are two independently generated mutants that contain TnphoA inserted at exactly the same position. These numbers were obtained using the 3E8 mutant. Similar results were obtained using the 6A6 mutant. (data not shown).

Isolation of other virulent genes

Based on the nucleotide and amino acid sequences described herein, isolation of additional virulence factor coding sequences was performed. Any pathogenic cell may serve as a source of nucleic acid suitable for cloning such a virulence gene. These sequences were identified as a coding protein showing structures, properties and activities associated with pathogenicity.

In one particular example of such an isolation method, any of the nucleotide sequences described herein may be used in conjunction with a conventional nucleic acid hybridization assay method. Such hybridization methods and assay procedures are well known in the art (Benton and Davis Science 196: 180: 1977; Grunstein and Hogness, Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 1997; Berger and Kimmel, A Guide to Molecular Cloning Techniques, 1987, Academic Press, New York; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. In one particular embodiment, all or part of the sequence 33A9 (described herein) can be used as a probe to test a recombinant plant DNA library for genes that show sequence identity to the 33A9 gene (Figures 5 and 6A-B). Hybridization sequences were detected by plaque hybridization and plaque hybridization according to a suitable standard method.

• ·

Alternatively, using all or part of the 33A9 polypeptide amino acid sequence, it is possible to design 33A9-specific oligonucleotide probes that include degenerate nucleotide probes (that is, a mixture of all possible coding sequences for a given amino acid sequence). These oligonucleotides can be based on the sequence of either the DNA strand and any suitable region of sequence 33A9 (Figures 5 and 6A to B; SEQ ID NOS: 102 and 103) of the 33A9 protein. General methods for designing and preparing such probes are described, for example, in Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1995. 1997 and November 3, 1997; Berger and Kimmel, A Guide to Molecular Cloning Techniques, 1987, Academic Press, New York. These oligonucleotides can be used to isolate the 33A9 gene, by using them as probes capable of hybridizing to the complementary 33A9 sequences, or as primers for various amplification methods, such as polymerase chain reaction (PCR) cloning strategies. If necessary, combinations of different detectably labeled oligonucleotide probes can be used to test a recombinant DNA library. Such libraries were prepared according to methods well known in the art, for example as described in Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927.

No. 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 1997, or commercially available.

herein, also sequentially used as primers in an amplification cloning strategy, for example using PCR. PCR methods are well known in the art, such as described in PCR Technology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and a

3.

As discussed above, oligonucleotides may be specific

Applications, Innis et al., Eds., Academic Press, Inc., New.

York, 1990; Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997. The primers were to allow cloning of the amplified product into a suitable vector, for example by including suitable restriction sites at the 5 'and 3' ends of the amplified fragment (as described herein). If necessary, the nucleotide sequences can be isolated using the PCR method of RACE or rapid amplification of the ends of the cDNA (see, for example, Innis et al., Ochman et al., 1993, A Guide to Methods and Applications, eds., Innis. (MA, States, DJ, 1990). By this method, oligonuketide primers based on the desired sequence were oriented 3 'and 5' and are used to generate overlapping PCR fragments. These overlapping 3 ' and 5 ' ends of RACE products combine to form intact full-length cDNA. This method is described in Ochman et al., 1993, A Guide to Methods and Applications, eds., Innis, M.A., States, D.J., 1990, and Frohman et al., Proc. Nati. Acad. Sci. USA 85: 8998, 1988.

Partial virulence sequences such as tag sequences can also be used as hybridization probes to identify full-length sequences as well as to test databases suitable for identifying previously undefined related virulence genes. For example, sequences 36A4, 25A12, and 33C7 have been extended to contigs 2507, 1126 and 1344.

The formation of sequence relatedness to the pathogenic polypeptide can be accomplished 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 assessed according to any method described herein, for example, the functional or immunological properties of its encoded product.

• ·

Once a suitable sequence has been identified, it is cloned according to standard methods and can be used, for example, to test substances that reduce the virulence of a pathogen.

Expression of the polypeptide

Generally, a polypeptide of the invention can be produced by transforming a suitable host cell with all or a portion of a nucleic acid molecule encoding the polypeptide or fragment thereof in a suitable expression vehicle.

It will be understood by those skilled in the art of molecular biology that any of the expression systems may be used to obtain the recombinant protein. Choosing the right cell is not critical to the invention. The polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (such as Saccharomyces cerevisiae, insect cells such as Sf21 cells or mammalian cells such as NIH 3T3, HeLa or COS cells are preferred). are available from a wide variety of sources (e.g., American Type Culture Collection, Rocckland, MD; also described in Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997. The method of transformation or transfection and the choice of expression tool depends on the host system selected, for example, as described in Ausubel et al., Methods of Screening of

Infection or

US Pat. No. 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997, and expression tools are described, for example, in Clonning Vectors: A Laboratory Manual (PH Pouwels). et al., 1985, Supp. 1987).

• v • »♦ ♦ • • • • • • • • •

One particular bacterial expression system suitable for polypeptide production is the E. coli microorganism pET expression system (Novagen, Inc., Madison, WI). According to this expression system, the DNA encoding the polypeptide is introduced into a pET vector with an orientation such that it is expressed. Since the gene encoding such a polypeptide is driven by T7 regulatory signals, expression of the polypeptides is achieved by inducing expression of T7 RNA polymerase in the host cell. This is typically accomplished using host strains that express T7 RNA polymerase in response to IPTG induction. After production, the recombinant polypeptide is isolated according to standard methods well known in the art.

Another bacterial expression system for producing polypeptides is the pGEX expression system (Pharmacia). This system uses the GST gene fusion system, which has been designed for the strong expression of genes or gene fragments as fusion proteins with rapid purification and obtaining functional gene products. The protein is fused to the carboxyl terminus of glutathione-S-transferase protein from Schistosoma japonica and is often purified from bacterial lysates by affinity chromatography using glutathione Sepharose 4B. Fusion proteins can be obtained under mild conditions by eluting glutathione. Cleavage of the glutathione S-transferase region from the fusion protein can be accomplished by the presence of sites that recognize site-specific proteases upstream of this region. For example, proteins expressed in plasmids pGEX-2T can be cleaved by thrombin. Proteins expressed in pGEX-3X can be cleaved by Factor Xa.

After isolation of the recombinant polypeptides of the invention, it is isolated using, for example, affinity chromatography. In one example of the invention, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention can be attached to a column and used to isolate a recombinant polypeptide. Lysis and fractionation of polypeptide-bearing cells prior to affinity chromatography can be accomplished using standard methods (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997).

After isolation, the recombinant protein can, if necessary, be further purified, for example, by high performance liquid chromatography (see, for example, Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

Polypeptides of the invention, particularly short peptide fragments, may also be produced by chemical synthesis (for example, the method described in Solid Phase Peptide Synthesis, 2 ^nd ed., 1984, Pierce Chemical Co., Rockford, IL).

These general methods of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).

Antibodies

To generate antibodies, the coding sequences of the polypeptide of the invention can be expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67: 31-40, 1988). The fusion protein was purified on glutathione Sepharose beads, eluted with glutathione, digested with thrombin (at an established cleavage site), and purified to the degree necessary for immunization of rabbits. Primary immunization was performed with Freund's complete adjuvant and subsequent immunizations were performed with Freond's incomplete adjuvant. Antibody titers were monitored by Western blot analysis and immunoprecipitation using a thrombin cleaved protein fragment of the GST fusion protein. Immune sera are purified based on their affinity using CNBr-Sepharose-associated protein. Specificity w • • φ 4 4 - 4 • 44

Antisera were determined using a panel of unrelated GST proteins.

As another or additional immunogen to the GST fusion proteins corresponding to the relatively unique immunogenic regions of the polypeptides of the invention, the gastric gastric hemocyanin (KHL) can be generated and attached by introducing Cterminal lysine. Antisera to each of these peptides are similarly purified based on affinity for BSA-conjugated peptides, and their specificity is tested in ELISA and western blotting using peptide conjugates and western blotting and immunoprecipitation using a polypeptide expressed as a GST fusion protein.

Alternatively, monoclonal antibodies that specifically bind to any of the polypeptides of the invention are prepared according to standard hybridoma technology (Kohler et al., Nature 256: 495, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; Ausubel et al., Methods of Screening U.S. Pat. No. 08 / 411,560, 08 / 852,927, and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997). The monoclonal antibodies produced were also tested for specific recognition by Western blotting or immunoprecipitation analysis (methods described in Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and 08 / 962,750) March 25, 1995, May 7, 1997, and November 3, 1997). Antibodies that specifically recognize a polypeptide of the invention are considered useful in the invention. Such antibodies can be used, for example, in an immunoassay. Alternatively, monoclonal antibodies can be prepared using the polypeptide of the invention described above from a phage display library (Vaughan et al., Nature Biotech 14: 309-314, 1996). Antibodies of the invention are produced using fragments of the polypeptide of the invention that lie outside the framework regions and are likely to be antigenic according to criteria such as high frequency of charged residues. In one specific example, such fragments were generated by standard PCR methods and cloned into the pGEX expression vector (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and

No. 08 / 962,750, filed March 25, 1995, May 7, 1997;

November 1997). The fusion proteins are expressed in an E. coli microorganism and purified using a glutathione agarose affinity matrix as described by Ausubel et al., Methods of Screening Compounds for the prevention of pathogenicity, USSN 08 / 411,560, 08 / 852,927 and No. 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997.

In order to minimize the potential problems of low affinity and specificity of antisera, two or three such fusions were generated for each protein and each fusion was injected into at least two rabbits. Antisera was produced by serial injections, with at least three doses being preferred.

Antibodies against any of the peptides described herein can be used to treat bacterial infections.

a number of P. aeruginosa have been identified which can therefore be used

Orientation tests

As mentioned above, virulent factors of the microorganism are involved in pathogenicity and which are used to test substances that reduce virulence of the organism as well as other microbial pathogens. For example, the invention provides methods for testing agents to identify those that potentiate (agonist) or block (antagonist) the action of a polypeptide.

• ·

or expressing a gene of a nucleic acid sequence of the invention. The test method may include high throughput methods. Any number of methods that include such screening tests are available. In one method, a candidate substance at various concentrations is added to the culture medium of pathogenic cells that express one of the nucleic acid sequences of the invention. Gene expression is then measured, for example, by standard northen transfer analysis (Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927 and

No. 08 / 962,750 (filed March 25, 1995, May 7, 1997, and November 3, 1997) using a hybridization probe which is any suitable fragment prepared from a nucleic acid molecule. The level of gene expression in the presence of a candidate substance is compared to the amount measured in the control culture medium lacking said molecule. A substance that promotes downregulation of pathogenic factor expression is believed to be useful in the present invention. Such a molecule can be used, for example, as a therapeutic agent for treating the pathogenicity of an infectious organism.

Alternatively, if necessary, the effect of the agents can be measured as the power of polypeptide production using the same general approach and standard immunological methods such as Western blotting or immunoprecipitation with pathogenic factor specific antibodies. For example, immunoassays may be used to detect the expression of at least one of the polypeptides of the invention in a pathogenic microorganism. Polyclonal and monoclonal antibodies (produced as described above) that are capable of binding to such a polypeptide can be used in any standard immunoassay format (e.g., ELISA, western blot or RIA assay) to measure the potency of the pathogenicity of the polypeptide. A substance that induces a decrease in the expression of a pathogenic polypeptide is considered to be partially **. . :: :::

• <<

.: ·. ·· «· ·· ·· ·· available. Such a molecule can again be used, for example, as a therapeutic in combating the pathogenicity of an infectious organism.

Alternatively or additionally, agents that specifically bind to and inhibit a pathogenic polypeptide of the invention may be sought. The efficacy of such a substance depends on its ability to interact with the pathogenic polypeptide. Such an interaction can often be tested using any of a variety of standard binding methods and functional assays (for example, those described in Ausubel et al., Methods of Screening Compounds for the prevention of infection or pathogenicity, USSN 08 / 411,560, 08). Nos. 852,927 and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997). For example, a substance can be tested using standard in vitro assays for interaction and binding of a polypeptide of the invention and its ability to modulate pathogenicity.

In one particular example of the invention, a substance that binds to a pathogenic polypeptide can be identified using a chromatography-based method. The recombinant polypeptide of the invention can be purified from the cells by standard methods to express the polypeptide (e.g., the polypeptide described above) and can be immobilized on a column. The solution of the substances then passes through the column and the pathogen-specific polypeptide-specific substance is identified by its ability to bind to the pathogenic polypeptide and immobilize on the column. In order to isolate the substance, the column is washed to remove non-specific bound molecules and the substance is then released from the column and collected. Substances isolated by this method (or other suitable method) may, if necessary, be further purified (for example, by high performance liquid chromatogarchy). In addition, candidate substances may be tested for their ability to make the pathogen less virulent. The substances isolated by this approach can also be used, for example, as therapeutics for treating or preventing a pathogenic infectious disease, or both. Substances which have been determined to bind, for example, a length of 529 by monitoring nm in acidic with the amount measured does not contain said can include, but are not limited to, pathogenic polypeptides with an affinity of less than or equal to 10 mM. especially useful.

The substances are further evaluated for their ability to inhibit the virulence of Pseudomonas cells by monitoring the effect of the substance on phenazine production (e.g. pyocyanine). In one approach, the agent is added to the culture medium of pathogenic cells at various concentrations. Pyocyanine is then measured by any standard method, absorbance of pyocyanine in a wave solution (Essar et al., J. Bacteriol. 172: 884, 1990). In order to maximize pyocyanine production in liquid culture for quantification purposes, cells were cultured in conditioned KA soil (King et. Al., J. Lab. Clin. Med. 44: 301, 1954) by the addition of 100 μCl FeCl ₃ . The strength of pyocyanine production in the presence of the substance is compared in the control culture medium of that molecule. A substance that induces downregulation of pyocyanine expression is considered useful in the present invention. Such a molecule can be used, for example, as a therapeutic agent in combating the pathogenicity of an infectious organism. Similar methods have also been used to test other suitable phenazines that limit pyorubin, aeruginosin A, myxin and tubermycin A. Other phenazines are described in Turner and Messenger (Advances in Microbial Physiology 27: 211-1275,

1986), Sorensen and Joseph, Pseudomonas aeruginosa as an opportunistic pathogen, Campa, M., ed., Plenum Press, NY, 1993), Ingram and Blackwood., Advances in applied microbiology 13: 267, 1970) and Gerber, CRC Handbook of Microbiology, Laskin, AI, and Lachevalier, eds., 2 ^nd edition, vol. 5, Chemical Rubber Co., Cleveland, Ohio, 1984, p. 573-576).

Potential antagonists include organic molecules, peptides, peptide mimics, and antibodies that bind to the nucleic acid sequence or polypeptide of the invention and thereby inhibit or extinguish their activity. Potential • Anatgonists also include small molecules that bind to and occupy the binding site of the polypeptide, thereby preventing binding to the cell binding molecules so as to prevent normal biological activity. Other potential antagonists include nonsense molecules.

Each of the DNA sequences described herein can also be used in the discovery and development of antipathogenic agents (e.g., antibiotics). The encoded protein after expression can be used as a target for testing antibacterial drugs. In addition, DNA sequences encoding the amino-terminal regions of the encoded protein or Shine-Delgarno or other translational mRNA sequences can be used to construct antisense sequences that direct the expression of the coding sequence.

The invention further provides the use of a polypeptide, polynucleotide or inhibitor of the invention to abolish the initial physical interactions between a pathogen in a mammalian host responsible for infection. The molecules of the invention may be used to prevent adhesion and colonization of bacteria to mammalian extracellular matrix proteins, to extracellular matrix proteins in wounds, to block mammalian cell invasion, or to block the normal course of pathogenesis.

Antagonists and agonists can be used, for example, to inhibit and treat various bacterial infections.

The substances identified in any of the described assays can be considered useful in protecting against the development of a pathogenic infection in any standard animal model (e.g., a burn test) and can be used as anti-pathogenic therapeutics (e.g., antibiotics).

Test substances and extracts

Substances capable of reducing pathogenic virulence are identified in large libraries of natural and synthetic products (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Experts in discovery and development

It is clear that the correct source of the extracts to be tested is not critical to the screening tests of the invention. Any number of chemical extracts or substances can be tested using the methods described herein. Examples of such extracts or substances include, but are not limited to, plant, fungal, prokaryotic or animal extracts, fermentation broths and synthetic substances, as well as modifications to existing substances. A variety of methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemicals, including, but not limited to, sugars, lipids, peptides, and nucleic acids. Synthetic fabric libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, natural substance libraries are available in the form of bacterial, fungal, plant and animal extracts from various sources including Biotics (Sussex, UK), Xanova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL). ) and PharmaMar, USA (Cambridge, Mass.). If necessary, further, synthetically and naturally produced libraries are produced according to methods known in the art, for example, by standard extraction and fractionation methods. Further, if necessary, any library or substance is often treated using standard chemical, physical or biochemical methods.

Those skilled in the art of drug discovery and discovery will appreciate that methods of dereplication (e.g., taxonomic dereplication, biological dereplication and chemical dereplication, or any combination thereof) or the elimination of replicates or repetition of materials already known for their autopathogenic activity may be used whenever necessary. .

When the crude extract has been found to have anti-pathogenic or antivirulent activity or binding activity, further fractionation of the positively-led extracts is necessary to isolate the chemical components responsible for the observed effect. Thus, the aim of the extraction, fractionation and purification process is to carefully characterize and identify the chemical in the crude extract that exhibits anti-pathogenic activity. Methods of fractionating and purifying such heterogeneous extracts are well known in the art. If necessary, substances that appear useful as agents for treating pathogenicity are chemically treated according to methods known in the art.

Pharmaceutical Therapeutic and Plant Protection Agents The invention describes simple methods for identifying substances (including peptides, small inhibitor molecules and mimics) capable of inhibiting pathogenicity or virulence of a pathogen). Chemicals that exhibit certain values useful in medicine or agriculture using the methods described herein can be used as medicaments, plant protection agents, or as information for structuring existing antipathogenic agents, for example, in designing drug structures. Such methods are useful in testing substances that have an effect on various pathogens, including bacteria, viruses, fungi, annelids, protozoa. Examples of pathogenic bacteria include, but are not limited to, Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus, Bacteroides, Bartonella, Bortella, Brucella,

Calymmatobacterium, Campylobacter, Citrobecter, Clostridium, Cornyebacterium, Enterobacter, Escherichia,

Haemophilus, Hafnia, Helicobacter, Klebsiella

Listeria, Morganella, Moraxella, Proteus,

Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas, Vibrio and Yersinia.

In the case of therapeutic use, the composition or agent identified using the methods described herein may be administered systemically, for example, in the form of pharmaceutically acceptable buffers, such as saline. The treatment may be carried out directly, for example by treating animals but not limited to nematodes, flatworms and

Francisella, Legionella,

Providence, directly, for example by treatment with an antagonist, also neutralizes the polypeptide. intravenous, which violate, suppress, attenuate or biological features associated with pathogenic

Preferred routes of administration include, for example, intraperitoneal, intramuscular, or intradermal injections that provide a continuous and sustained level of drug to a patient. Treatment of human patients or animals is performed using a therapeutically effective amount of an antipathogenic agent in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences, E.W. Martin. The amount of antipathogenic agent to be administered (for example, an antibiotic) varies depending on the route of administration, the age, body weight of the patient and the type of disease and the extent of the disease. Generally, the amount is in the same range as used with other agents in the treatment of other microbial diseases, although in certain cases a lower amount is needed due to the increased specificity of the agent. The agent is administered at a dose that inhibits microbial proliferation. For example, when administered systemically, an amount of 0.1 ng to 10 g / kg body weight is typically administered.

For agricultural use, the compositions and agents identified using the methods described herein can be applied as a spray applied chemical or dusted with plant leaves. Typically, such agents are applied to the surface of the plant prior to being infected with the pathogen to prevent infection. Seeds, bulbs, roots, tubers and sub-bulbs are also treated to prevent pathogenic attack after planting with control pathogens that exist in the soil of the planting site. Soil that is planted with vegetables, ornamental flowers, shrubs or trees can be treated with chemical fuming pesticides that kill various microbial pathogens. The treatment is preferably performed several days or weeks before discontinuation. The chemicals can be applied either mechanically, for example by tractor or manually. Furthermore, chemicals identified using test methods can be used as disinfectants.

Overview of the drawings

Figure 1 is a schematic diagram showing the physical map of cosmid B148 (SEQ ID NO: 1) and the orientation of the identified open reading frame (ORF).

Figure 2 depicts the nucleotide sequence of cosmid B148 (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), ORF1242c (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);

ORF3965C (SEQ ID NO: 28), ORF3218 (SEQ ID NO: 30),

ORF3568c (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 ( ORF9991 (SEQ ID NO: 82), ORF10765C (SEQ ID NO: 84), ORF10475 (SEQ ID NO: 86),

ORE11095C (SEQ ID NO: 88), ORF11264 (SEQ ID NO: 90), ORF11738 (SEQ ID NO: 92), ORF12348c (SEQ ID NO: 94), ORF12314c (SEQ ID NO: 94);

NO: 96) ORF13156C (SEQ ID NO: 98), ORF12795 (SEQ ID NO NO: 100), ORF13755c (SEQ ID NO: 210), ORF13795C (SEQ ID NO: 212), ORF14727C (SEQ ID NO: 214), ORF13779 (SEQ ID NO: 216), ORF14293C (SEQ ID NO: 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) , ORF17793

(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), ORF623c (SEQ ID NO: 256), ORF21210c (SEQ ID NO: 258), ORF21493c (SEQ.

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. NO:

282), ORF24222 (SEQ ¹ 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), ORF28118C (SEQ ID NO: 322), ORF28129c (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), ORF34385) (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), ORF40706C (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), ORF41589 (SEQ ID NO: 434) ORF42172c (SEQ: ID NO: 436) a ORF42233C (SEQ ID NO : 151 ) · Picture no. 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. 11), ORF594 (SEQ ID NO : 13), QRF1040 (SEQ ID NO: 15) ORF1640c (SEQ ID NO: 17), ORF2228c (SEQ ID NO: 19), ORF2068c (SEQ ID NO : 21), ORF1997 (SEQ ID NO NO: 23) , ORF2558C (SEQ ID NO: 25 ), ORF2929C (SEQ ID NO : 27), ORF3965C (SEQ NO ID: 29) , ORF3218 (SEQ ID NO: 31), ORF3568c (SEQ ID NO: 33), ORF4506C (SEQ ID NO : 35), ORF3973 (SEQ ID NO NO: 37), ORF4271 (SEQ ID NO 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 NO ID: 53) , ORF6325 (SEQ ID NO: 55), ORF7 567 c (SEQ ID NO: 57), ORF7180 (SEQ ID NO : 59), ORF7501 (SEQ ID NO i 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 ( ORF9991 (SEQ ID NO: 83), ORF10765C (SEQ ID NO: 85), ORF10475 (SEQ ID NO: 87),

ORF11095C (SEQ ID NO: 89 ORF11264 (SEQ : D NO: 91), ORF11738 (SEQ ID NO: 92) , ORF12348c (SEQ NO ID: 95) , ORF12314C (SEQ ID NO: 97) ORF13156c (SEQ ID NO: 99), ORF12795 (SEQ i NO : 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 NO: 221), ORF14360 (SEQ ID NO: 223), ORF15342c (SEQ ID NO: 225) ORF15260C (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 and 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), ORF623c (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 NO: 271), ORF23228 (SEQ ID NO : 273), ORF23367 (SEQ ID NO NO: 275) ORF25103c (SEQ ID NO: 277) i, 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 1 (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 NO: 317), ORF28033C (SEQ ID NO: 319), ORF29701C (SEQ ID NO: 321),

• ·

ORF28118C (SEQ ID NO: 323), • · · · · · • · ID • · NO: • · · · 325), ORF28129c (SEQ 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), ORF34385) (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), ORF36739 (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 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: 431), ORF41284 (SEQ ID NO: 433), ORF41598 (SEQ ID ORF42172c (SEQ ID NO: 435) 437) a ORF42233C In the picture (SEQ ID NO: 152). No. 5 shows the nucleotide sequence (SEQ ID NO:

102) a coding protein encoded by sequence 33A9.

Figure 6A shows the deduced amino acid sequence (SEQ ID NO: 103) of the protein encoded by sequence 33A9.

• • •

Figure 6B shows the nucleotide sequences of several ORFs 1 to 10 (SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, and 198) identified in sequence 33A9 and their amino acid sequences (ORF 1) to 10, SEQ ID NOs: 199, 200,

201, 202, 203, 204, 205, 206, 207, and 208).

Figure 7 shows a physical map of the 34B12 EcoRI fragment, the map indicating the positions of the three ORFs: ORF1 (L-S), ORF2, and ORF1S. It also shows a nucleotide sequence corresponding to the incorporation of pho34B12 (SEQ ID NO: 104) containing ORF1 (LS) (SEQ ID NOs: 105 and 107), oRF2 (SEQ ID NOs: 106 and 108) and ORF1-S (SEQ ID NO: 159 and 209).

Figure 8 shows the deduced amino acid sequence of ORF1 (L-S) (SEQ ID NO: 107), which is shown in Figure 7.

Figure 9 shows the deduced amino acid sequence of ORF2 (SEQ ID NO: 108) shown in Figure 7.

Figure 10 shows the nucleotide sequence (SEQ ID NO: 109) corresponding to the incorporation of 36A4.

Figure 11 shows the deduced amino acid sequence of the peptide (SEQ ID NO: 110) encoded by sequence 36A4. The predicted peptide encoded by sequence 36A4 shows homology to the hrpM gene of Pseudomonas syringae (Loubens, et al., Mol. Microbiol. 10: 329-340, 1993).

Figure 12 shows the nucleotide sequence (SEQ ID NO: 111) of contig 2507 identified using nucleotide sequence 36A4.

Figure 13 shows the nucleotide sequence (SEQ ID NO: 112) corresponding to the incorporation of 23A2.

Figure 14A shows the deduced amino acid sequence of the peptide (SEQ ID NO: 113) encoded by sequence 23A2. The peptide predicted based on the sequence 23A2 is homologous to the known protein of 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), included in GenBank: L11616.

Figure 14B shows the nucleotide sequence (SEQ ID NO: 148) and the predicted partial amino acid sequence of PA14 mexA and mexB (SEQ ID NOs: 149 and 150).

Figure 15 shows the nucleotide sequence (SEQ ID NO: 114) of the PAO1 phenazine operon identified by the tag 3E8 sequence.

Figure 16A shows the nucleotide sequence (SEQ ID NO: 115) of the tag 3E8 sequence.

Figure 16B shows the nucleotide sequences flanking the tag 3E8 sequence (SEQ ID NO: 160).

Figure 17 shows the amino acid sequence of 3E8 PHZA (SE ID NO: 116).

Figure 18A shows the deduced amino acid sequence of 3E8 PHZB (SEQ ID NO: 117).

Figure 18B shows the deduced partial amino acid sequence of 3E8 PHZA (SEQ ID NO: 161).

Figure 18C shows the deduced partial amino acid sequence of 3E8 PHZB (SEQ ID NO: 162).

Figure 18D shows the deduced partial amino acid sequence of 3E8 PHZC (SEQ ID NO: 163).

Figure 18E shows the nucleotide sequence (SEQ ID NO: 164) and predicted partial amino acid sequence (SEQ ID NO: 165) of PA14phzP.

Figure 19 shows the nucleotide sequence (SEQ ID NO: 118) of the tag 34H4 sequence.

Figure 20 shows the nucleotide sequence (SEQ ID NO: 119) of the tag 33C7 sequence.

Figure 21 shows the nucleotide sequence (SEQ ID NO. 120) of the tag sequence of 25a2.3.

Figure 22 shows the nucleotide sequence (SEQ ID NO: 121) of the tag 8C12 sequence.

• · · · · ·

On Picture no. 23 Yippee displayed nucleotide sequence (SEQ ID NO: 122) sequence tag 2A8. On Picture no. 24 Yippee displayed nucleotide sequence (SEQ ID NO: 123 124 125 , 126, 127 and 128) sequences tag 41A5, 50E12,

35A9, pho23, 16G12, and 25FlTnpůoA.

Figure 24B shows the nucleotide sequence (SEQ ID NO: 166) and predicted amino acid sequence (SEQ ID NO: 167) of PA14phol5.

Figure 24C shows the nucleotide sequence (SEQ ID NO: 168) of PA1450E12 encoding YgdP _Pa (SEQ ID NO: 169) and PtsP _Pa (SEQ ID NO: 170).

Figure 24D shows the nucleotide sequence (SEQ ID NO: 171) of PA1435A9 encoding mtrR _Pa (SEQ ID NO: 172).

Figure 24E shows the nucleotide sequence (SEQ ID NO: 173) of PA1425F1 encoding ORFT (SEQ ID NO: 174), ORFU (SEQ ID NO: 175), and Dj IA _Pa (SEQ ID NO: 176).

Figure 25 shows the nucleotide sequence (SEQ ID NO: 12 9) of the phnn and phnB genes of Pseudomonas aeruginosa PAO1 and PA14.

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 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 Pseudomonas aeroginosa strain 8830.

Figure 30 shows the nucleotide sequence (SEQ ID NO: 134) of the algD gene of Pseudomonas aeroginosa strain 8830.

Figure 31 shows the nucleotide sequence (SEQ ID NO

NO: 135) contig 1126 identified using 25A12.

·· ··

Figure 32 shows a physical map of contig 1344 (SEQ ID NO: 136) identified using 33C7, which illustrates three established ORFs: ORFA (SEQ 154) and ORFC (SEQ ID NO)

ORFB (SEQ ID NO: 153). Also shown are the amino acid sequences ORFA (SEQ ID NO: 156), ORFB (SEQ ID NO: 157) and ORFC (SEQ ID NO: 158) encoded by their ORF.

Figure 33 shows the nucleotide sequence (SEQ ID NO: 137) of the 1G2 tag sequence.

Figures 34A-D are graphs showing complementation of the phenotype of worm pathogenic TnphoA mutants using the C. elegans slow kill assay.

Figure 34A is a graph showing that the non-pathogenic phenotype of mutant 12A1 (empty rhomboids) can be completely complemented at wild-type (full square) PA14 level with the lasR gene from PA01 under the control of the constitutive lacZ in trans promoter in strain 12A1, pKDT17) rings). The reconstructed lasR mutant PA14 lasR-G (open squares) is not pathogenic as 12A1 (open diamonds). The results of the experiment using adult one day old are shown.

Figure 34B is a graph showing the complementation of the pho 15 phenotype with prolonged killing. The phol5 (pECdsbA) (open diamonds) and phol5 (pPAdsbA) strains carry the dsbA gene from E. coli and P. aeruginosa in trans position under the control of the constitutive lacZ promoter.

Figure 34C is a graph where the 25F1 phenotype was only partially restored by the 25F1 (pORF338) and 25F1 (p3-ORF) strains carrying plasmids containing orf338 and orf33α-orf224-djlA _Pa .

Figure 34D is a graph showing complementation of the 50E12 operon by the orf159-ptsP _Pa operon. Strain 50el2 (pUCP18) similar to the 12A1 mutant, does not kill the worms even after 63 hours. Both strains 50E12 (pMT205-lac) and 50E12 (pMT206-nat) expressing the putative operon orf159-ptsP _Pa were able to kill C.elegans.

50E12 (pMT205-lac) the transcription of orf159-ptsP _{Pa is} regulated by the constitutive lacZ promoter 99 fc I whereas the 50E12 (pMT206nat) operon is driven by its natural promoter.

Each point represents the mean value + standard deviation of 3-4 replicants. Unless otherwise stated in the experiments, synchronized L4 worms were used. For each complement analysis, at least two experiments were performed.

Figure 35 is a schematic diagram of an anthalinate synthase complex encoded by phnA and phnB genes that catalyze the conversion of chorism to anthranilate. The anthranilate serves as a precursor of pyocyanine production in P. aeruginosa, strain PA01 (Essar et al., J, Bacteriol. 172: 884-900, 1990). The double arrows indicate the inclusion of multiple undefined steps leading to the conversion of anthranilate to pyocyanine.

Figure 35B is a diagram showing the generation of the AphnAphnB mutant by a 1602 bp deletion within the phnA and phnB genes.

Figure 35C is a graph showing the effect of the phnAphnB mutant on rapid killing of C. elegans. Rapid killing assays were performed using wild type PA14 strain, TnphoA mutant 3E8, or AphnAphnB strain. Worm mortality was recorded 3 hours after initial bacterial exposure and the defect in rapid killing in the AphnAphnB strain was comparable to that of another phenazine mutant 3E8.

DETAILED DESCRIPTION OF THE INVENTION

Example 1:

It describes any nucleic acid sequence that can be isolated as described herein or is often isolated based on homology or PCR amplification assays using the nucleic acid sequences of the invention. The invention further provides polypeptides that are modified by methods that eliminate their pathogenic activity (as tested, for example, as described herein). Such changes may include certain mutations, deletions, insertions, or post-translational modifications, or may include the inclusion of any of the polypeptides of the invention as one component of a larger fusion protein. The invention further provides polypeptides that have lost their pathogenicity.

Example 2:

Describes any protein that is substantially identical to a polypeptide of the invention. Such homologues include other substantially naturally occurring polypeptides, as well as allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to any of the nucleic acid sequences under very stringent conditions or less are preferred under conditions of low stringency (e.g., washes) 2x SSC at 40 ° C (probe length of at least 40 nucleotides) and proteins that specifically bind to the antiserum of the invention.

Example 3:

It includes analogs of any naturally occurring polypeptide of the invention. Analogs differ from naturally according to the invention with differences in post-translational modification.

occurring polypeptides in the amino acid sequence,

The analogs of the invention will generally exhibit at least 85%, more preferably 90% and most preferably 95% or even 99% identity to all or part of the naturally occurring sequence of the invention. The length of the sequence being compared is at least 15 amino acid residues, preferably at least 25 amino acid residues and more preferably more than 35 amino acid residues. To determine the degree of identity, a BLAST program with an early probability value of e ^-3 and e ¹⁰⁰ may be used, indicating a closely related sequence. Modifications include chemical derivatization of polypeptides in vivo and in vitro, for example, acetylation, carboxylation, phosphorylation, or glycosylation. Such treatments may occur during polypeptide synthesis or during processing or after treatment with the isolated modification enzymes. Analogs may also differ from the naturally occurring polypeptides of the invention by changes in the primary sequence. These include genetic variations, both natural and induced (for example, those resulting from accidental mutagenesis by irradiation or exposure to ethammethylsulfate or site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Clonning: A Laboratory Manual (2d ed), CSH Press , 1989 or Ausubel et al., Methods of Screening Compounds, USSN 08 / 411,560, 08 / 852,927, and 08 / 962,750, filed March 25, 1995, May 7, 1997, and November 3, 1997. Included are: also cyclized peptides, molecules and analogs that contain residues other than L-amino acids such as Damino acids or non-naturally occurring or synthetic amino acids such as β or γ amino acids.

Example 4:

In addition to full-length polypeptides, fragments of any of the polypeptides of the invention are further described. The term "fragment" means at least five, preferably at least 20 continuous amino acids, preferably at least 30 amino acids, more preferably at least 60 to 80 or more continuous amino acids. Fragments of the invention may be generated by methods known in the art and may result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing).

Example 5:

Further disclosed are nucleotide sequences that allow specific detection of any nucleotide sequence of the invention. For example, the nucleic acid sequences or fragments thereof described herein can be used as probes to hybridize nucleotide sequences using standard hybridization methods under appropriate conditions. Sequences which hybridize to the coding sequence or its complement are considered useful according to the invention. Sequences that hybridize to a coding sequence of a nucleic acid of the invention or a complement thereof and that encode a polypeptide of the invention are also considered useful in the invention. The term "fragment" means a nucleic acid sequence, means at least 5 continuous nucleotides, preferably at least 10 continuous nucleotides, more preferably at least 20 to 30 continuous nucleotides, and most preferably at least 40 to 80 or more continuous nucleotides. Nucleic acid sequence fragments can be generated by methods known in the art.

Example 6:

Further disclosed is a method of inducing an immunological response in an individual, particularly a human, comprising inoculating the individual with, for example, any polypeptide (or fragment or analog or fusion protein thereof) of the invention to produce antibodies and / or T cell immune responses. infections (for example Pseudomonas aeruginosa). The invention further provides a method of inducing an immunological response in an individual comprising introducing a nucleic acid vector, thereby directing the expression of the polypeptides (or fragment or fusion protein thereof) described herein to induce an immunological response.

Example 7:

Further disclosed is a vaccine composition comprising the polypeptides or nucleic acid sequences of the invention. For example, a polypeptide of the invention can be used as an antigen in a host vaccine to produce specific antibodies that prevent bacterial invasion. For example, they block the production of phenazines. The invention further provides a vaccine formulation comprising an immunogenic recombinant polypeptide of the invention together with a suitable carrier.

Example 8:

Further disclosed are compositions (e.g., probes containing a nucleotide sequence), polypeptides, antibodies, and methods for diagnosing pathogenic conditions.

Claims (1)

PATENT CLAIMS An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO: 252. The isolated nucleic acid molecule of claim 1 comprising the sequence shown in SEQ ID NO: 252. An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO: 105. The isolated nucleic acid molecule of claim 3, comprising the sequence shown in SEQ ID NO: 105. An isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NO: 106. The isolated nucleic acid molecule of claim 5, comprising the sequence shown in SEQ ID NO: 106. A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 253. The substantially pure polypeptide of claim 7, wherein said amino acid sequence comprises the sequence set forth in SEQ ID NO: 253. A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 107. The substantially pure polypeptide of claim 9, wherein said amino acid sequence comprises the sequence set forth in SEQ ID NO: 107. A substantially pure polypeptide comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 108. The substantially pure polypeptide of claim 11, wherein said amino acid sequence comprises the sequence set forth in SEQ ID NO: 108. 13. A method of determining a substance capable of attenuating the expression of a pathogenic virulence factor, comprising: (a) obtaining a pathogenic cell expressing the nucleic acid molecule of claim 1; and (b) contacting said pathogenic cell with a candidate agent, reducing expression of said nucleic acid molecule following contact with said candidate agent to reduce expression of a pathogenic virulent factor. 14. Way according to claim 13, c i s E by that it is pathogenic: the cell infects a mammal. 15 Dec Way according to claim 13, characterized by i s E by said pathogenic the cell infects the plant. 16. Way identification Substances which is capable weaken expresi pathogenic virulent factor, v y z n and č u j i c i se til τι, that includes: (a) (b) obtaining a pathogenic cell expressing the nucleic acid molecule according to claim 3 and contacting said pathogenic cell with a candidate agent, attenuating the expression of said nucleic acid molecule following contact with a guided candidate agent determining the agent that attenuates the expression of a pathogenic virulent factor. The method of claim 16, wherein said pathogenic cell infects a mammal, according to claim 16, wherein said pathogenic cell infects a plant. 19. A method for identifying a substance capable of attenuating the expression of a pathogenic virulence factor, comprising (a) obtaining a pathogenic cell expressing the nucleic acid molecule of claim 5; 20 May 21. 22nd (a) (b) 23. (a) (b) 24. (a) contacting said pathogenic cell with a candidate agent, attenuating the expression of said nucleic acid molecule following contact with a controlled candidate agent that attenuates the expression of a pathogenic virulence factor. indicating the candidate's contact The method of claim 19, wherein said pathogenic cell infects a mammal. The method of claim 19, wherein said pathogenic cell infects the plant. A method for identifying a substance that binds a polypeptide, comprising: contacting the candidate agent with a substantially pure polypeptide comprising the amino acid sequence of claim 7 under conditions that allow binding and detection of binding of the candidate agent to the polypeptide. A method for identifying a polypeptide binding agent comprising the step of: to a substance with a substantially pure polypeptide comprising the amino acid sequence of claim 9 under conditions that allow binding, detecting binding of the candidate to the substance to the polypeptide. A method of identifying a substance that binds to a polypeptide by the substance of a polypeptide comprising the amino acid sequence of claim 11 under conditions that allow binding and (b) detecting the binding of the candidate substance to the polypeptide. 25. A method of treating a pathogenic infection in a mammal comprising (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 the polypeptides encoded by the nucleic acid molecule of claim 1 in said pathogen. characterized in that the candidate contact comprises: with substantially pure &gt; 26. (a) (b) 27 Mar: 28. (a) (b) 29. 30. (a) (b) 31. 32. (a) (b) the infection already includes: mammals, A method of treating pathogenic comprising identifying a mammal having a pathogenic infection and administering to said mammal a therapeutically effective amount of a composition that inhibits the expression or activity of the polypeptide encoded by the nucleic acid molecule of claim 3 in said pathogen. The method of claim 26, wherein said pathogen is Pseudomonas aeruginosa. 15. A method of treating a pathogenic infection comprising identifying a mammal having a pathogenic infection and administering to said mammal a therapeutically effective amount of a composition that inhibits the expression or activity of the polypeptide encoded by the nucleic acid molecule of claim 5 in said pathogen. The method of claim 28, wherein said pathogen is Pseudomonas aeruginosa. by A method of mammals, treating a pathogenic infection, comprising: identifying a mammal having a pathogenic infection and administering to said mammal a therapeutically effective amount of a composition that binds and inhibits the polypeptide encoded by the amino acid sequence of claim 5. The method of claim 30, wherein said pathogen is Pseudomonas aeruginosa. A method of treating a pathogenic infection in a mammal, comprising: determining a mammal having a pathogenic infection and administering to said mammal a therapeutically effective amount of a composition that binds and inhibits the polypeptide encoded by the amino acid sequence of claim 7. * ·····. ····· 94 · «· · ... · .... ·. ···· · ······ · ·· · · 9 9999 9999 99 99 99 33. The method of claim 32, wherein said pathogenic infection is caused by Pseudomonas aeruginosa. 34. A method of treating a pathogenic infection in a mammal comprising: (a) determining a mammal having a pathogenic infection; and (b) administering to said mammal a therapeutically effective amount of a composition that binds and inhibits the polypeptide encoded by the amino acid sequence of claim 9. 35. Method according to claim 34, confesses learning with that aeruginosa. pathogenic infection is caused by Pseudomonas 36. Method treatment pathogenic infection u mammals, characterized c í se by that includes (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 substantially pure polypeptide comprising the amino acid sequence of claim 11. 37. The method of claim 36, wherein said pathogenic infection is caused by Pseudomonas aeruginosa. 38. A method of identifying a substance that inhibits virulence of a cell Pseudomonas characterized in that: (and) includes: acquisition Pseudomonas cells (b) contact said cells with a candidate for substance a (C) detection presence of phenazines, where reduction of that phenazines relative to an untreated control cell is an indication that the agent inhibits virulence of said cell Pseudomonas. 39. The method of claim 38, wherein said cell is Pseudomonas aeruginosa. »» • · »· • • » • • r · 95 • · * * 9 • »· 4 10 · · * ·· 4 9 • · • · 40. Method according to claim 38, confesses č u j i c and with E by wherein said phenazine is detected spectroscopy. 41. Method according to claim 38, confesses č u j i c and with E by wherein said phenazine is pyocyanine • 42. Method according to claim 41, confesses č u j i c and with E by that is stated pyocyanin se detected measurement absorbance at 520 nm. 43. The method of claim 38, wherein said cell is present in said culture.