Atty. Docket 8883-0015 CLASS I LASSO PEPTIDES AS INHIBITORS OF THE BACTERIAL TYPE III SECRETION SYSTEM This application claims the benefit of U.S. Provisional Application No.63/627,764, filed ^^ January 31, 2024. This invention was made with Government support under Grant No. R01AI141511, awarded by the National Institutes of Health. The Government has certain rights in the invention. An XML file for a Sequence Listing XML is submitted herewith. ^^^ FIELD OF THE EMBODIMENTS This specification pertains to the field of using class I lasso peptides as inhibitors of the type III secretion system (T3SS) in bacteria. SUMMARY ^^^ Some embodiments of the invention include the following: [1] a lasso peptide for use in the prevention and/or treatment of a disease and/or infection caused by a Gram-negative bacterium, wherein the lasso peptide is selected from the group consisting of a lasso peptide of sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO. ^^^ 1] with 1 or 2 amino acid residues deleted or replaced by another amino acid residue, a lasso peptide of sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO. 1] with the 4th amino acid residue replaced by isoleucine (I) or leucine (L) and/or with the 17th amino acid residue replaced by valine (V) or leucine (L), siamycin I, ^^^ a lasso peptide of sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO. 2] with 1 or 2 amino acid residues deleted or replaced by another amino acid residue, a lasso peptide of sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO. 2] with the 4th amino acid residue replaced by leucine (L) or valine (V), and/or with the 17th amino acid residue replaced by isoleucine (I) or leucine (L), ^^^ aborycin, and combinations of these. [2] The lasso peptide of [1], wherein the lasso peptide is siamycin I and/or aborycin. [3] The lasso peptide of [1], wherein the lasso peptide is siamycin I. ^
Atty. Docket 8883-0015 [4] The lasso peptide of [1], wherein the lasso peptide is aborycin. [5] The lasso peptide of any one of [1] through [4], wherein the Gram-negative ^^ bacterium is of the Pseudomonadota phylum or the Gammaproteobacteria class. [6] The lasso peptide of any one of [1] through [4], wherein the Gram-negative bacterium is of the Pseudomonadales order, the Pseudomonadaceae family, or the Pseudomonas genus. ^^^ [7] The lasso peptide of any one of [1] through [4], wherein the Gram-negative bacterium is Pseudomonas aeruginosa. [8] The lasso peptide of any one of [1] through [4], wherein the Gram-negative ^^^ bacterium is of the Enterobacterales order, the Enterobacteriaceae family, or the Salmonella genus. [9] The lasso peptide of any one of [1] through [4], wherein the Gram-negative bacterium is Salmonella enterica. ^^^ [10] The lasso peptide of any one of [1] through [4], wherein the Gram-negative bacterium is Salmonella enterica serovar Typhimurium. [11] The lasso peptide of any one of [1] through [4], wherein the Gram-negative ^^^ bacterium is Salmonella enterica serovar Typhi. [12] The lasso peptide of any one of [1] through [11], wherein the Gram-negative bacterium comprises a type III secretion system (T3SS). ^^^ [13] The lasso peptide of [12], wherein the type III secretion system (T3SS) is within the Ysc T3SS family or within the Inv-Mxi-Spa T3SS family. [14] The lasso peptide of [12], wherein the type III secretion system (T3SS) is SPI-1 T3SS or Psc T3SS. ^
Atty. Docket 8883-0015 [15] The lasso peptide of any one of [1] through [14], wherein the lasso peptide inhibits a type III secretion system (T3SS) of the Gram-negative bacterium. ^^ [16] A method for preventing and/or treating a disease and/or an infection caused by a Gram-negative bacterium in an organism comprising: administering a lasso peptide to the organism^^^^^ preventing and/or treating the disease and/or the infection caused by the Gram-negative bacterium in the organism, ^^^ wherein the lasso peptide is selected from the group consisting of a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with 1, 2, 3, or 4 amino acid residues deleted or replaced by another amino acid residue, a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with the 4th amino acid residue replaced by isoleucine (I) or leucine (L) and/or with the ^^^ 17th amino acid residue replaced by valine (V) or leucine (L), siamycin I, a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with 1, 2, 3, or 4 amino acid residues deleted or replaced by another amino acid residue, a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with ^^^ the 4th amino acid residue replaced by leucine (L) or valine (V) and/or with the 17th amino acid residue replaced by isoleucine (I) or leucine (L), aborycin, and combinations of these. [17] The method of [16], wherein the lasso peptide is siamycin I and/or aborycin. ^^^ [18] The method of [16], wherein the lasso peptide is siamycin I. [19] The method of any one of [16] through [18], wherein the organism is an animal, a mammal, or a non-human animal. ^^^ [20] The method of any one of [16] through [18], wherein the organism is a human. [21] The method of any one of [16] through [20], wherein a pharmaceutical composition comprises the lasso peptide and a pharmaceutically acceptable carrier or diluent. ^
Atty. Docket 8883-0015 [22] The method of any one of [16] through [21], wherein the method is a prophylactic method comprising administering the lasso peptide to the organism prior to onset of the disease. ^^ [23] The method of any one of [16] through [21], wherein the method is a responsive method comprising administering the lasso peptide to the organism at the time of or after onset of the disease. ^^^ [24] A pharmaceutical composition for the prevention or treatment of a disease and/or infection caused by a Gram-negative bacterium, comprising a lasso peptide, wherein the lasso peptide is siamycin I and/or aborycin. [25] The pharmaceutical composition of [24], wherein the lasso peptide is siamycin I. ^^^ [26] Use of a lasso peptide in the manufacture of a medicament for the prevention or treatment of a disease and/or infection caused by a Gram-negative bacterium, wherein the lasso peptide is siamycin I and/or aborycin. ^^^ [27] The pharmaceutical composition of [26], wherein the lasso peptide is siamycin I. [28] A method of inhibiting the growth of a Gram-negative bacterium, comprising applying a lasso peptide to the Gram-negative bacterium^^^^^ inhibiting the growth of the Gram-negative bacterium, ^^^ wherein the lasso peptide is selected from the group consisting of a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with 1, 2, or 3 amino acid residues deleted or replaced by another amino acid residue, a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with the 4th amino acid residue replaced by isoleucine (I) or leucine (L) and/or with the ^^^ 17th amino acid residue replaced by valine (V) or leucine (L), siamycin I, a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with 1, 2, or 3 amino acid residues deleted or replaced by another amino acid residue, ^
Atty. Docket 8883-0015 a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with the 4th amino acid residue replaced by leucine (L) or valine (V) and/or with the 17th amino acid residue replaced by isoleucine (I) or leucine (L), aborycin, and combinations of these. ^^ [29] The method of [28], wherein the lasso peptide is siamycin I and/or aborycin. [30] The method of [28], wherein the lasso peptide is siamycin I. ^^^ [31] The method of [28], wherein the lasso peptide is aborycin I. [32] The method of any one of [28] through [31], wherein the Gram-negative bacterium is of the Pseudomonadota phylum or the Gammaproteobacteria class. ^^^ [33] The method of any one of [28] through [31], wherein the Gram-negative bacterium is of the Pseudomonadales order, of the Pseudomonadaceae family, of the Pseudomonas genus, or Pseudomonas aeruginosa. [34] The method of any one of [28] through [31], wherein the Gram-negative bacterium is ^^^ of the Enterobacterales order, of the Enterobacteriaceae family, of the Salmonella genus, Salmonella enterica, Salmonella enterica serovar Typhimurium, or Salmonella enterica serovar Typhi. [35] The method of any one of [28] through [31], wherein the Gram-negative bacterium is ^^^ of the Yersiniaceae family, of the Yersinia genus, Yersinia pestis, Yersinia enterocolitica, of the order Vibrionales, of the family Vibrionaceae, of the genus Vibrio, Vibrio parahaemolyticus, of the class Betaproteobacteria, of the order Burkholderiales, of the family Alcaligenaceae, of the genus Bordetella, Bordetella pertussis, of the family Burkholderiaceae, of the genus Burkholderia, Burkholderia ^^^ pseudomallei, of the genus Escherichia, Escherichia coli, of the genus Shigella, or Shigella flexneri. [36] The method of any one of [28] through [31], wherein the Gram-negative bacterium is of the Chlamydiota phylum, of the Chlamydiia class, of the Chlamydiales order, of ^
Atty. Docket 8883-0015 the Chlamydiaceae family, of the Chlamydia genus, Chlamydia trachomatis, or Chlamydia pneumonia. [37] The method of any one of [28] through [36], wherein the lasso peptide inhibits a type ^^ III secretion system (T3SS) of the Gram-negative bacterium. [38] The method of any one of [28] through [37], wherein the lasso peptide is applied to the Gram-negative bacterium in vivo. ^^^ [39] The method of any one of [28] through [38], wherein the lasso peptide inhibits the growth of the Gram-negative bacterium in an organism and optionally wherein the organism is a non-human animal or a human. ^^^ [40] A peptide selected from the group consisting of a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with 1, 2, 3, or 4 amino acid residues deleted or replaced by another amino acid residue and a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with ^^^ 1, 2, 3, or 4 amino acid residues deleted or replaced by another amino acid residue. [41] The peptide of [40], wherein the peptide sequence is CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with the 4th amino acid residue replaced by isoleucine (I) or leucine (L) and/or with the 17th amino acid residue ^^^ replaced by valine (V) or leucine (L). [42] The peptide of [40], wherein the peptide sequence is CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO. 2] with the 4th amino acid residue replaced by leucine (L) or valine (V) and/or with the 17th amino acid residue replaced ^^^ by isoleucine (I) or leucine (L). [43] A pharmaceutical composition comprising a therapeutically effective amount of the peptide of any one of [40] through [42] and a pharmaceutically acceptable carrier or diluent. ^
Atty. Docket 8883-0015 [44] The peptide of any one of [40] through [42] for use as a medicament. [45] A method of inhibiting a type III secretion system (T3SS) of a Gram-negative ^^ bacterium, comprising applying a lasso peptide to the Gram-negative bacterium^^^^^ inhibiting the type III secretion system (T3SS) of the Gram-negative bacterium, wherein the lasso peptide is selected from the group consisting of ^^^ a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with 1, 2, or 3 amino acid residues deleted or replaced by another amino acid residue, a peptide sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1] with the 4th amino acid residue replaced by isoleucine (I) or leucine (L) and/or with the 17th amino acid residue replaced by valine (V) or leucine (L), ^^^ siamycin I, a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with 1, 2, or 3 amino acid residues deleted or replaced by another amino acid residue, a peptide sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2] with the 4th amino acid residue replaced by leucine (L) or valine (V) and/or with the 17th ^^^ amino acid residue replaced by isoleucine (I) or leucine (L), aborycin, and combinations of these. [46] The method of [45], wherein the lasso peptide is siamycin I and/or aborycin. ^^^ [47] The method of any one of [45] through [46], wherein the Gram-negative bacterium is of the Pseudomonadota phylum or the Gammaproteobacteria class. [48] The method of any one of [45] through [46], wherein the Gram-negative bacterium is of the Pseudomonadales order, of the Pseudomonadaceae family, of the ^^^ Pseudomonas genus, or Pseudomonas aeruginosa. [49] The method of any one of [45] through [46], wherein the Gram-negative bacterium is of the Enterobacterales order, of the Enterobacteriaceae family, of the Salmonella ^
Atty. Docket 8883-0015 genus, Salmonella enterica, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, or Salmonella enterica serovar Paratyphi. BRIEF DESCRIPTION OF THE DRAWINGS ^^ Figure 1A shows relative light units (RLU) for P. aeruginosa exoT-lux grown under T3SS- inducing conditions in the presence of DMSO (dimethyl sulfoxide) or 20 µg/mL of the SNE013-15 extract, with luminescence monitored after 4 hours as a readout of T3SS activity. The averages and ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Figure 1B shows relative fluorescence units (RFU) for P. aeruginosa PAO1 grown in LB ^^^ (lysogeny broth) media in the presence of DMSO, 20 µg/mL SNE013-15, or 20 µg/mL carbenicilin, with cell viability measured using resazurin. The averages and standard errors of the mean of three independent experiments are shown. Figure 1C shows relative fluorescence units (RFU) for P. aeruginosa Xen41 as grown in LB in the presence of DMSO or 20 µg/mL SNE013-15, with luciferase activity measured. The ^^^ averages and standard errors of the mean of three independent experiments are shown. Figure 2 shows relative light units (RLU) for P. aeruginosa exoU-Hibit grown under T3SS- inducing conditions for 4 hours in the presence of discrete fractions of the Streptomyces albovinaceus fraction SNE013 or in the presence of the known T3SS inhibitor MBX1641, used as a positive control. T3SS activity was assessed by measuring luminescence produced by an ExoU- ^^^ Hibit reporter protein secreted into the culture supernatant. RLU produced by supernatants of P. aeruginosa exoU-Hibit treated with DMSO was set at 100%. The averages of three independent experiments are shown, and the data were fit with nonlinear curves. Figure 3 shows the predicted structure of siamycin I. Figure 4A shows relative light units (RLU) for P. aeruginosa exoU-Hibit grown under ^^^ T3SS-inducing conditions for 4 hours. That is, T3SS activity was assessed by measuring luminescence produced by an ExoU-Hibit reporter protein secreted into the culture supernatant. The effect of different concentrations (in mg/mL) of siamycin I, extract SNE013-20-10+12G, and MBX1641 on secretion of ExoU-Hibit is shown. Relative light units (RLU) produced by supernatants of P. aeruginosa exoU-Hibit treated with DMSO was set at 100%. The known T3SS ^^^ inhibitor MBX1641 was used as a positive control. The data were fit with nonlinear curves. Figure 4B shows relative light units (RLU) for P. aeruginosa exoU-Hibit grown under T3SS-inducing conditions for 4 hours. That is, T3SS activity was assessed by measuring luminescence produced by an ExoU-Hibit reporter protein secreted into the culture supernatant. The average of three independent experiments in the presence of different concentrations (in µM) of ^
Atty. Docket 8883-0015 purified siamycin I is shown^^^^^^IC50 was 7.3 µM. Relative light units (RLU) produced by supernatants of P. aeruginosa exoU-Hibit treated with DMSO was set at 100%. The known T3SS inhibitor MBX1641 was used as a positive control. The data were fit with nonlinear curves. Figure 5A shows relative light units (RLU) for wild type (WT) S. enterica serovar ^^ Typhimurium harboring a NanoLuc-myc® tagged SipA plasmid that was grown in 0.3 M NaCl LB supplemented with 50 µg/mL streptomycin to induce the T3SS in a high protein-binding microplate for three hours at 37°C in the presence of DMSO or SNE013-20-10+12G. The plates were washed with phosphate-buffered saline (PBS), and luminescence was measured as a readout of relative secretion after addition of NanoGlo® Luciferase Assay reagent. The average of three independent ^^^ experiments ± standard error of the mean in the presence of different concentrations (in mg/mL) of SNE013-20-10+12G is shown. Figure 5B shows results for overnight cultures of wild type (WT) or non-motile ^fliC S. Typhimurium were spotted onto motility agar (0.3% agar in LB) containing 0.30 µM siamycin I, 0.128 mg/ml SNE013-20-10+12G, or equivalent volume DMSO. The plates were incubated at 37°C ^^^ for 5 hours and rates of bacterial migration from the point of inoculation were measured. The results were normalized to the DMSO-treated WT sample. The average of three independent experiments ± standard error of the mean is shown. p<0.001, paired Student t-test. Figure 6 shows the survival rate (probability of survival) of Galleria mellonella larvae at various time post injection (PI). Galleria mellonella larvae at the last instar developmental stage ^^^ were injected in the left pro-leg with DMSO carrier control, MBX1641, or the Streptomyces albovinaceus extract SNE013-20-10+12G, and with phosphate buffered saline (PBS) or 8-12 WT (wild type) P. aeruginosa PAO1 (WT) or ^pscF P. aeruginosa PAO1 (^pscF) colony forming units. The ^pscF strain is deficient for T3SS activity and is used as a negative control. Larval melanization (death) was monitored over 24 hours post injection (PI). N=20 larvae per group. **, ^^^ p = 0.0064 Log-rank (Mantel-Cox test). For example, injection with PBS or injection with PBS + SNE013-20-10+12G resulted in a 100% survival rate at 24 hours. Injection with ^pscF + DMSO resulted in a 95% survival rate at 17 hours and a 95% survival rate at 24 hours. Injection with WT + DMSO resulted in a 95% survival rate at 17 hours and a declining survival rate at longer times as shown in this Fig.6. Injection with WT + MBX1641 resulted in a 95% survival rate at 22 hours ^^^ and a 95% survival rate at 24 hours. Injection with WT + SNE013-20-10+12G resulted in a 95% survival rate at 19 hours, an 85% survival rate at 20 hours, and an 85% survival rate at 24 hours. ^
Atty. Docket 8883-0015 DETAILED DESCRIPTION The bacterial type III secretion system (T3SS) enables acute infections caused by pathogens including the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp) pathogen Pseudomonas ^^ aeruginosa (P. aeruginosa). In 2019, >300,000 deaths worldwide were associated with antimicrobial resistant Pseudomonas, and this number is projected to increase if no new antimicrobial drugs are developed. The bacterial type III secretion system (T3SS) is critical for Pseudomonas to cause ventilator- associated pneumonia as well as eye and wound infections. ^^^ The type III secretion system (T3SS) may represent a drug target. The type III secretion system (T3SS) is externally accessible to small molecules. T3SS inhibitors could possibly be used as treatments and/or prophylaxis for these infections. Because the T3SS is largely found in pathogens and not beneficial bacteria, T3SS inhibitors might have an advantage over classic antibiotics that perturb the beneficial human microbiome. ^^^ A high-throughput screening pipeline to identify natural product extracts with validated T3SS inhibitory activity using a novel library derived from marine and terrestrial bacteria, 20% of which come from microbial genera not studied for small molecules, was carried out. This library has been used to discover novel metabolites with activity against lung cancer, insects, and metabolic disorders, but has not been mined for T3SS inhibitory activity and represents an untapped resource. ^^^ As discussed in the following, an extract from Streptomyces albovinaceus was found to contain class I lasso peptides with T3SS inhibitory activity. This bioactive extract protects an invertebrate model organism from P. aeruginosa infection, despite having no published activity against Gram- negative bacteria. That is, a natural products library was screened for extracts that inhibit the Pseudomonas aeruginosa type III secretion system (T3SS). An extract containing class I lasso ^^^ peptides that inhibits T3SS activity in vitro and protects Galleria mellonella larvae from Pseudomonas-induced death was identified. As discussed in the following, purified siamycin I, a class I lasso peptide, inhibited Pseudomonas T3SS activity with an IC50 of 7 ^M. The T3SS is a factor (protein complex) used by many Gram-negative bacterial pathogens to cause acute infection. As many Gram-negative pathogens are becoming more resistant to current ^^^ antibiotics, new antimicrobials are needed. Pseudomonas aeruginosa is resistant to many antibiotics and has been identified by the World Health Organization as a target for new antimicrobials. An embodiment can act as an antimicrobial agent to prevent or treat Pseudomonas infection. ^
Atty. Docket 8883-0015 Although class I lasso peptides have previously been shown to have no activity against Gram-negative bacteria, Class I lasso peptides were not before tested for activity against T3SS activity in Gram-negative pathogens. Pseudomonas is not known to be resistant to class I lasso peptides. As described in the following, it was found that class I lasso peptides can inhibit the ^^ activity of the Pseudomonas T3SS. Class I lasso peptides may be used to prevent or treat infection, for example, Pseudomonas or Salmonella infection. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same ^^^ meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the provided compositions, suitable methods and materials are described below. Each publication, patent application, patent, and other reference mentioned herein is herein incorporated by reference in its entirety as if each such publication, patent application, ^^^ patent, and other reference had been individually incorporated. In case of an inconsistency, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the methods and compositions discussed herein will be apparent from the following written description, drawings, and claims. ^^^ It is understood that wherever embodiments are described herein with the language “comprising”, then otherwise analogous embodiments, described in terms of “containing” “consisting of”, and/or “consisting essentially of” are also provided. However, when used in the claims as transitional phrases, each should be interpreted separately and in the appropriate legal and factual context (e.g., in claims, the transitional phrase “comprising” is considered more of ^^^ an open-ended phrase while “consisting of” is more exclusive and “consisting essentially of” achieves a middle ground). As used herein, the terms "approximately" and "about," as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, ^^^ 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). For example, when used in the context of an amount of a given compound in a composition, "about" may mean +/-10% of the recited value. For instance, a ^
Atty. Docket 8883-0015 composition including a compound having about 40% of a given compound may include 30-50% of the compound. The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include each of the following: both A and B^ A or B^ A (alone)^ and B (alone). Likewise, the term “and/or” ^^ as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following: A, B, ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated ^^^ herein, and each separate value is incorporated into the specification as if it were individually recited herein. Where embodiments of the disclosure are described in terms of a Markush group or another grouping of alternatives, the disclosed composition or method encompasses not only the entire group listed as a whole but also encompasses each member of the group individually and all ^^^ possible subgroups of the main group and also encompasses the main group absent one or more of the group members. The disclosed methods and compositions also envisage the explicit exclusion of one or more of any of the group members in the disclosed compositions or methods. Unless otherwise indicated or indicated by the context, a singular form is to be understood as also encompassing the plural form, and a plural form is to be understood as also encompassing ^^^ the singular form. An embodiment may include one or more described elements and may optionally include one or more additional elements that are not specifically described. An embodiment may be essentially free of or completely free of non-described elements^^that is, non-described elements may optionally be essentially omitted or completely omitted from an embodiment. ^^^ A lasso peptide is an oligopeptide that includes an N-terminal macrocyclic ring through which a linear C-terminal tail can be threaded. Lasso peptides are considered to be rotaxanes. Lasso peptides are a member of the class of amino acid based lasso structures. The lasso peptide macrocyclic ring can, for example, be formed from about 7, 8, or 9 amino acid residues^^^^^^ example, the ring can be formed by an isopeptide bond between the N-terminal amine of the first ^^^ amino acid residue of the peptide and the carboxylate chain of an aspartate or glutamate amino acid residue. For example, the C-terminal tail can be from about 7 to about 15 amino acid residues long. In a class I lasso peptide, the lasso tail can be constrained by a disulfide bond between a tail cysteine residue and a ring cysteine residue. The structure of a lasso peptide can make the lasso peptide resistant to thermally-induced unfolding and/or to degradation by proteases. ^
Atty. Docket 8883-0015 Alanine (A), valine (V), leucine (L), and isoleucine (I) are amino acids having hydrophobic (nonpolar) aliphatic side chains. Alanine (A) has a methyl side chain and a hydropathy index of ^^ ^^!aline (V) has an isopropyl side chain and a ^"^^^^^^^"^^^^^^^^^^#^$^^^eucine (L) has an isobutyl side chain and a ^"^^^^^^^"^^^^^^^^^^%^ ^^^^^^isoleucine (I) has a sec-butyl side chain and a ^^ hydropathy index of 4.5. A Gram-negative bacterium has a cell envelope that includes a peptidoglycan cell wall between and inner cytoplasmic membrane and an outer membrane. The outer membrane can act as a protective barrier against some antibiotics, detergents, and lysozyme. The outer leaflet of the outer membrane includes a lipopolysaccharide of which the lipid A component can trigger a toxic ^^^ reaction and/or septic shock in a host organism when the bacteria are lysed. The type III secretion system (T3SS) is a bacterial secretion system that is found in certain species of Gram-negative bacteria, for example, in the Pseudomonas genus and the Salmonella genus, as well as in the Shigella, Escherichia , Vibrio, Burkholderia, Yersinia, and Chlamydia genera. Many bacteria possessing the T3SS are pathogens of plants, animals, non-human animals, ^^^ and/or humans. The T3SS is a protein complex that includes about 30 different proteins. The T3SS has the form of a hollow needle with a base. That is, the T3SS has an inner membrane ring located in the inner membrane of the bacterium, a connector located in the periplasm of the bacterium, outer membrane rings located in the outer membrane of the bacterium, a hollow needle itself (located extracellularly outside of the bacterium), and a tip (of the hollow needle, located extracellularly ^^^ outside of the bacterium). The inner membrane ring, connector, and outer membrane rings can be considered to form the base of the T3SS, and an inner rod connects the needle to the base. The hollow needle is about 60-80 nm in length, 8 nm in external diameter, and 3 nm in internal diameter (of its hollow region). Proteins (effector proteins) can be passed from the cytoplasm of the bacterium through the hollow needle and into the cytoplasm of a host cell. T3SS genes can be as ^^^ operons. Such operons can be located on the bacterial chromosome or on a plasmid of the bacterium. For example, in Salmonella bacteria most T3SS genes are gathered in a chromosomal region, termed the Salmonella pathogenicity island (SPI). Induction of secretion by the T3SS is triggered by contact of the hollow needle with a host cell. Effector proteins can have several effects on the host cell. For example, an effector protein can promote the host cell to engulf the bacterium ^^^ (uptake of the bacterium by the host cell), so that the bacterium can then replicate and propagate infection. Streptomyces albovinaceus is a species of bacteria of the genus Streptomyces of the family Streptomycetaceae of the order Streptomycetales of the class Actinomycetia of the phylum Actinomycetota of the domain Bacteria. ^
Atty. Docket 8883-0015 Pseudomonas aeruginosa is a species of bacteria of the genus Pseudomonas of the family Pseudomonadaceae of the order Pseudomonadales of the class Gammaproteobacteria of the phylum Pseudomonadota of the domain Bacteria. Bacteria of the phylum Pseudomonadota, including Pseudomonas aeruginosa, are Gram-negative bacteria. Lineages of Pseudomonas aeruginosa ^^ include those genetically characterized by the model strains PAO1, PA14, and PA7. Pseudomonas aeruginosa is an encapsulated aerobic–facultatively anaerobic, rod-shaped bacterium, which is^ citrate, catalase, and oxidase positive and can aggregate into biofilms. A strain of Pseudomonas aeruginosa may be motile or not. Numerous bacteria of the Pseudomonadota phylum, including Pseudomonas aeruginosa, are pathogenic and can cause disease in plants, animals, and/or humans. ^^^ Numerous strains of Pseudomonas aeruginosa are multidrug resistant and are resistant to antibiotics. For example, a strain of Pseudomonas aeruginosa may be resistant to carbapenems, polymyxins, and tigecycline, as well as other antibiotics. Pseudomonas aeruginosa can affect immunocompromised as well as immunocompetent patients^^^^^^^^^^^^^^ Pseudomonas aeruginosa can affect cystic fibrosis, non-cystic fibrosis bronchiectasis, and elderly patients. Pseudomonas ^^^ aeruginosa can act as an opportunistic or as a specialized pathogen. For example, Pseudomonas aeruginosa can infect the airway, urinary tract, gastrointestinal tract, ear, outer ear, eye, bones, skin, burns, and wounds, and causes blood infections. For example, Pseudomonas aeruginosa can cause and/or be associated with diseases and conditions such as pneumonia, community acquired pneumonia, ventilator-associated pneumonia, bronchopneumonia, septic shock, sepsis syndromes, ^^^ blood infections, ecthyma gangrenosum, osteomyelitis, urinary track infections, gastrointestinal infections, such as necrotising enterocolitis, soft tisssue and skin infections, eye infections, “swimmer’s ear”, dermatitis, “hot-tub rash”, hemorrhage, necrosis, infection of a lung, infection of a kidney. Pseudomonas aeruginosa can release toxins such as virulence factor exotoxin A, the exoenzyme, ExoU, and pyoverdine, and the release of the intracellular contents of Pseudomonas ^^^ aeruginosa can induce an immunologic response in the host or patient. For example, another pathogen of the Pseudomonas genus is the plant pathogen Pseudomonas syringae. Salmonella enterica is a species of bacteria of the genus Salmonella of the family Enterobacteriaceae of the order Enterobacterales of the class Gammaproteobacteria of the phylum Pseudomonadota of the domain Bacteria. Subspecies of Salmonella enterica include arizonae ^^^ (IIIa), diarizonae (IIIb), houtenae (IV), salamae (II), indica (VI), and enterica (I). Salmonella enterica includes numerous serovars. Examples of the enterica subspecies of Salmonella enterica, i.e., serovars of Salmonella enterica subsp. enterica, include the serovars Typhi, Enteritidis, Paratyphi, Typhimurium, and Choleraesuis. As other bacteria of the phylum Pseudomonadota, Salmonella enterica is a Gram-negative bacterium. Bacteria of the Salmonella genus are ^
Atty. Docket 8883-0015 facultative anaerobes and are rod-shaped. Some bacteria of the Salmonella genus can invade different cell types, such as epithelial cells, M cells, macrophages, and/or dendritic cells. Numerous bacteria of the Enterobacterales order, including Salmonella enterica, are pathogenic and can cause disease in plants, animals, and/or humans. Some of such disease caused by Salmonella enterica can ^^ be termed salmonellosis. For example, Salmonella enterica subsp. enterica, serovar Typhi can cause typhoid fever, and Salmonella enterica subsp. enterica, serovar Paratyphi can cause ^^^^^"^^^^^^^^!^^^^^"^^^^^^^^!^^^^^^^^^^^^"^^^^^^^^!^^^^^^^&^^^^&^^!^^"^'^^^^^^^^^^^^^^&^^^!^^^ In typhoid fever, the Salmonella enterica bacteria can invade the bloodstream, invade organs, and/or secrete endotoxins. Salmonella enterica can be spread by contaminated food and/or water. ^^^ Infection with Salmonella enterica can result in intestinal inflammation (enteritis) with diarrhea, fever, vomiting, and abdominal cramps. In some cases, infection with Salmonella enterica can result in sepsis, infection of the blood stream, and osteomyelitis. For example, another pathogen of the Salmonella genus is the species^Salmonella bongori. Yersinia pestis, Yersinia enterocolitica, Vibrio parahaemolyticus, Bordetella pertussis, ^^^ Burkholderia pseudomallei, Escherichia coli, and Shigella flexneri are Gram-negative bacteria that each have a type III secretion system (T3SS) that is in the Ysc family or in the Inv-Mxi-Spa family. Chlamydia trachomatis and Chlamydia pneumonia are Gram-negative bacteria that each have a type III secretion system (T3SS). ^^^ Embodiments In some embodiments, a method of inhibiting the growth of a Gram-negative bacterium includes applying a lasso peptide to the Gram-negative bacterium. The lasso peptide can be applied to the Gram-negative bacterium while the Gram-negative bacterium is within a cell, such as a host cell. The cell or host cell in which the Gram-negative bacterium resides can be in vitro, for ^^^ example, in a culture, or can be in vivo, for example, in an infected animal, non-human animal, or human. For example, the lasso peptide can be a class I lasso peptide, such as siamycin I (of the 21 amino acid sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1]) or aborycin (of the 21 amino acid sequence CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO.2]). Aborycin has the same amino acid sequence as siamycin I, except that in aborycin the 4th amino acid valine (V) and the 17th ^^^ amino acid isoleucine (I) of siamycin I are switched with respect to each other. For example, the lasso peptide can have the amino acid (peptide or oligopeptide) sequence of siamycin I with 1, 2, 3, 4, or more amino acid residues deleted or replaced by another amino acid residue, for example, 2, 3, 4, or more amino acid residues can be switched with respect to each other. For example, the lasso peptide can have the amino acid sequence of siamycin I with the 4th amino acid residue replaced by ^
Atty. Docket 8883-0015 isoleucine (I) or leucine (L) and/or with the 17th amino acid residue replaced by valine (V) or leucine (L). For example, the lasso peptide can have amino acid (peptide or oligopeptide) sequence of aborycin with 1, 2, 3, 4, or more amino acid residues deleted or replaced by another amino acid residue, for example, 2, 3, 4, or more amino acid residues can be switched with respect to each ^^ other. For example, the lasso peptide can have the amino acid sequence of aborycin with the 4th amino acid residue replaced by leucine (L) or valine (V) and/or with the 17th amino acid residue replaced by isoleucine (I) or leucine (L). The lasso peptide can be in a threaded form (tail at or through the ring), or the lasso peptide can be in an unthreaded form (tail not at and not through the ring). A single type of lasso peptide can be used in the method, or a combination of lasso peptides ^^^ can be used in the method. For example, siamycin I, aborycin, or both siamycin I and aborycin can be used in the method. In the method, the lasso peptide can inhibit a type III secretion system (T3SS) of the Gram-negative bacterium. For example, the T3SS can be the P. aeruginosa Psc T3SS, which belongs to the Ysc T3SS family. For example, the T3SS can be the Salmonella enterica SPI-1 T3SS, which belongs to the Inv-Mxi-Spa T3SS family. ^^^ For example, in an embodiment of a method of inhibiting the growth of a Gram-negative bacterium, the Gram-negative bacterium can be of the Pseudomonadota phylum or the Gammaproteobacteria class. For example, the Gram-negative bacterium can be of the Pseudomonadales or of the Enterobacterales order. For example, the Gram-negative bacterium can be of the Pseudomonadaceae or of the Enterobacteriaceae family. For example, the Gram-negative ^^^ bacterium can be of the Pseudomonas or the Salmonella genus. For example, the Gram-negative bacterium can be Pseudomonas aeruginosa, such as of a lineage genetically characterized by the model strain PAO1, PA14, or PA7. For example, the Gram-negative bacterium can be Salmonella enterica, such as Salmonella enterica subsp. enterica, and such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, or Salmonella enterica serovar Paratyphi. ^^^ In some embodiments, the lasso peptide is used in the prevention and/or treatment of a disease and/or infection caused by a Gram-negative bacterium, for example, in the prevention of the disease and/or infection in an organism. For example, the organism can be a multicellular organism, an animal, a non-human animal, a mammal, a non-human mammal, a human, a subject, and/or a patient. For example, the lasso peptide can be administered to the organism. With such ^^^ administration of the lasso peptide to the organism, the lasso peptide may be applied to (e.g., contacted with or exposed to) the Gram-negative bacterium, which may reside within a (host) cell of the organism to inhibit the growth of the Gram-negative bacterium. Growth encompasses the growth of a single bacterial cell, e.g., growth in cell size of the bacterium, and encompasses cell division as reproduction, e.g., binary fission of a parent cell into two daughter cells. For example, ^
Atty. Docket 8883-0015 inhibition of growth of Gram-negative bacteria in the organism can result in the prevention and/or treatment of a disease and/or infection caused in the organism by the Gram-negative bacteria. Preventing a disease can be a prophylactic method^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^&^^^(^^ administered before onset of the disease, either before or after exposure to the Gram-negative ^^ bacterium. Treating a disease can be a responsive method, for example, the lasso peptide can be administered at the same time as or after onset of the disease, after exposure to the Gram-negative bacterium. For example, the lasso peptide can be administered as part of a pharmaceutical composition to the organism. In some embodiments of a method, the lasso peptide is applied to a Gram-negative ^^^ bacterium to inhibit a type III secretion system (T3SS) of the Gram-negative bacterium. For example, the lasso peptide can be applied to the Gram-negative bacterium in vivo, such as in an organism, such as a multicellular organism. For example, the lasso peptide can be applied to the Gram-negative bacterium in vitro. ^^^ In some embodiments, the lasso peptide or a pharmaceutical composition including the lasso peptide can be administered to the organism, for example, systemically or locally, for example, orally, nasally, intraperitoneally, parenterally, or topically, and for example, by an oral, nasal (e.g., inhalation or insufflation), injection, intravenous, intramuscular, subcutaneous, injection into tissue, or topical route. ^^^ In some embodiments, a pharmaceutical composition includes a therapeutically effective amount of a lasso peptide and a pharmaceutically acceptable carrier or diluent. For example, the lasso peptide may be administered orally in combination with a pharmaceutically acceptable carrier or vehicle such as an inert diluent or an assimilable edible carrier. For example, the lasso peptide may be enclosed in a hard or a soft shell gelatin capsule, ^^^ may be compressed into a tablet, or may be incorporated directly with the food of a patient's diet. The lasso peptide may be combined with one or more excipients and used in an administrative form, such as an ingestible pill, a tablet, a buccal tablet, a troche, a wafer, a capsule, a sustained- release preparation or device, a time release pill, a time release tablet, a time release capsule, an elixir, a suspension, a syrup, or the like. Such an administrative form may include a binder such as ^^^ gum tragacanth, acacia, com starch, ^^^)^^^^^^^^an ^^&^^^^^^^^*&^^^^^^^&^^&^*^^^^^^^^^^^^^^ disintegrating agent such as corn starch, potato starch, or ^^)^^^&^^&^^^^^^^*(^^&^^^^^*&^^^^ ^^)^^^^*^^^^^^^^^^^ a carrier or filler such as clay, microcrystalline cellulose, silica, alumina, or a ^^^"^^^^^^^^+^^^a sweetening and/or a flavoring agent. A capsule administrative form may include, in addition to the above, a liquid, such as water, ethanol, an alcohol, an oil, a vegetable oil, ^
Atty. Docket 8883-0015 propylene glycol, a polyethylene glycol, glycerol, a glyceryl ester, and/or triacetin. For example, a solid administrative form, such as a tablet, pill, or capsule may have a coating, such as an enteric coating, gelatin, wax, shellac, or a sugar. For example, a syrup or elixir may include a preservative, such as a methylparaben or a propylparaben, a surfactant, or a dye. For example, the lasso peptide ^^ may be administered nasally in combination with a pharmaceutically acceptable carrier or vehicle such as a fine inert powder. For example, the lasso peptide may be administered intravenously or intraperitoneally by infusion or injection. An infusible or injectable administrative form can be prepared as a solution of the lasso peptide in water or saline, optionally mixed with a nontoxic surfactant, or can be prepared as a dispersion or suspension in an alcohol, oil, glycerol, polyethylene ^^^ glycols, triacetin, and/or a surfactant. An isotonic agent, such as sodium chloride, can be included. Such an infusible or injectable administrative form can include a preservative to prevent the growth of microorganisms, for example, under ordinary conditions of storage and use. For example, the lasso peptide may be applied topically. A topical administrative form, for example, a paste, gel, ointment, or soap, can be prepared as solution, dispersion, or suspension of the lasso peptide with a ^^^ dermatologically acceptable carrier, which may include water, ethanol, an alcohol, an oil, a vegetable oil, propylene glycol, a polyethylene glycol, glycerol, a glyceryl ester, a synthetic polymer, a fatty acid, a fatty acid salts and/or ester, a fatty alcohols, a modified cellulose, or a mineral or modified mineral. A topical administrative form can be prepared as a liquid for spraying onto the skin or as a liquid that impregnates an absorbent pad, bandage, or dressing to be applied to ^^^ the skin. Any material in an administrative form should be pharmaceutically acceptable and substantially non-toxic in the amounts included. For example, the unit dosage form (administrative form) may include from about 0.1 to about 10000 mg, from about 1 to about 5000 mg, from about 10 to 1000 mg, from about 1 to 100 mg, or about 100 mg of lasso peptide per unit dosage form. For example, the unit dosage form may ^^^ be at least or no more than 0.1, 1, 10, 25, 50, 100, 300, 500, 1000, 1500, 5000, or 10000 mg of lasso peptide per unit dosage form. For example, the concentration of the lasso peptide in an administrative form can be from about 0.001 - 25% by weight, from about 0.01 - 10% by weight, from about 0.05 to about 7% by weight, from about 0.1 - 5% by weight, or about 0.2 - 2% by weight. ^^^ A therapeutically effective dose can be determined empirically, for example, in a cell culture assay or in an animal model. For example, the information obtained can then be used to determine useful doses and routes for administration in humans. For example, a suitable dose may be in the range of from about 0.001 to about 100 mg/kg/day (milligrams of lasso peptide per kilogram of body weight per day), from about 0.01 to about 50 mg/kg/day, or from about 0.1 to about 10 ^
Atty. Docket 8883-0015 mg/kg/day for the patient. For example, a suitable dose may be at least or no more than 0.001, 0.01, 0.1, 1, 10, 50, or 100 mg/kg/day. For example, a suitable dose may be about 0.1 mg/kg/day, 1 mg/kg/day, 10 mg/kg/day, 20 mg/kg/day, 50 mg/kg/day, or 100 mg/kg/day. The suitable dose, in milligrams of lasso peptide per kilogram of body weight per day, can be achieved by administering ^^ the unit dosage form having an appropriate amount of lasso peptide at appropriate intervals or continuously through a day or part of a day. For example, for a 75 kg body weight patient, a dose of 20 mg/kg/day can be achieved by orally administering a unit dosage form of 1500 mg of the lasso peptide once per day, a unit dosage form of 500 mg of the lasso peptide three times per day, or a unit dosage form of 500 mg of the lasso peptide five times per day, or by continuously ^^^ intravenously administering the lasso peptide at a rate of 62.5 mg per hour. The lasso peptide can be administered to achieve peak plasma concentrations in the organism to which the lasso peptide is administered of, for example, from about 0.5 to about 75 ^M, from about 1 to 50 ^M, from about 2 to about 30 ^M, or from about 5 to about 25 ^M. For example, peak plasma concentrations can include at least or no more than 0.2, 0.5, 1, 2, 5, 7.3, 7.5, 10, 20, 30, 50, 75, 100, or 200 ^M. For ^^^ example, during a course of treatment, plasma levels may be from about 1 to 100 ^M or from about 2 to about 25 ^M. Examples In these Examples, it is shown that extracts from Streptomyces albovinaceus and class I ^^^ lasso peptides, such as siamycin I and/or aborycin, can inhibit the growth of Gram-negative bacteria, such as Pseudomonas aeruginosa and Salmonella enterica, and protect, through prevention, prophylaxis, treatment, or responsive treatment, an animal from disease caused by Gram-negative bacteria. ^^^ Extract SNE013 from Streptomyces albovinaceus has T3SS inhibitory activity. A P. aeruginosa strain expressing an exoT-luxCDABE transcriptional reporter was used to screen for T3SS inhibitors1.6080 extracts from a novel library derived from marine and terrestrial bacteria were screened in triplicate.1006 extracts were found to have T3SS inhibitory activity. Of these, 320 were prioritized for follow-up studies based on fold-inhibition and previously known bioactivity. Hits ^^^ from our primary screen may be T3SS inhibitors, general antibiotics, or luciferase inhibitors. Therefore, the ability of these 320 extracts to inhibit luciferase activity in the P. aeruginosa PAO1 Xen41 strain (Perkin Elmer) encoding a constitutively-expressed luciferase operon was assessed. Of the 320 extracts tested in triplicate, 207 were found to inhibit luciferase activity. The remaining 113 hits were tested in triplicate for general antibiotic activity using the alamarBlue cell viability reagent ^
Atty. Docket 8883-0015 (Thermo Fisher). A total of 27 extracts were found to inhibit P. aeruginosa growth and were excluded from follow-up studies. The remaining 86 extracts were prioritized based on fold- inhibition of exoT promoter-driven luciferase activity from the primary screen as well as little to no activity in the alamarBlue or Xen41 luciferase assays. ^^ As an orthogonal assay for T3SS inhibitory activity, a strain of P. aeruginosa PA103 carrying a T3SS secreted protein reporter, in which the ExoU T3SS effector protein is fused to the HiBiT small luciferase subunit, was used. When ExoU-HiBiT is secreted into the culture supernatant through the T3SS, addition of the LgBiT large luciferase subunit and luciferase substrate allows bioluminescence production proportional to the amount of ExoU-HiBiT secretion ^^^ and, therefore, T3SS activity. The top 69 extracts of the 86 identified from primary and secondary screening in the ExoU-HiBiT secretion assay were tested in triplicate, and 13 extracts with no activity were found. The remaining 56 exhibited T3SS inhibitory activity and were categorized into five tiers based on their aggregate data over all four assays. One of the top tier extracts was SNE013-15 derived from Streptomyces albovinaceus isolated from South Carolina. SNE013-15 ^^^ inhibited exoT promoter activity by two-fold, and therefore inhibited Pseudomonas aeruginosa T3SS gene expression, but did not exhibit luciferase or general antibiotic activity (see Figs.1A-1C). Extract SNE013 from Streptomyces albovinaceus contains bioactive lasso peptides. In order to identify the bioactive molecules in the SNE013 extract, the original Streptomyces albovinaceus ^^^ culture was regrown, and bioactivity-guided fractionation was carried out. Several fractions able to inhibit secretion of the ExoU-HiBiT T3SS reporter protein ^^^^^^^^^^^^^^^^ that is, discrete fractions of the Streptomyces albovinaceus fraction SNE013 contained (exhibited) T3SS inhibitory activity, with SNE013-20-10+12G being the most active (see, Fig.2). Mass spectrometry and NMR were used to determine that this fraction (SNE013-20- ^^^ 10+12G) contained either aborycin or siamycin I, two class I lasso peptides with identical amino acid composition and slightly different sequences. Aborycin or siamycin I only vary in the position of an isoleucine (I) and a valine (V) residue. That is, aborycin has the 21 amino acid residue sequence (standard one-letter symbols (abbreviations) of amino acid residues shown) CLGIGSCNDFAGCGYAVVCFW [SEQ ID NO. 2] with isoleucine (I) as the 4th amino acid residue ^^^ and with valine (V) as the 17th amino acid residue. Siamycin I has 21 amino acid residue sequence CLGVGSCNDFAGCGYAIVCFW [SEQ ID NO.1], the same as that of aborycin, except that in siamycin I a valine (V) is the 4th amino acid residue and isoleucine (I) is the 17th amino acid residue. The predicted structure of siamycin I is shown in Fig.3. Aborycin and siamycin I have previously been found to have antibiotic activity against Gram-positive bacteria, but not against Gram-negative ^
Atty. Docket 8883-0015 bacteria2, 3. Siamycin I has been reported to inhibit HIV fusion to the host cell membrane4. Siamycin I was available for purchase. Siamycin I exhibited a similar inhibition of ExoU-HiBiT secretion as did SNE013-20-10+12G (see, Fig.4A). The IC50 of siamycin I in this assay was 7.3 µM, on par with other classes of previously identified T3SS inhibitors (see, Fig.4B)5. That is, ^^ the lasso peptide siamycin I has T3SS inhibitory activity. Extract SNE013 from Streptomyces albovinaceus inhibits the Salmonella SPI-1 T3SS but does not inhibit flagellar motility. The P. aeruginosa T3SS belongs to the Ysc T3SS family, while the Salmonella enterica SPI-1 T3SS belongs to the Inv-Mxi-Spa T3SS family6. To determine if the ^^^ SNE013 extract can inhibit T3SSs from both the Ysc and Inv-Mxi-Spa families, a strain of Salmonella enterica serovar Typhimurium encoding a T3SS secretion reporter consisting of a translation fusion between the SipA T3SS effecter protein and a NanoLuc-myc® tag7 was used. When the SipA-NanoLuc protein is secreted into the culture supernatant through the T3SS, addition of a luciferase substrate allows bioluminescence production proportional to the amount of SipA- ^^^ NanoLuc secretion and, therefore, T3SS activity. The SNE013-20-10+12G extract inhibited the Salmonella enterica SPI-1 T3SS in a dose-dependent manner, suggesting broad activity across multiple T3SS families (see, Fig 5A). SNE013-20-10+12G did not inhibit bacterial motility (see, Fig.5B), suggesting that SNE013-20-10+12G is specific to the T3SS and does not inhibit the flagellar apparatus, which is evolutionarily related to the T3SS6, 8. That is, the SNE013-20-10+12G ^^^ fraction from extract SNE013 from Streptomyces albovinaceus inhibited Salmonella enterica SPI-1 T3SS activity but did not inhibit flagellar motility. Extract SNE013 from Streptomyces albovinaceus protects Galleria mellonella larvae from P. aeruginosa-induced death. Invertebrates serve as excellent models for P. aeruginosa ^^^ pathogenesis^. Larvae of the Galleria mellonella (G. mellonella) Greater Wax Moth die within 24 hours of P. aeruginosa injection^^, and this pathology is largely dependent on the Pseudomonas T3SS^^. SNE013-20-10+12G or the MBX1641 positive control was injected into Galleria larvae in the 6th instar phase simultaneously with P. aeruginosa PAO1, and melanization was monitored as an indicator of larval death (Fig. 6). A T3SS-deficient ,pscF P. aeruginosa mutant was used as a ^^^ negative control. SNE013-20-10+12G injected with PBS alone did not kill larvae. The Streptomyces albovinaceus extract SNE013-20-10+12G significantly protected Galleria mellonella larvae from P. aeruginosa-induced death. Taken together, these data indicate that siamycin I and possibly aborycin, the lasso peptides isolated from Streptomyces albovinaceus, are T3SS inhibitors that block P. aeruginosa acute infection. ^
Atty. Docket 8883-0015 The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the invention. All examples ^^ presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. ^^^ 22 ^
Atty. Docket 8883-0015 Citations: 1. Aiello D, Williams JD, Majgier-Baranowska H, Patel I, Peet NP, Huang J, Lory S, Bowlin TL, Moir DT. Discovery and characterization of inhibitors of Pseudomonas aeruginosa type III ^^&^^^^^^^^^^^^^^&^^(^^)^^^^^^^^^^^^^^^^$^^^^-#^-^.^/ -99. Epub 20100222. doi: ^^ 10.1128/AAC.01598-^/^^0*(1^^^0123.^$^^45/^$^^01^23.^01^$ 5%54/^ 2. Shao M, Ma J, Li Q, Ju J. Identification of the Anti-Infective Aborycin Biosynthetic Gene Cluster from Deep-Sea-Derived Streptomyces sp. SCSIO ZS0098 Enables Production in a 6^^^^^^^)^*^^6^^^^^1^^^3^*)^^^$^^/^^4^$^^^7^*(^$^^/^$$^^^^^^.^^^^%%/^+^^^4^$^^$7. PubMed ^^^ 0123.^%^4/--45^^01^23.^01^5#^/5^%^ 3. Tan S, Ludwig KC, Muller A, Schneider T, Nodwell JR. The Lasso Peptide Siamycin-I Targets Lipid II at the Gram-0^^^^^!^^^^^^^8*^^^&^^^^^8^^^^^^^^^^^^$^^/^^#^-^./55-74. Epub 20190503. doi: 10.1021/acschembio.9b00157. PubMed PMID: 31026131. ^^^ 4. Lin PF, Samanta H, Bechtold CM, Deminie CA, Patick AK, Alam M, Riccardi K, Rose RE, White RJ, Colonno RJ. Characterization of siamycin I, a human immunodeficiency virus fusion ^^^^(^^^^^^^^^^^^&^^(^^)^^^^^^^^^^^^^^^^^//5^#^^^^.^%%-8. doi: 10.1128/AAC.40.1.133. PubMed 0123.^ 4 4 /#^^01^23.^01^^5%^4^^ ^^^ 5. Lam HN, Lau T, Lentz A, Sherry J, Cabrera-Cortez A, Hug K, Lalljie A, Engel J, Lokey RS, Auerbuch V. Developing Cyclic Peptomers as Broad-Spectrum Type III Secretion System Inhibitors in Gram-9^)^^^!^^^^&^^^^^^^^^^^^^&^^(^^)^^^^^^^^^^^^^^^^$^$^^5-^4^.^^^5/^$^^^7^*(^$^$^^5^4^^ doi: 10.1128/AAC.01690-$^^^0*(1^^^0123.^%% 4-#%-^^01^23.^01^ %4%$%4^ ^^^ 6. Troisfontaines P, Cornelis GR. Type III secretion: more systems than you think. Physiology ^^^^^^^^^^^^$^^-^$^.%$5-39. doi: 10.1152/physiol.00011.2005. PubMed PMID: 16174872. 7. Westerhausen S, Nowak M, Torres-Vargas CE, Bilitewski U, Bohn E, Grin I, Wagner S. A ^^^ NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by (^&^^^^^^^^"^^^222^^^&^^^^^^^^"^^^^^^^1^^^1^&^^(^^^^^$^$^^^^%^5^.^$#^-54. Epub 20200304. doi: 10.1111/mmi.14490. PubMed PMID: 32068313. ^
Atty. Docket 8883-0015 8. Deng W, Marshall NC, Rowland JL, McCoy JM, Worrall LJ, Santos AS, Strynadka NCJ, Finlay BB. Assembly, structure, function and regulation of type III secretion systems. Nat Rev Microbiol. $^^4^^-^5^.%$%-37. Epub 20170410. doi: 10.1038/nrmicro.2017.20. PubMed PMID: 28392566. ^^ 9. Jander G, Rahme LG, Ausubel FM. Positive correlation between virulence of Pseudomonas ^^^*)^^^^^^^*^^^^^^^^^^^&^^^^^^^^^^&^^^^:^^^&^^^^^^^^$^^^^^ $^^%^.% #%-5. doi: 10.1128/JB.182.13.3843-% #-^$^^^^^0*(1^^^0123.^^^ -^^^%^^01^23.^01^/#--/^ ^^^ 10. Ramarao N, Nielsen-Leroux C, Lereclus D. The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis. J Vis Exp.2012(70):e4392. Epub 20121211. ^^^.^^^^%4/^+#%/$^^0*(1^^^0123.^$%$4^-^/^^01^23.^01^%-54^5-^ 11. Ngo TD, Ple S, Thomas A, Barette C, Fortune A, Bouzidi Y, Fauvarque MO, Pereira de ^^^ Freitas R, Francisco Hilario F, Attree I, Wong YS, Faudry E. Chimeric Protein-Protein Interface Inhibitors Allow Efficient Inhibition of Type III Secretion Machinery and Pseudomonas aeruginosa ;^^*^^^&^^^^^8^2^^^&^^3^^^^$^^/^-^^^^.^ #%-54. Epub 20190927. doi: 10.1021/acsinfecdis.9b00154. PubMed PMID: 31525902.^ ^