The faecal microbiome of the Australian silver gull contains phylogenetically diverse ExPEC, aEPEC and Escherichia coli carrying the transmissible locus of stress tolerance.

Wildlife are implicated in the dissemination of antimicrobial resistance, but their roles as hosts for Escherichia coli that pose a threat to human and animal health is limited. Gulls (family Laridae) in particular, are known to carry diverse lineages of multiple-antibiotic resistant E. coli, including extra-intestinal pathogenic E. coli (ExPEC). Whole genome sequencing of 431 E. coli isolates from 69 healthy Australian silver gulls (Chroicocephalus novaehollandiae) sampled during the 2019 breeding season, and without antibiotic selection, was undertaken to assess carriage in an urban wildlife population. Phylogenetic analysis and genotyping resolved 123 sequence types (STs) representing most phylogroups, and identified diverse ExPEC, including an expansive phylogroup B2 cluster comprising 103 isolates (24 %; 31 STs). Analysis of the mobilome identified: i) widespread carriage of the Yersinia High Pathogenicity Island (HPI), a key ExPEC virulence determinant; ii) broad distribution of two novel phage elements, each carrying sitABCD and iii) carriage of the transmissible locus of stress tolerance (tLST), an element linked to sanitation resistance. Of the 169 HPI carrying isolates, 49 (48 %) represented diverse B2 isolates hosting FII-64 ColV-like plasmids that lacked iutABC and sitABC operons typical of ColV plasmids, but carried the serine protease autotransporter gene, sha. Diverse E. coli also carried archetypal ColV plasmids (52 isolates; 12 %). Clusters of closely related E. coli (<50 SNVs) from ST58, ST457 and ST746, sourced from healthy gulls, humans, and companion animals, were frequently identified. In summary, anthropogenically impacted gulls host an expansive E. coli population, including: i) putative ExPEC that carry ColV virulence gene cargo (101 isolates; 23.4 %) and HPI (169 isolates; 39 %); ii) atypical enteropathogenic E. coli (EPEC) (17 isolates; 3.9 %), and iii) E. coli that carry the tLST (20 isolates; 4.6 %). Gulls play an important role in the evolution and transmission of E. coli that impact human health.

Genomic analysis of diverse environmental Acinetobacter isolates identifies plasmids, antibiotic resistance genes, and capsular polysaccharides shared with clinical strains.

Acinetobacter baumannii, an important pathogen known for its widespread antibiotic resistance, has been the focus of extensive research within its genus, primarily involving clinical isolates. Consequently, data on environmental A. baumannii and other Acinetobacter species remain limited. Here, we utilized Illumina and Nanopore sequencing to analyze the genomes of 10 Acinetobacter isolates representing 6 different species sourced from aquatic environments in South Australia. All 10 isolates were phylogenetically distinct compared to clinical and other non-clinical Acinetobacter strains, often tens of thousands of single-nucleotide polymorphisms from their nearest neighbors. Despite the genetic divergence, we identified pdif modules (sections of mobilized DNA) carrying clinically important antimicrobial resistance genes in species other than A. baumannii, including carbapenemase oxa58, tetracycline resistance gene tet(39), and macrolide resistance genes msr(E)-mph(E). These pdif modules were located on plasmids with high sequence identity to those circulating in globally distributed A. baumannii ST1 and ST2 clones. The environmental A. baumannii isolate characterized here (SAAb472; ST350) did not possess any native plasmids; however, it could capture two clinically important plasmids (pRAY and pACICU2) with high transfer frequencies. Furthermore, A. baumannii SAAb472 possessed virulence genes and a capsular polysaccharide type analogous to clinical strains. Our findings highlight the potential for environmental Acinetobacter species to acquire and disseminate clinically important antimicrobial resistance genes, underscoring the need for further research into the ecology and evolution of this important genus.IMPORTANCEAntimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

Genomic surveillance for antimicrobial resistance - a One Health perspective.

Antimicrobial resistance (AMR) - the ability of microorganisms to adapt and survive under diverse chemical selection pressures - is influenced by complex interactions between humans, companion and food-producing animals, wildlife, insects and the environment. To understand and manage the threat posed to health (human, animal, plant and environmental) and security (food and water security and biosecurity), a multifaceted 'One Health' approach to AMR surveillance is required. Genomic technologies have enabled monitoring of the mobilization, persistence and abundance of AMR genes and mutations within and between microbial populations. Their adoption has also allowed source-tracing of AMR pathogens and modelling of AMR evolution and transmission. Here, we highlight recent advances in genomic AMR surveillance and the relative strengths of different technologies for AMR surveillance and research. We showcase recent insights derived from One Health genomic surveillance and consider the challenges to broader adoption both in developed and in lower- and middle-income countries.

Antimicrobial susceptibility and molecular characteristics of Mycoplasma bovis isolated from cases of bovine respiratory disease in Australian feedlot cattle.

To date, antimicrobial susceptibility has not been reported for Australian Mycoplasma bovis isolates. This study determined minimal inhibitory concentrations (MICs) for 12 different antimicrobials against Australian M. bovis isolates and used whole genome sequencing to screen those showing high macrolide MICs for point mutations in target genes. Most lung tissue/swab samples from bovine respiratory disease cases (61/76, 80.3%) tested positive for M. bovis. A set of 50 representative isolates (50/61, 82.0%) that showed adequate growth, was used for MIC testing. Uniformly, low MIC values were confirmed for enrofloxacin (≤ 4 µg/mL), florfenicol (≤ 8 µg/mL), gamithromycin (≤ 2 µg/mL), spectinomycin (≤ 4 µg/mL), tetracycline (≤ 8 µg/mL), tiamulin (≤ 4 µg/mL), and tulathromycin (≤ 0.5 µg/mL). A small proportion (10%) of isolates exhibited high MICs (≥ 32 µg/mL) for tildipirosin, tilmicosin, tylosin, and lincomycin, which were above the epidemiological cut-off values for each antimicrobial (≥ 4 µg/mL). These isolates, originating from three Australian states, underwent whole genome sequencing/multilocus sequencing typing and were compared with the reference strain PG45 to investigate mutations that might be linked with the high macrolide/lincosamide MICs. All five belonged to ST52 and two macrolide associated mutations were identified within the 23 S rRNA gene (A2058G in two sequenced isolates and G748A in all sequenced isolates). Four additional 23 S rRNA gene mutations did not appear to be linked to macrolide resistance. Whilst the majority of Australian M. bovis isolates appear susceptible to the tested antimicrobials, emerging macrolide resistance was detected in three Australian states and requires continued monitoring.

Genomic Analysis of Carbapenem-Resistant Comamonas in Water Matrices: Implications for Public Health and Wastewater Treatments.

Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired blaGES-5, blaOXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All blaGES-5- and blaOXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to "last-resort drugs," such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and inorganic substrates is well documented, some species are also opportunistic pathogens. However, data regarding AMR in Comamonas spp. are limited. Here, through the genomic analyses of 39 carbapenem-resistant Comamonas isolates, we make several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encoding carbapenemase blaGES-5 or extended-spectrum ß-lactamase blaOXA alleles. Given the importance of Comamonas species in potential wastewater bioaugmentation and bioremediation strategies, as well as their status as emerging pathogens, the acquisition of critically important antibiotic resistance genes on genomic islands warrants future monitoring.

Genomic analysis of Elizabethkingia species from aquatic environments: Evidence for potential clinical transmission.

Elizabethkingia species are ubiquitous in aquatic environments, colonize water systems in healthcare settings and are emerging opportunistic pathogens with reports surfacing in 25 countries across six continents. Elizabethkingia infections are challenging to treat, and case fatality rates are high. Chromosomal bla B , bla GOB and bla CME genes encoding carbapenemases and cephalosporinases are unique to Elizabethkingia spp. and reports of concomitant resistance to aminoglycosides, fluoroquinolones and sulfamethoxazole-trimethoprim are known. Here, we characterized whole-genome sequences of 94 Elizabethkingia isolates carrying multiple wide-spectrum metallo-ß-lactamase (bla B and bla GOB) and extended-spectrum serine­ß-lactamase (bla CME) genes from Australian aquatic environments and performed comparative phylogenomic analyses against national clinical and international strains. qPCR was performed to quantify the levels of Elizabethkingia species in the source environments. Antibiotic MIC testing revealed significant resistance to carbapenems and cephalosporins but susceptibility to fluoroquinolones, tetracyclines and trimethoprim-sulfamethoxazole. Phylogenetics show that three environmental E. anophelis isolates are closely related to E. anophelis from Australian clinical isolates (∼36 SNPs), and a new species, E. umeracha sp. novel, was discovered. Genomic signatures provide insight into potentially shared origins and a capacity to transfer mobile genetic elements with both national and international isolates.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.

Multidrug-Resistant Lineage of Enterotoxigenic Escherichia coli ST182 With Serotype O169:H41 in Airline Waste.

Enterotoxigenic Escherichia coli (ETEC) is the primary aetiologic agent of traveller's diarrhoea and a significant cause of diarrhoeal disease and death in developing countries. ETEC O169:H41 strains are known to cause both traveller's diarrhoea and foodborne outbreaks in developed countries and are cause for concern. Here, whole-genome sequencing (WGS) was used to assemble 46 O169:H41 (ST182) E. coli draft genomes derived from two airplane waste samples sourced from a German international airport. The ST182 genomes were compared with all 84 publicly available, geographically diverse ST182 genomes to construct a core genome-based phylogenetic tree. ST182 isolates were all phylogroup E, the majority serotype O169:H41 (n = 121, 93%) and formed five major clades. The airplane waste isolates differed by an average of 15 core SNPs (range 0-45) but their accessory genome content was diverse. While uncommon in other ST182 genomes, all airplane-derived ST182 isolates carried: (i) extended-spectrum ß-lactamase gene bla CTX-M- 15 notably lacking the typical adjacent ISEcp1; (ii) qnrS1 and the S83L mutation in gyrA, both conferring resistance to fluoroquinolones; and (iii) a class 1 integron structure (IS26-intI1 Δ 648-dfrA17-aadA5-qacEΔ1-sul1-ORF-srpC-padR-IS6100-mphR-mrx-mphA-IS26) identified previously in major extraintestinal pathogenic E. coli STs but not in ETEC. ST182 isolates carried ETEC-specific virulence factors STp + CS6. Adhesin/invasin tia was identified in 89% of aircraft ST182 isolates (vs 23%) and was located on a putative genomic island within a hotspot region for various insertions including PAI I536 and plasmid-associated transposons. The most common plasmid replicons in this collection were IncFII (100%; F2:A-:B-) and IncB/O/K/Z (89%). Our data suggest that potentially through travel, E. coli ST182 are evolving a multidrug-resistant profile through the acquisition of class 1 integrons and different plasmids.

Protein cleavage influences surface protein presentation in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a significant cause of pneumonia and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model organism where extensive 'omics' studies have been conducted to gain insight into how minimal genome self-replicating organisms function. An N-terminome study undertaken here identified 4898 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Notably, almost half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events predominately targeting tryptic-like sites, though cleavages at negatively charged residues in P1' (D and E) with lysine or serine/alanine in P2' and P3' positions also occurred frequently. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Of the 160 surface proteins identified, 134 were also targets of endo-proteolytic processing. These processing events are likely to have profound implications for how the host immune system recognises and responds to M. pneumoniae.

Genomic analysis of trimethoprim-resistant extraintestinal pathogenic Escherichia coli and recurrent urinary tract infections.

Urinary tract infections (UTIs) are the most common bacterial infections requiring medical attention and a leading justification for antibiotic prescription. Trimethoprim is prescribed empirically for uncomplicated cases. UTIs are primarily caused by extraintestinal pathogenic Escherichia coli (ExPEC) and ExPEC strains play a central role in disseminating antimicrobial-resistance genes worldwide. Here, we describe the whole-genome sequences of trimethoprim-resistant ExPEC and/or ExPEC from recurrent UTIs (67 in total) from patients attending a regional Australian hospital from 2006 to 2008. Twenty-three sequence types (STs) were observed, with ST131 predominating (28 %), then ST69 and ST73 (both 7 %). Co-occurrence of trimethoprim-resistance genes with genes conferring resistance to extended-spectrum ß-lactams, heavy metals and quaternary ammonium ions was a feature of the ExPEC described here. Seven trimethoprim-resistance genes were identified, most commonly dfrA17 (38 %) and dfrA12 (18 %). An uncommon dfrB4 variant was also observed. Two blaCTX-M variants were identified - blaCTX-M-15 (16 %) and blaCTX-M-14 (10 %). The former was always associated with dfrA12, the latter with dfrA17, and all blaCTX-M genes co-occurred with chromate-resistance gene chrA. Eighteen class 1 integron structures were characterized, and chrA featured in eight structures; dfrA genes featured in seventeen. ST131 H30Rx isolates possessed distinct antimicrobial gene profiles comprising aac(3)-IIa, aac(6)-Ib-cr, aph(3')-Ia, aadA2, blaCTX-M-15, blaOXA-1 and dfrA12. The most common virulence-associated genes (VAGs) were fimH, fyuA, irp2 and sitA (all 91 %). Virulence profile clustering showed ST131 H30 isolates carried similar VAGs to ST73, ST405, ST550 and ST1193 isolates. The sole ST131 H27 isolate carried molecular predictors of enteroaggregative E. coli/ExPEC hybrid strains (aatA, aggR, fyuA). Seven isolates (10 %) carried VAGs suggesting ColV plasmid carriage. Finally, SNP analysis of serial UTI patients experiencing worsening sequelae demonstrated a high proportion of point mutations in virulence factors.

Duplication and diversification of a unique chromosomal virulence island hosting the subtilase cytotoxin in Escherichia coli ST58.

The AB5 cytotoxins are important virulence factors in Escherichia coli. The most notable members of the AB5 toxin families include Shiga toxin families 1 (Stx1) and 2 (Stx2), which are associated with enterohaemorrhagic E. coli infections causing haemolytic uraemic syndrome and haemorrhagic colitis. The subAB toxins are the newest and least well understood members of the AB5 toxin gene family. The subtilase toxin genes are divided into a plasmid-based variant, subAB1, originally described in enterohaemorrhagic E. coli O113:H21, and distinct chromosomal variants, subAB2, that reside in pathogenicity islands encoding additional virulence effectors. Previously we identified a chromosomal subAB2 operon within an E. coli ST58 strain IBS28 (ONT:H25) taken from a wild ibis nest at an inland wetland in New South Wales, Australia. Here we show the subAB2 toxin operon comprised part of a 140 kb tRNA-Phe chromosomal island that co-hosted tia, encoding an outer-membrane protein that confers an adherence and invasion phenotype and additional virulence and accessory genetic content that potentially originated from known virulence island SE-PAI. This island shared a common evolutionary history with a secondary 90 kb tRNA-Phe pathogenicity island that was presumably generated via a duplication event. IBS28 is closely related [200 single-nucleotide polymorphisms (SNPs)] to four North American ST58 strains. The close relationship between North American isolates of ST58 and IBS28 was further supported by the identification of the only copy of a unique variant of IS26 within the O-antigen gene cluster. Strain ISB28 may be a historically important E. coli ST58 genome sequence hosting a progenitor pathogenicity island encoding subAB.

The Diverse Functional Roles of Elongation Factor Tu (EF-Tu) in Microbial Pathogenesis.

Elongation factor thermal unstable Tu (EF-Tu) is a G protein that catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome inside living cells. Structural and biochemical studies have described the complex interactions needed to effect canonical function. However, EF-Tu has evolved the capacity to execute diverse functions on the extracellular surface of both eukaryote and prokaryote cells. EF-Tu can traffic to, and is retained on, cell surfaces where can interact with membrane receptors and with extracellular matrix on the surface of plant and animal cells. Our structural studies indicate that short linear motifs (SLiMs) in surface exposed, non-conserved regions of the molecule may play a key role in the moonlighting functions ascribed to this ancient, highly abundant protein. Here we explore the diverse moonlighting functions relating to pathogenesis of EF-Tu in bacteria and examine putative SLiMs on surface-exposed regions of the molecule.

Formylated N-terminal methionine is absent from the Mycoplasma hyopneumoniae proteome: Implications for translation initiation.

N-terminal methionine excision (NME) is a proteolytic pathway that cleaves the N-termini of proteins, a process that influences where proteins localise in the cell and their turnover rates. In bacteria, protein biosynthesis is initiated by formylated methionine start tRNA (fMet-tRNAfMet). The formyl group is attached by formyltransferase (FMT) and is subsequently removed by peptide deformylase (PDF) in most but not all proteins. Methionine aminopeptidase then cleaves deformylated methionine to complete the process. Components of NME, particularly PDF, are promising therapeutic targets for bacterial pathogens. In Mycoplasma hyopneumoniae, a genome-reduced, major respiratory pathogen of swine, pdf and fmt are absent from its genome. Our bioinformatic analysis uncovered additional enzymes involved in formylated N-terminal methionine (fnMet) processing missing in fourteen mycoplasma species, including M. hyopneumoniae but not in Mycoplasma pneumoniae, a major respiratory pathogen of humans. Consistent with our bioinformatic studies, an analysis of in-house tryptic peptide libraries confirmed the absence of fnMet in M. hyopneumoniae proteins but, as expected fnMet peptides were detected in the proteome of M. pneumoniae. Additionally, computational molecular modelling of M. hyopneumoniae translation initiation factors reveal structural and sequence differences in areas known to interact with fMet-tRNAfMet. Our data suggests that some mycoplasmas have evolved a translation process that does not require fnMet.

Escherichia coli ST302: Genomic Analysis of Virulence Potential and Antimicrobial Resistance Mediated by Mobile Genetic Elements.

aEPEC are associated with persistent diarrhea, and diarrheal outbreaks in both humans and animals worldwide. They are differentiated from typical EPEC by the lack of bundle-forming pili, and from EHEC by the lack of phage-mediated stx toxins. However, phylogenetic analyses often associate aEPEC with EHEC, promoting the hypothesis that aEPEC are the progenitors of EHEC, which is supported by aEPEC conversion to EHEC by stx-carrying phages. While aEPEC can cause disease outright, the potential to acquire stx, one of the most potent bacterial toxins known, merits close monitoring. Escherichia coli ST302 (O108:H9, O182:H9, O45:H9) are aEPEC that have been isolated from diarrheic human, pig and rabbit hosts, as well as in healthy pigs, however, no study to date has focused on E. coli ST302 strains. Through WGS and hybrid assembly we present the first closed chromosome, and two circularized plasmids of an ST302 strain - F2_18C, isolated from a healthy pig in Australia. A phylogenetic analysis placed E. coli ST302 strains in proximity to EHEC ST32 (O145:H28) strains. Public databases were interrogated for WGSs of E. coli ST302 strains and short-read gene screens were used to compare their virulence-associated gene (VAG) and antimicrobial resistance gene (ARG) cargo. E. coli ST302 strains carry diverse VAGs, including those that typically associated with extraintestinal pathogenic E. coli (ExPEC). Plasmid comparisons showed that pF2_18C_FIB shared homology with EHEC virulence plasmids such as pO103 while pF2_18C_HI2 is a large multidrug resistance IncHI2:ST3 plasmid. A comparison of 33 HI2:ST3 plasmids demonstrated that those of Australian origin have not acquired resistances to extended-spectrum beta-lactams, colistin, fosfomycin or rifampicin, unlike those originating from Asia. F2_18C was shown to carry two additional pathogenicity islands - ETT2, and the STEC-associated PAI CL 3, plasmid-associated heavy metal resistance genes, as well as several unoccupied stx-phage attachment sites. This study sheds light on the virulence and AMR potential of E. coli ST302 strains and informs AMR genomic surveillance.

N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen.

Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function.

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae.

Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.

Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.

Non-proteolytic functions of microbial proteases increase pathological complexity.

Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

Cilium adhesin P216 (MHJ_0493) is a target of ectodomain shedding and aminopeptidase activity on the surface of Mycoplasma hyopneumoniae.

MHJ_0493 (P216) is a highly expressed cilium adhesin in Mycoplasma hyopneumoniae. P216 undergoes cleavage at position 1074 in the S/T-X-F↓-X-D/E-like motif (1072)T-N-F↓Q-E(1076) generating N-terminal and C-terminal fragments of 120 kDa (P120) and 85 kDa (P85) on the surface of M. hyopneumoniae. Here we show that several S/T-X-F↓X-D/E-like motifs exist in P216 but only (1072)T-N-F↓Q-E(1076) and (1344)I-T-F↓A-D-Y(1349) were determined to be bona fide processing sites by identifying semitryptic peptides consistent with cleavage at the phenylalanine residue. The location of S/T-X-F↓-X-D/E-like motifs within or abutting regions of protein disorder greater than 40 consecutive amino acids is consistent with our hypothesis that site access influences the cleavage efficiency. Approximately 20 cleavage fragments of P216 were identified on the surface of M. hyopneumoniae by LC-MS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LC-MS/MS analysis of semitryptic peptides within P216 identified novel cleavage sites. Moreover, detection of a series of overlapping semitryptic peptides that differed by the loss a single amino acid at their N-terminus is consistent with aminopeptidase activity on the surface of M. hyopneumoniae. P120 and P85 and their cleavage fragments bind heparin and cell-surface proteins derived from porcine epithelial-like cells, indicating that P216 cleavage fragments retain the ability to bind glycosaminoglycans.

P159 from Mycoplasma hyopneumoniae binds porcine cilia and heparin and is cleaved in a manner akin to ectodomain shedding.

Mycoplasma hyopneumoniae colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of M. hyopneumoniae. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F↓X-D/E-like motifs located at positions (233)F↓Q(234) and (981)F↓Q(982), generating P27, P110, and P52. An unrelated cleavage site (738)L-K-V↓G-A-A(743) in P110 shows sequence identity with a cleavage site (L-N-V↓A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LC-MS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LC-MS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1(P159)-F4(P159)), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes.