Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa preprotein (P135) that is cleaved into three fragments identified here as P45 683

SummaryMycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (K D~76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with K Ds of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.

Chlamydia psittaci is an avian pathogen capable of spill-over infections to humans. A parrot C. psittaci strain was recently detected in an equine reproductive loss case associated with a subsequent cluster of human C. psittaci infections. In this study, we screened for C. psittaci in cases of equine reproductive loss reported in regional New South Wales, Australia during the 2016 foaling season. C. psittaci specific-PCR screening of foetal and placental tissue samples from cases of equine abortion (n = 161) and foals with compromised health status (n = 38) revealed C. psittaci positivity of 21.1% and 23.7%, respectively. There was a statistically significant geographical clustering of cases ~170 km inland from the mid-coast of NSW (P < 0.001). Genomic analysis and molecular typing of C. psittaci positive samples from this study and the previous Australian equine index case revealed that the equine strains from different studs in regional NSW were clonal, while the phylogenetic analysis revealed that the C. psittaci strains from both Australian equine disease clusters belong to the parrot-associated 6BC clade, again indicative of spill-over of C. psittaci infections from native Australian parrots. The results of this work suggest that C. psittaci may be a more significant agent of equine reproductive loss than thought. A range of studies are now required to evaluate (a) the exact role that C. psittaci plays in equine reproductive loss; (b) the range of potential avian reservoirs and factors influencing infection spill-over; and (c) the risk that these equine infections pose to human health.

ABSTRACTMycoplasma hyopneumoniaecauses enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146that mimic P50P146, P40P146, and P85P146were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146generated fromM. hyopneumoniaestrain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin ofM. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by whichM. hyopneumoniaeregulates protein topography.IMPORTANCEVaccines used to controlMycoplasma hyopneumoniaeinfection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface ofM. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50P146, P40P146, and P85P146. Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface ofM. hyopneumoniae.

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site (761)L-N-V↓A-V-S(766) in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.

T heileria orientalis is a vector-borne hemoprotozoan that infects cattle and buffalo and is generally spread by ticks of the Haemaphysalis genus (1-3). Historically, this organism has been referred to as Theileria sergenti, Theileria buffeli, or the T. orientalis/T. sergenti/T. buffeli complex; however, the name T. sergenti is now considered invalid (4), and T. orientalis is commonly used to refer to all (5). From a clinical perspective, T. orientalis can cause anemia, lethargy, jaundice, fever, abortion, and mortality in cattle (6). Clinical infection can also result in decreased milk production in dairy cattle (7). T. orientalis is a conditional pathogen, and while pathogenic forms are largely limited to Eastern Asia and Australasia (5, 6, 8-10), it is frequently detected in asymptomatic animals; these benign forms are globally spread (5,(11)(12)(13)(14). Analysis of the most common genotyping locus (p32), encoding the variable major piroplasm surface protein (MPSP), currently identifies 11 distinct T. orientalis genotypes (13,15,16). Of these genotypes, type 2 (Ikeda) and to a lesser extent type 1 (Chitose) are typically found in association with clinical disease (6,9,10,(17)(18)(19)(20)(21)(22). The presence of pathogenic and benign forms of T. orientalis greatly complicates its clinical diagnosis, with standard blood film analysis unable to identify the pathogenic genotypes.Multiple conventional PCR (cPCR) assays have been published for the identification of T. orientalis in blood samples (9,21,23,24). The most commonly cited assays detect and genotype T. orientalis by amplifying unique regions of the MPSP gene (21). These assays use two universal primers to detect T. orientalis infection and specific forward primers to identify the Ikeda, Chitose, and Buffeli genotypes. While this method is highly sensitive and has been validated in prior studies (19,21), it is not multiplexed and is highly susceptible to PCR inhibitors (6), which are often found in nucleotide extractions obtained from blood (25,26). To overcome inhibition, undiluted and diluted nucleotide extracts can be examined in parallel to prevent false-negative results (6,19,27). However, if multiple reactions per sample are required to determine the presence of infection, the procedure becomes both expensive and time-consuming. Furthermore, cPCR does not give an accurate representation of parasite load and therefore cannot provide an indication of the severity of T. orientalis infection. A recently developed multiplexed tandem PCR (28) is able to discriminate between four genotypes of T. orientalis, but it is only semiquantitative and therefore cannot be used to define a clinical threshold.Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one-and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity.

BackgroundTheileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries. This paper provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite.MethodsTo identify possible vectors for disease, ticks, biting flies and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife, and mosquitoes and biting flies were collected in traps in close proximity to outbreak herds. Ticks were identified via DNA barcoding of the mitochondrial cytochrome oxidase I gene. Barcoded ticks were pooled according to species or phylogenetic group and tested for the presence of T. orientalis and the genotypes Ikeda, Chitose and Buffeli using real-time PCR.ResultsDNA barcoding and phylogenetic analysis identified ticks from the following species: Haemaphysalis longicornis, Ixodes holocyclus, Ixodes cornuatus, Ixodes hirsti, and Bothriocroton concolor. Additional Haemaphysalis, Ixodes and Bothriocroton spp. were also identified. Of the ticks investigated, only H. longicornis ticks from cattle carried theilerial DNA, with the genotypes Ikeda, Chitose and Buffeli represented. Mosquitoes collected in close proximity to outbreak herds included; Aedes camptorhynchus, Aedes notoscriptus, Coquillettidia linealis, Culex australicus, and Culex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquitoes. The haematophagous flies tested negative.ConclusionsThis is the first demonstration of a potential vector for T. orientalis in the current Australasian disease outbreak.