Construction of Mycoplasma hyopneumoniae P97 Null Mutants.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

Transposon Mutagenesis of Foodborne Pathogenic Escherichia coli.

The Enterobacteriaceae, and in particular, Escherichia coli including foodborne pathotypes are particularly amenable to transposon mutagenesis. Here we describe the use of mini-Tn5 and Mu d1(Ap lac) to generate transposon inserts for analysis of enterohemorrhagic Escherichia coli EDL933. We also discuss how to array the library in 96-well plates and sequence individual clones for further analysis.

Analysis of swine antigen-specific antibody responses to Mycoplasma hyopneumoniae infection determined by protein microarray.

Pigs harbor several different species of mycoplasmas, of which Mycoplasma hyopneumoniae presents the most significant economic impact on the swine industry. While ELISAs are the predominant diagnostic assay to measure antibody responses during infection with M. hyopneumoniae, the assay itself is only a rough estimate of the total antibody response. It lends little information on pathogen-wide antigen-specific responses. In addition, antibody responses to M. hyopneumoniae as measured by ELISA are slow to develop in infected swine. Our goal was to determine if a protein microarray could be more sensitive and informative of the serological responses of pigs to M. hyopneumoniae infection. The gene sequences of approximately 50 M. hyopneumoniae surface proteins or protein fragments were cloned, mutated to remove UGA codons, expressed in Escherichia coli and purified. The arrays were used to interrogate pig sera from various sources. Sera from naturally-infected swine gave some variability in antigen-specific responses, but, unexpectedly, the responses against the C-terminal portion of the major adhesin P97 was weak in all animals, including those that were experimentally infected. In two of four 118-day experimentally-infected caesarian-derived colostrum-deprived pigs, the strongest antibody responses occurred on days 30 and 54 against members of the P97/P102 paralog families. Our Day 0 results in the other two animals indicate that although thought to be mycoplasma free by all known criteria (serology and PCR), they may have harbored an inapparent Mycoplasma infection. In summary, the protein microarray has the potential to identify new targets for assay development to enhance sensitivity of antibody-based assays.

Development and optimization of a cell-associated challenge model for Mycoplasma hyorhinis in 7-week-old cesarean-derived, colostrum-deprived pigs.

Mycoplasma hyorhinis (MHR) causes polyserositis and lameness in grower pigs. While herd-specific vaccines for this bacterium are being marketed, there are currently no licensed, commercially available vaccines for MHR. The objective of this study was to develop a challenge model in cesarean-derived, colostrum-deprived (CDCD) pigs using cell-associated MHR that results in both severe pericarditis and lameness, in order to evaluate suitable vaccine candidates. We investigated administering MHR to 7-week-old pigs over 3 d using 3 different routes compared to administering MHR on a single day using 1 of 3 routes. Pigs were monitored for 21 d for signs of lameness and well-being. At the end of the study, pigs were examined for evidence of polyserositis and arthritis associated with Mycoplasma. Results indicate that clinical manifestation of disease depended more on the route of administration than on the total dose given. A single intravenous (IV) administration of MHR resulted in extensive polyserositis, while a single intranasal (IN) administration showed little to no signs of disease. A single intraperitoneal (IP) administration did not induce the same level of polyserositis as observed in the IV group, but did result in an increased incidence of lameness. Furthermore, pigs administered MHR by IP (Day 0), IV (Day 1), and IN (Day 2) on 3 consecutive days showed a more robust disease manifestation, which resulted in both polyserositis and lameness. Optimization of this group showed that elimination of the 3rd-day IN challenge had no detrimental effect on clinical outcomes. The consecutive day administration of cell-associated MHR will allow polyserositis and lameness to be simultaneously evaluated in future vaccine trials.

Mycoplasma hyorhinis (MHR) cause une polysérosite et de la boiterie chez les porcs en croissance. Alors que pour ce microorganisme des vaccins spécifiques de troupeaux sont disponibles, il n'y a actuellement aucun vaccin homologué commercialement disponible pour MHR. L'objectif de la présente étude était de développer un modèle d'infection chez des porcs obtenus par césarienne et privés de colostrum en utilisant du MHR associé à des cellules qui causait une péricardite sévère et de la boiterie, afin d'évaluer des vaccins candidats appropriés. Nous avons évalué l'administration de MHR à des porcs âgés de 7 semaines sur 3 jours en utilisant trois voies d'administration différentes comparativement à l'administration de MHR en une seule journée en utilisant une des trois routes. Les porcs ont été suivis pendant 21 jours pour des signes de boiterie et de bien-être. À la fin de l'étude, les porcs ont été examinés pour des évidences de polysérosite et d'arthrite associées avec Mycoplasma. Les résultats indiquaient que la manifestation clinique de la maladie était plus dépendante de la voie d'administration que du nombre total de doses administré. L'administration intraveineuse (IV) d'une dose unique de MHR a résulté en une polysérosite généralisée, alors qu'une dose unique par voie intranasale (IN) ne causa que peu ou pas de signes de maladie. L'administration d'une dose unique par voie intrapéritonéale (IP) n'a pas induit le même degré de polysérosite que celui observé dans le groupe IV, mais on nota une augmentation de l'incidence de boiterie. De plus, les porcs auxquels on administra MHR par voie IP (Jour 0), IV (Jour 1), et IN (Jour 2) pendant 3 jours consécutifs ont montré des manifestations plus fortes de la maladie, ce qui résultat en polysérosite et boiterie. L'optimisation pour ce groupe a démontré que l'élimination de l'inoculation IN au troisième jour n'avait aucun effet délétère sur les résultats cliniques. L'administration de MHR associé à des cellules pendant des jours consécutifs permettra d'évaluer simultanément la polysérosite et la boiterie lors d'essais futurs de vaccins.(Traduit par Docteur Serge Messier).

Cholesterol exacerbates Mycoplasma hyopneumoniae-induced apoptosis via stimulating proliferation and adhesion to porcine alveolar macrophages.

Mycoplasma hyopneumoniae (M. hyo) is the agent of porcine enzootic pneumonia, a disease that causes considerable economic losses in the swine industry. Induction of apoptosis in porcine alveolar macrophages is an important pathogenic mechanism of M. hyo. Cholesterol has been reported to influence cell adherence and cell invasion of Mycoplasma gallisepticum and Mycoplasma fermentans leading to apoptosis, but the role of cholesterol on the apoptotic inducing activity of M. hyo remains unknown. In this study, we found a positive correlation between cholesterol level and M. hyo infection in porcine serum and lung tissue. Cholesterol exacerbated M. hyo-induced apoptosis in porcine alveolar macrophages (PAMs) in a dosage-dependent manner, which was associated with increased hydrogen peroxide (H2O2) and nitric oxide (NO) production, up-regulated TNF-α mRNA expression, and activated caspase-3. The pathogenicity-enhancing effect of cholesterol was related to increased M. hyo proliferation with an up-regulation of M. hyo genes responsible for DNA and protein synthesis, which led to improved M. hyo adherence to PAMs, presumably via increased mRNA expression of adhesin genes. In conclusion, cholesterol promotes the apoptotic effect of M. hyo through stimulating proliferation and enhancing its adherence to PAMs. Hence, the study gives new insights into the role of cholesterol on the PAM - M. hyo interations.

Use of signature-tagged mutagenesis to identify genes associated with colonization of sheep by E. coli O157:H7.

Outbreaks of Escherichia coli O157:H7 in the United States due to contaminated foods are a public health issue and a continuing problem. The major reservoir for these organisms is the gastrointestinal tract of ruminants where they are a member of the resident microbiota. Several factors that contribute to the colonization of cattle have been identified, but a systematic screen of genes that might contribute to the colonization and persistence phenotype in mature ruminants has not been reported. Using a sheep model of persistence, signature tagged mutagenesis (STM) was used to screen 1326 mutants for a persistence-negative phenotype of E. coli O157:H7. We identified 9 genes by STM that appeared to be required for colonization and/or survival in sheep. Three of the genes had functions associated with central metabolism (thiK, ftrA and nrdB), one was involved with LPS formation (wbdP), one encodes a non-LEE encoded effector protein (nleB) and one was a methyltransferase encoded on a prophage (Z2389). The remaining three genes did not have homology with any known genes. Six sheep given ΔwbdP and 2 sheep each were given mutants (ΔthiK (Z1745), ΔftrA (Z2164) and Z2389). The ΔwbdP mutant was recovered from the feces of 4/6 sheep at 6 days pi with a mean number of 1.42log10CFU/g feces compared to 4.6log10CFU/g feces for the wild type strain. This difference was significant (P<0.001) over the time course of the experiment (days 6-23). Both ΔthiK and ΔftrA mutants were recovered from 1 of 2 sheep at 9 days PI by enrichment procedures (<50CFU/g feces) whereas mutant Z2389 was not recovered from either animal past 2 days pi. The roles of all of these gene products require further study to determine how the persistence phenotype of a given strain of E. coli O157:H7 interacts with host factors.

Cross reactivity among the swine mycoplasmas as identified by protein microarray.

Mycoplasmas are cell wall-less bacteria that infect a variety of animals in a species-specific manner. In swine, Mycoplasma hyopneumoniae is the most virulent and presents the most disease and economic problems to the swine industry. Serological assays are commonly used to assess colonization and disease, but antigenic cross-reactivity between M. hyopneumoniae and other mycoplasma species, most notably Mycoplasma hyorhinis, Mycoplasma hyosynoviae and Mycoplasma flocculare, is a concern. The extent of cross-reactivity has not been thoroughly investigated. These studies were designed to identify M. hyopneumoniae proteins that are recognized by rabbit hyperimmune sera raised against the other swine mycoplasmas. Our results indicate extensive cross-reactivity between M. flocculare and M. hyopneumoniae, which explains previous reports seen with ELISA assays. Only three of the thirty-nine M. hyopneumoniae proteins tested showed no cross reactivity with the other three swine mycoplasmas, mhp182 (42kDa C-terminal fragment), mhp638 and mhp684 (C-terminal fragment). Two proteins, mhp384 and mhp511, were cross-reactive with hyperimmune sera generated against three of the four species. None of the anti-M. hyorhinis hyperimmune sera reacted to any of the M. hyopneumoniae proteins. These results suggest that inapparent M. flocculare infections could produce positive responses in M. hyopneumoniae serological assays due to cross-reactivity, and that M. hyosynoviae infections are less likely to do so and M. hyorhinis infections unlikely to affect assay results.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

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.

Multilocus sequence typing of Mycoplasma bovis reveals host-specific genotypes in cattle versus bison.

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and the bacterium most frequently isolated from the polymicrobial syndrome known as bovine respiratory disease complex. Recently, M. bovis has emerged as a significant health problem in bison, causing necrotic pharyngitis, pneumonia, dystocia and abortion. Whether isolates from cattle and bison comprise genetically distinct populations is unknown. This study describes the development of a highly discriminatory multilocus sequencing typing (MLST) method for M. bovis and its use to investigate the population structure of the bacterium. Genome sequences from six M. bovis isolates were used for selection of gene targets. Seven of 44 housekeeping genes initially evaluated were selected as targets on the basis of sequence variability and distribution within the genome. For each gene target sequence, four to seven alleles could be distinguished that collectively define 32 sequence types (STs) from a collection of 94 cattle isolates and 42 bison isolates. A phylogeny based on concatenated target gene sequences of each isolate revealed that bison isolates are genetically distinct from strains that infect cattle, suggesting recent disease outbreaks in bison may be due to the emergence of unique genetic variants. No correlation was found between ST and disease presentation or geographic origin. MLST data reported here were used to populate a newly created and publicly available, curated database to which researchers can contribute. The MLST scheme and database provide novel tools for exploring the population structure of M. bovis and tracking the evolution and spread of strains.

Antibody responses of swine following infection with Mycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and M. flocculare.

Several mycoplasma species possessing a range of virulence have been described in swine. The most commonly described are Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, and Mycoplasma flocculare. They are ubiquitious in many pig producing areas of the world, and except for M. hyopneumoniae, commercial antibody-based assays are lacking for most of these. Antibody cross-reactivity among these four mycoplasma species is not well characterized. Recently, the use of pen-based oral fluids for herd surveillance is of increasing interest. Thus, this study sought to measure pig antibody responses and the level of cross-reactivity in serum and pen-based oral fluids after challenge with four species of swine mycoplasmas. Four groups of four mycoplasma-free growing pigs were separately inoculated with the different mycoplasma species. Pen-based oral fluids and serum samples were collected weekly until necropsy. Species-specific Tween 20 ELISAs were used to measure antibody responses along with four other commercial M. hyopneumoniae ELISAs. Animals from all groups seroconverted to the challenge species of mycoplasma and no evidence of cross-contamination was observed. A delayed antibody response was seen with all but M. hyorhinis-infected pigs. Cross-reactive IgG responses were detected in M. hyopneumoniae- and M. flocculare-infected animals by the M. hyorhinis Tween 20 ELISA, while sera from M. hyosynoviae and M. flocculare-infected pigs were positive in one commercial assay. In pen-based oral fluids, specific anti-M. hyopneumoniae IgA responses were detected earlier after infection than serum IgG responses. In summary, while some antibody-based assays may have the potential for false positives, evidence of this was observed in the current study.

Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232.

BACKGROUND: Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome. RESULTS: Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping. CONCLUSIONS: Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

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.

Analysis of autoinducer-2 quorum sensing in Yersinia pestis.

The autoinducer-2 (AI-2) quorum-sensing system has been linked to diverse phenotypes and regulatory changes in pathogenic bacteria. In the present study, we performed a molecular and biochemical characterization of the AI-2 system in Yersinia pestis, the causative agent of plague. In strain CO92, the AI-2 signal is produced in a luxS-dependent manner, reaching maximal levels of 2.5 µM in the late logarithmic growth phase, and both wild-type and pigmentation (pgm) mutant strains made equivalent levels of AI-2. Strain CO92 possesses a chromosomal lsr locus encoding factors involved in the binding and import of AI-2, and confirming this assignment, an lsr deletion mutant increased extracellular pools of AI-2. To assess the functional role of AI-2 sensing in Y. pestis, microarray studies were conducted by comparing Δpgm strain R88 to a Δpgm ΔluxS mutant or a quorum-sensing-null Δpgm ΔypeIR ΔyspIR ΔluxS mutant at 37°C. Our data suggest that AI-2 quorum sensing is associated with metabolic activities and oxidative stress genes that may help Y. pestis survive at the host temperature. This was confirmed by observing that the luxS mutant was more sensitive to killing by hydrogen peroxide, suggesting a potential requirement for AI-2 in evasion of oxidative damage. We also show that a large number of membrane protein genes are controlled by LuxS, suggesting a role for quorum sensing in membrane modeling. Altogether, this study provides the first global analysis of AI-2 signaling in Y. pestis and identifies potential roles for the system in controlling genes important to disease.

Transcriptome analysis of acyl-homoserine lactone-based quorum sensing regulation in Yersinia pestis [corrected].

The etiologic agent of bubonic plague, Yersinia pestis, senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen Y. pseudotuberculosis uses quorum sensing system to regulate motility, the role of quorum sensing in Y. pestis has been unclear. In this study we performed transcriptional profiling experiments to identify Y. pestis quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.

Loop-mediated isothermal amplification for rapid and convenient detection of Mycoplasma hyopneumoniae.

Loop-mediated isothermal amplification (LAMP), a novel method of gene amplification, was employed in this study for detecting Mycoplasma hyopneumoniae in the respiratory tract or lungs of swine. The pathogen can be detected in LAMP reactions containing as few as 10 fg purified target DNA (10 copies of M. hyopneumoniae genome) within 30 min, which was comparable to real-time PCR. After 30-min reaction at 63 °C, the addition of a certain amount of dye (SYBR Green I and hydroxyl naphthol blue at a proper ratio) into the LAMP reaction system makes the results easily determined as positive or negative by visual inspection. In addition, the LAMP was able to distinguish between M. hyopneumoniae and other closely-related mycoplasma strains, indicating a high degree of specificity. The LAMP assay was more simple and cheap, since the reaction could be completed under isothermal conditions and less laboratorial infrastructure are required. And, it was proven reliable for M. hyopneumoniae diagnosis of nasal swab and lung samples from the field.

Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

UNLABELLED: Mycoplasma hyopneumoniae causes 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 P50(P146), P40(P146), and P85(P146) that 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 sequence (672)ATEF↓QQ(677), 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 F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain 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 of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. IMPORTANCE: Vaccines used to control Mycoplasma hyopneumoniae infection 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 of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F↓X-D/E-like sites generating P50(P146), P40(P146), and P85(P146). 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 of M. hyopneumoniae.

Mycoplasma hyopneumoniae Surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs.

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.

Sequence TTKF ↓ QE defines the site of proteolytic cleavage in Mhp683 protein, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae.

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 pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.

Identification of biologically significant genes from combinatorial microarray data.

High-throughput microarray technology has enabled the simultaneous measurement of the abundance of tens of thousands of gene-expression levels, opening up a new variety of opportunities in both basic and applied biological research. In the wealth of genomic data produced so far, the analysis of massive volume of data sets has become a challenging part of this innovative approach. In this study, a series of microarray experimental data from Yersinia pestis (Y. pestis), the etiologic agent of plague in humans, were analyzed to investigate the effect of the treatments with quorum-sensing signal molecules (autoinducer-2 and acyl-homoserine lactones) and mutation (ΔypeIR, ΔyspIR, and ΔluxS) on the variation of gene-expression levels. The gene-expression data have been systematically analyzed to find potentially important genes for vaccine development by means of a coordinated use of statistical learning algorithms, that is, principal component analysis (PCA), gene shaving (GS), and self-organizing map (SOM). The results achieved from the respective methods, the lists of genes identified as differentially expressed, were combined to minimize the risk that might arise when using a single method. The commonly detected genes from multiple data mining methods, which reflect the linear/nonlinear dimensionality and similarity measure in gene-expression space, were taken into account as the most significant group. In conclusion, tens of potentially biologically significant genes were identified out of over 4000 genes of Y. pestis. The "active" genes discovered in this study will provide information on bacterial genetic targets important for the development of novel vaccines.