Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection.

The unfolded protein response (UPR) plays a crucial role in Mycoplasma hyopneumoniae (M. hyopneumoniae) pathogenesis. We previously demonstrated that M. hyopneumoniae interferes with the host UPR to foster bacterial adhesion and infection. However, the underlying molecular mechanism of this UPR modulation is unclear. Here, we report that M. hyopneumoniae membrane protein Mhp271 interacts with host GRP78, a master regulator of UPR localized to the porcine tracheal epithelial cells (PTECs) surface. The interaction of Mhp271 with GRP78 reduces the porcine beta-defensin 2 (PBD-2) production, thereby facilitating M. hyopneumoniae adherence and infection. Furthermore, the R1-2 repeat region of Mhp271 is crucial for GRP78 binding and the regulation of PBD-2 expression. Intriguingly, a coimmunoprecipitation (Co-IP) assay and molecular docking prediction indicated that the ATP, rather than the substrate-binding domain of GRP78, is targeted by Mhp271 R1-2. Overall, our findings identify host GRP78 as a target for M. hyopneumoniae Mhp271 modulating the host UPR to facilitate M. hyopneumoniae adherence and infection.

Reannotation of Mycoplasma hyopneumoniae hypothetical proteins revealed novel potential virulence factors.

Mycoplasma hyopneumoniae is a bacterium that inhabits the swine respiratory tract, causing porcine enzootic pneumonia, which generates significant economic losses to the swine industry worldwide. The knowledge on M. hyopneumoniae biology and virulence have been significantly increased by genomics studies. However, around 30% of the predicted proteins remained of unknown function so far. According to the original annotation, the genome of M. hyopneumoniae 7448, a Brazilian pathogenic strain, had 693 coding DNA sequences, 244 of which were annotated as coding for hypothetical or uncharacterized proteins. Among them, there may be still several genes coding for unknown virulence factors. Therefore, this study aimed to functionally reannotate the whole set of 244 M. hyopneumoniae 7448 proteins of unknown function based on currently available database and bioinformatic tools, in order to predict novel potential virulence factors. Predictions of physicochemical properties, subcellular localization, function, overall association to virulence and antigenicity are provided. With that, 159 out of the set of 244 proteins of unknown function had a putative function associated to them, allowing identification of novel enzymes, membrane transporters, lipoproteins, DNA-binding proteins and adhesins. Furthermore, 139 proteins were generally associated to virulence, 14 of which had a function assigned and were differentially expressed between pathogenic and non-pathogenic strains of M. hyopneumoniae. Moreover, all extracellular or cytoplasmic membrane predicted proteins had putative epitopes identified. Overall, these analyses improved the functional annotation of M. hyopneumoniae 7448 genome from 65% to 87% and allowed the identification of new potential virulence factors.

Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence.

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson's index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

Influence of Mycoplasma hyopneumoniae natural infection on the respiratory microbiome diversity of finishing pigs.

Mycoplasma (M.) hyopneumoniae interacts with the respiratory microbiota and facilitates colonization of other pathogens. The present study investigated the pulmonary and nasal microbiota of M. hyopneumoniae-infected and M. hyopneumoniae-free pigs. Sixty-six pigs from three commercial herds were selected at the end of the finishing phase: 44 originated from two M. hyopneumoniae-positive herds and 22 from a M. hyopneumoniae-negative farm. At the slaughterhouse, samples of nasal turbinate (NT) and bronchus-alveolar lavage fluid (BALF) were collected. DNA was extracted with a commercial kit and the infection status was confirmed by qPCR. All samples from the same herd were pooled, and next-generation sequencing based on the hypervariable region V3-V4 of the 16 s bacterial rDNA was performed. Data analysis included the taxonomic analysis, Alpha diversity indexes, and Principal coordinates analysis (Pcoa) using Jaccard, Bray-Curtis, Weighted Unifrac, and Unweighted Unifrac distances. All pigs from the infected herds tested PCR positive for M. hyopneumoniae, whereas all pigs from the negative farm were negative. There was a greater diversity of microorganisms in BALF when compared to NT samples in all the farms. BALF samples from infected animals showed higher abundance of M. hyopneumoniae than NT samples and a predominance of Pasteurella multocida among the main species identified, which was also abundant in the M. hyopneumoniae-free herd. PCoa diagrams indicated that for most of the samples, dissimilarity on bacterial composition was observed, regardless of infection status and sample type. Therefore, the lung microbiota was modulated by M. hyopneumoniae infection, which could play a role in the pathogenesis of M. hyopneumoniae-disease.

The role of co-infections in M. hyopneumoniae outbreaks among heavy fattening pigs: a field study.

Little is known about how co-infections and genotype dynamics affect Mycoplasma hyopneumoniae infection in fattening pigs. This study was aimed at assessing the role of co-infections in M. hyopneumoniae outbreaks, their influence on the presence of M. hyopneumoniae genotypes and their impact on consequent lung lesions. Tracheobronchial swabs (TBS) from 300 finishers were collected from 10 farms at the onset of enzootic pneumonia outbreaks and 1 month later, sampling of 3 groups per farm: Group A showed clinical signs first, Group B was housed near Group A, and Group C was located in a different building. Pigs' lungs were scored at the slaughterhouse. TBS were tested for the main pathogens involved in respiratory diseases, and samples positive for M. hyopneumoniae were genotyped by multiple-locus variable-number tandem repeat analysis (MLVA). Pigs in Group A showed the highest prevalence and load of M. hyopneumoniae. A positive association was detected between M. hyopneumoniae and Mycoplasma hyorhinis, whereas Actinobacillus pleuropneumoniae was more frequent when the M. hyopneumoniae load was higher. Nevertheless, co-infection had no effect on lung lesion scores. The presence of multiple MLVA types (mixed infections) increased in time only in pigs from Group C and was positively associated with porcine reproductive and respiratory syndrome virus infection. Lung lesions were more severe in pigs with at least one TBS positive for M. hyopneumoniae and in pigs with a history of mixed infections. The central role of M. hyopneumoniae and relevance of mixed infections suggest that increased biosecurity might be beneficial for lung lesion sequelae.

The influence of regulatory elements on Mycoplasma hyopneumoniae 7448 transcriptional response during oxidative stress and heat shock.

BACKGROUND: The comprehension of genome organization and gene modulation is essential for understanding pathogens' infection mechanisms. Mycoplasma hyopneumoniae 7448 genome is organized in transcriptional units (TUs), which are flanked by regulatory elements such as putative promoters, terminators and repetitive sequences. Yet the relationship between the presence of these elements and bacterial responses during stress conditions remains unclear. Thus, in this study, in silico and RT-qPCR analyses were associated to determine the effect of regulatory elements in gene expression regulation upon heat shock and oxidative stress conditions. METHODS AND RESULTS: Thirteen TU's organizational profiles were found based on promoters and terminators distribution. Differential expression in genes sharing the same TUs was observed, suggesting the activity of internal regulatory elements. Moreover, 88.8% of tested genes were differentially expressed under oxidative stress in comparison to the control condition, being 81.3% of them surrounded by their own regulatory elements. Similarly, under heat shock, 44.4% of the genes showed regulation when compared to control condition, being 75.0% of them surrounded by their own regulatory elements. CONCLUSIONS: Altogether, this data suggests the activity of internal regulatory elements in gene modulation of M. hyopneumoniae 7448 transcription.

NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear. RESULTS: In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains. CONCLUSIONS: NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.

Development of a Multi-Epitope Vaccine for Mycoplasma hyopneumoniae and Evaluation of Its Immune Responses in Mice and Piglets.

Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing Mycoplasma pneumonia of swine (MPS), brings massive economic losses worldwide. Genomic variability and post-translational protein modification can enhance the immune evasion of Mhp, which makes MPS prone to recurrent outbreaks on farms, even with vaccination or other treatments. The reverse vaccinology pipeline has been developed as an attractive potential method for vaccine development due to its high efficiency and applicability. In this study, a multi-epitope vaccine for Mhp was developed, and its immune responses were evaluated in mice and piglets. Genomic core proteins of Mhp were retrieved through pan-genome analysis, and four immunodominant antigens were screened by host homologous protein removal, membrane protein screening, and virulence factor identification. One immunodominant antigen, AAV27984.1 (membrane nuclease), was expressed by E. coli and named rMhp597. For epitope prioritization, 35 B-cell-derived epitopes were identified from the four immunodominant antigens, and 10 MHC-I and 6 MHC-II binding epitopes were further identified. The MHC-I/II binding epitopes were merged and combined to produce recombinant proteins MhpMEV and MhpMEVC6His, which were used for animal immunization and structural analysis, respectively. Immunization of mice and piglets demonstrated that MhpMEV could induce humoral and cellular immune responses. The mouse serum antibodies could detect all 11 synthetic epitopes, and the piglet antiserum suppressed the nuclease activity of rMhp597. Moreover, piglet serum antibodies could also detect cultured Mhp strain 168. In summary, this study provides immunoassay results for a multi-epitope vaccine derived from the reverse vaccinology pipeline, and offers an alternative vaccine for MPS.

Genotype diversity of Mycoplasma Hyopneumoniae in Chinese swine herds based on multilocus sequence typing.

BACKGROUND: Between 2018 and 2020, 989 clinical specimens from pigs showing clinical signs of a variety of swine diseases in 27 provinces in China were sampled and submitted for further testing. Nested PCR targeting the 16S rRNA gene of Mycoplasma hyopneumoniae and subsequent sequencing were used to analyse these specimens. Mycoplasma hyopneumoniae-positive samples were assayed by multilocus sequence typing (MLST). The aim of the study was to reveal the distribution of M. hyopneumoniae and determine the genotypes of M. hyopneumoniae in pig herds in China based on MLST. RESULTS: Among these 989 samples, 199 samples were M. hyopneumoniae-positive. The M. hyopneumoniae positivity rate was 7.2% (35/494) in 2018, 18.4% (38/207) in 2019, and 43.8% (126/288) in 2020. In total, 47 samples were successfully assayed by MLST. Sixteen new M. hyopneumoniae sequence types from 9 provinces were recorded in the present study. CONCLUSIONS: This is the first report on sample positivity rates and molecular typing results for M. hyopneumoniae in swine herds in China. MLST has revealed high genotype diversity among M. hyopneumoniae from different provinces of China.

Evidences of differential endoproteolytic processing on the surfaces of Mycoplasma hyopneumoniae and Mycoplasma flocculare.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetic similar bacteria that colonize the swine respiratory tract. However, while M. hyopneumoniae is a pathogen that causes porcine enzootic pneumonia, M. flocculare is a commensal. Adhesion to the respiratory epithelium is mediated by surface-displayed adhesins, and at least some M. hyopneumoniae adhesins are post-translational proteolytically processed, producing differential proteoforms with differential adhesion properties. Based on LC-MS/MS data, we assessed differential proteolytic processing among orthologs of the five most abundant adhesins (p97 and p216) or adhesion-related surface proteins (DnaK, p46, and ABC transporter xylose-binding lipoprotein) from M. hyopneumoniae strains 7448 (pathogenic) and J (non-pathogenic), and M. flocculare. Both surface and cytoplasmic non-tryptic cleavage events were mapped and compared, and antigenicity predictions were performed for the resulting proteoforms. It was demonstrated that not only bona fide adhesins, but also adhesion-related proteins undergo proteolytical processing. Moreover, most of the detected cleavage events were differential among M. hyopneumoniae strains and M. flocculare, and also between cell surface and cytoplasm. Overall, our data provided evidences of a complex scenario of multiple antigenic proteoforms of adhesion-related proteins, that is differential among M. hyopneumoniae strains and M. flocculare, altering the surface architecture and likely contributing to virulence and pathogenicity.

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2.

BACKGROUND: Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets. RESULTS: The high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets. CONCLUSIONS: Above all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.

Hydrogen peroxide production and myo-inositol metabolism as important traits for virulence of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia. In our previous work, we reconstructed the metabolic models of this species along with two other mycoplasmas from the respiratory tract of swine: Mycoplasma hyorhinis, considered less pathogenic but which nonetheless causes disease and Mycoplasma flocculare, a commensal bacterium. We identified metabolic differences that partially explained their different levels of pathogenicity. One important trait was the production of hydrogen peroxide from the glycerol metabolism only in the pathogenic species. Another important feature was a pathway for the metabolism of myo-inositol in M. hyopneumoniae. Here, we tested these traits to understand their relation to the different levels of pathogenicity, comparing not only the species but also pathogenic and attenuated strains of M. hyopneumoniae. Regarding the myo-inositol metabolism, we show that only M. hyopneumoniae assimilated this carbohydrate and remained viable when myo-inositol was the primary energy source. Strikingly, only the two pathogenic strains of M. hyopneumoniae produced hydrogen peroxide in complex medium. We also show that this production was dependent on the presence of glycerol. Although further functional tests are needed, we present in this work two interesting metabolic traits of M. hyopneumoniae that might be directly related to its enhanced virulence.

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.

Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples.

BACKGROUND: Porcine enzootic pneumonia is a worldwide problem in swine production. The infected host demonstrates a respiratory disease whose etiologic agent is Mycoplasma hyopneumoniae (Mhp). A total of 266 lung samples with Mycoplasma-like lesions were collected from two slaughterhouses. We analyzed the genetic profile of Mhp field samples using 16 genes that encode proteins involved in the mechanisms of bacterial pathogenesis and/or the immune responses of the host. Bioinformatic analyses were performed to classify the Mhp field samples based on their similarity according to the presence of the studied genes. RESULTS: Our results showed variations in the frequency of the 16 studied genes among different Mhp field samples. It was also noted that samples from the same farm were genetically different from each other and samples from different regions could be genetically similar, which is evidence of the presence of different genetic profiles among the Mhp field strains that circulate in Brazilian swine herds. CONCLUSION: This work demonstrated the genetic diversity of several Mhp field strains based on 16 selected genes related to virulence and/or immune response in Brazil. Our findings demonstrate the difference between Mhp field strains could influence the virulence, and we hypothesize that the most frequent genes in Mhp field strains could possibly be used as vaccine candidates. Based on our results, we suspect that Mhp genetic variability may be associated with the frequency of genes among the field strains and we have demonstrated that some Mhp field samples could not have many important genes described in the literature.

Surface proteins mhp390 (P68) contributes to cilium adherence and mediates inflammation and apoptosis in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia (EP) and responsible for major economic losses in global swine industry. After colonization of the respiratory epithelium, M. hyopneumoniae elicits a general mucociliary clearance loss, prolonged inflammatory response, host immunosuppression and secondary infections. Until now, the pathogenesis of M. hyopneumoniae is not completely elucidated. This present study explores the pathogenicity of mhp390 (P68, a membrane-associated lipoprotein) by elucidating its multiple functions. Microtitrer plate adherence assay demonstrated that mhp390 is a new cilia adhesin that plays an important role in binding to swine tracheal cilia. Notably, mhp390 could induce significant apoptosis of lymphocytes and monocytes from peripheral blood mononuclear cells (PBMCs), as well as primary alveolar macrophages (PAMs), which might weaken the host immune response. In addition, mhp390 contributes to the production of proinflammatory cytokines, at least partially, via the release of IL-1ß and TNF-α. To the best of our knowledge, this is the first report of the multiple functions of M. hyopneumoniae mhp390, which may supplement known virulence genes and further develop our understanding of the pathogenicity of M. hyopneumoniae.

Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity.

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.

Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma Hyopneumoniae by a Baculovirus Expression System.

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province.

In Argentina, enzootic pneumonia (EP) is highly prevalent and different genetic types of Mycoplasma hyopneumoniae have been identified. However, there is a lack of information about prevalence and other epidemiological aspects of EP in Mendoza province. A multiple Locus variable-number tandem repeat analysis (MLVA) targeting P97 R1, P97 R1A and P146 R3 loci was used to assess the genetic diversity of M. hyopneumoniae from clinical specimens recovered from pigs from five herds located in different districts of Mendoza province. M. hyopneumoniae could be typed from 27 bronchoalveolar lavages (BAL) specimens, and eight different MLVA types were identified. This is the first report about diversity of M. hyopneumoniae in Mendoza. Results obtained in this work allow drawing a better picture of the genetic diversity of this pathogen in Argentina.

Global analysis of sRNA target genes in Mycoplasma hyopneumoniae.

BACKGROUND: Small RNAs (sRNAs) are noncoding molecules that regulate different cellular activities in several bacteria. The role of sRNAs in gene expression regulation is poorly characterized in the etiological agent of porcine enzootic pneumonia Mycoplasma hyopneumoniae. We performed a global analysis of the sRNAs, sRNA target genes and regulatory elements previously identified in their genome and analyzed the expression of some sRNAs and their target genes by quantitative RT-PCR (qPCR) in three different culture conditions. RESULTS: Seven of the 145 sRNA target genes are organized as monocistronic genes (mCs) while the other 138 sRNA target genes are organized into transcriptional units (TU). The identification of transcriptional regulatory elements (promoter motif, DNA repeat sequence or intrinsic terminator) was verified in 116 of the 145 sRNA target genes. Moreover, the 29 sRNA target genes without regulatory elements revealed the presence of at least one regulatory element in the boundaries of the TU or in other internal genes of the TU. We verified that 16 sRNAs showed differential expression, seven in heat shock condition and 14 in oxidative stress condition. Analysis of the differential expression of the sRNA target genes showed that the tested sRNAs possibly regulate gene expression. The sRNA target genes were up- or down-regulated possibly in response to sRNA only under oxidative stress condition. Moreover, the sRNA target genes are involved in diverse processes of the cell, some of which could be linked to transcription processes and cell homeostasis. CONCLUSION: Our results indicate that bacterial sRNAs could regulate a number of targets with various outcomes, and different correlations between the levels of sRNA transcripts and their target gene mRNAs were found, which suggest that the regulation of gene expression via sRNAs may play an important role in mycoplasma.

Utilization of macrophage extracellular trap nucleotides by Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.