Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation.

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo­nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SIGNIFICANCE: In this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

Ribonucleotide reductase as a therapeutic target for drug repurposing as anthelmintics.

Visceral cestodiases, like echinococcoses and cysticercoses, are zoonoses of worldwide distribution and are responsible for public health problems in many countries, especially in underdeveloped regions. Current treatments have low efficiency and there are few drugs currently in use for chemotherapy, making the development of new anthelmintics an urgent matter. The nucleotide salvage pathways are the only ones available for nucleotide synthesis in cestodes and other parasitic helminths, and, here, we used in silico approaches to assess the potential of the enzymes in these pathways as targets for drug repurposing as anthelminthics. First, a genomic survey allowed to identify a repertoire of 28 enzymes of the purine and pyrimidine salvage pathways from the cestode Echinococcus granulosus sensu stricto. Regarding purines, the parasite relies on salvaging free bases rather than salvaging nucleosides. Pyrimidines, on the other hand, can be salvaged from both bases and nucleosides. Druggability of the parasite enzymes was assessed, as well as the availability of commercial inhibitors for them. Druggable enzymes were then ranked according to their potential for drug repurposing and the 17 most promising enzymes were selected for evolutionary analyses. The constructed phylogenetic trees allowed to assess the degree of conservation among ortholog enzymes from parasitic helminths and their mammalian hosts. Positive selection is absent in all assessed flatworm enzymes. A potential target enzyme for drug repurposing, ribonucleotide reductase (RNR), was selected for further assessment. RNR 3D-modelling showed structural similarities between the E. granulosus and the human orthologs suggesting that inhibitors of the human RNR should be effective against the E. granulosus enzyme. In line with that, E. granulosus protoscolices treated in vitro with the inhibitor hydroxyurea had their viability and DNA synthesis reduced. These results are consistent with nucleotide synthesis inhibition and confirm the potential of a nucleotide salvage inhibitors for repurposing as an anthelmintic.

Differential domains and endoproteolytic processing in dominant surface proteins of unknown function from Mycoplasma hyopneumoniae and Mycoplasma flocculare.

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia (PEP), a chronic respiratory disease that leads to severe economic losses in the pig industry. Swine infection and PEP development depend on the adhesion of the pathogen to the swine respiratory tract and the host immune response, but these and other disease determinants are not fully understood. For instance, M. hyopneumoniae has a large repertoire of proteins of unknown function (PUFs) and some of them are abundant in the cell surface, where they likely mediate so far unknown pathogen-host interactions. Moreover, these surface PUFs may undergo endoproteolytic processing to generate larger repertoires of proteoforms to further complicate this scenario. Here, we investigated the five PUFs more represented on the surface of M. hyopneumoniae pathogenic strain 7448 in comparison with their orthologs from the nonpathogenic M. hyopneumoniae J strain and the closely related commensal species Mycoplasma flocculare. Comparative in silico analyses of deduced amino acid sequences and proteomic data identified differential domains, disordered regions and repeated motifs. We also provide evidence of differential endoproteolytic processing and antigenicity. Phylogenetic analyses were also performed with ortholog sequences, showing higher conservation of three of the assessed PUFs among Mycoplasma species related to respiratory diseases. Overall, our data point out to M. hyopneumoniae surface-dominant PUFs likely associated with pathogenicity.

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.

Pathogenicity & virulence of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae: is the etiological agent of porcine enzootic pneumonia (EP), a disease that impacts the swine industry worldwide. Pathogen-induced damage, as well as the elicited host-response, contribute to disease. Here, we provide an overview of EP epidemiology, control and prevention, and a more in-depth review of M. hyopneumoniae pathogenicity determinants, highlighting some molecular mechanisms of pathogen-host interactions relevant for pathogenesis. Based on recent functional, immunological, and comparative "omics" results, we discuss the roles of many known or putative M. hyopneumoniae virulence factors, along with host molecules involved in EP. Moreover, the known molecular bases of pathogenicity mechanisms, including M. hyopneumoniae adhesion to host respiratory epithelium, protein secretion, cell damage, host microbicidal response and its modulation, and maintenance of M. hyopneumoniae homeostasis during infection are described. Recent findings regarding M. hyopneumoniae pathogenicity determinants also contribute to the development of novel diagnostic tests, vaccines, and treatments for EP.

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.

Differential responses to stress of two Mycoplasma hyopneumoniae strains.

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40-60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.

Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP.