A serum NMR metabolomic analysis of the Corynebacterium pseudotuberculosis infection in goats.

Caseous lymphadenitis (CLA), an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants, is highly prevalent worldwide. Economic losses have already been associated with the disease, and little is known about the host-pathogen relationship associated with the disease. The present study aimed to perform a metabolomic study of the C. pseudotuberculosis infection in goats. Serum samples were collected from a herd of 173 goats. The animals were classified as controls (not infected), asymptomatic (seropositives but without detectable CLA clinical signs), and symptomatic (seropositive animals presenting CLA lesions), according to microbiological isolation and immunodiagnosis. The serum samples were analyzed using nuclear magnetic resonance (1H-NMR), nuclear Overhauser effect spectroscopy (NOESY), and Carr-Purcell-Meiboom-Gill (CPMG) sequences. The NMR data were analyzed using chemometrics, and principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed to discover specific biomarkers responsible for discrimination between the groups. A high dissemination of the infection by C. pseudotuberculosis was observed, being 74.57% asymptomatic and 11.56% symptomatic. In the evaluation of 62 serum samples by NMR, the techniques were satisfactory in the discrimination of the groups, being also complementary and mutually confirming, demonstrating possible biomarkers for the infection by the bacterium. Twenty metabolites of interest were identified by NOESY and 29 by CPMG, such as tryptophan, polyunsaturated fatty acids, formic acid, NAD+, and 3-hydroxybutyrate, opening promising possibilities for the use of these results in new therapeutic, immunodiagnosis, and immunoprophylactic tools, as well as for studies of the immune response against C. pseudotuberculosis. KEY POINTS: • Sixty-two samples from healthy, CLA asymptomatic, and symptomatic goats were screened • Twenty metabolites of interest were identified by NOESY and 29 by CPMG • 1H-NMR NOESY and CPMG were complementary and mutually confirming.

Transcriptome profile of Corynebacterium pseudotuberculosis in response to iron limitation.

BACKGROUND: Iron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches. RESULTS: Transcriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction. CONCLUSION: Based on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.

Prediction of new vaccine targets in the core genome of Corynebacterium pseudotuberculosis through omics approaches and reverse vaccinology.

Corynebacterium pseudotuberculosis is the etiologic agent of veterinary relevance diseases, such as caseous lymphadenitis, affecting different animal species causing damage to the global agribusiness. So far, there are no completely effective treatment methods to overcome the impacts caused by this pathogen. Several genomes of the species are deposited on public databases, allowing the execution of studies related to the pan-genomic approach. In this study, we used an integrated in silico workflow to prospect novel putative targets using the core genome, a set of shared genes among 65 C. pseudotuberculosis strains. Subsequently, through RNA-Seq data of the same abiotic stresses in two strains, we selected only induced genes to compose the reverse vaccinology workflow based in two different strategies. Our results predicted six probable antigens in both analysis, which indicates that they have a strong potential to be used in further studies as vaccine targets against this bacterium.

Binding studies of a putative C. pseudotuberculosis target protein from Vitamin B12 Metabolism.

Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.

Rapidly evolving changes and gene loss associated with host switching in Corynebacterium pseudotuberculosis.

Phylogenomics and genome scale positive selection analyses were performed on 29 Corynebacterium pseudotuberculosis genomes that were isolated from different hosts, including representatives of the Ovis and Equi biovars. A total of 27 genes were identified as undergoing adaptive changes. An analysis of the clades within this species and these biovars, the genes specific to each branch, and the genes responding to selective pressure show clear differences, indicating that adaptation and specialization is occurring in different clades. These changes are often correlated with the isolation host but could indicate responses to some undetermined factor in the respective niches. The fact that some of these more-rapidly evolving genes have homology to known virulence factors, antimicrobial resistance genes and drug targets shows that this type of analysis could be used to identify novel targets, and that these could be used as a way to control this pathogen.

Inhibition of thioredoxin A1 from Corynebacterium pseudotuberculosis by polyanions and flavonoids.

In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens. The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.

Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

OBJECTIVE: Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. RESULTS: Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

Structure and interaction of Corynebacterium pseudotuberculosis cold shock protein A with Y-box single-stranded DNA fragment.

Cold shock proteins (Csps) function to preserve cell viability at low temperatures by binding to nucleic acids and consequently control gene expression. The mesophilic bacterium Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis in animals, and infection in livestock is a considerable economic burden worldwide. In this report, the structure of cold shock protein A from Cp (Cp-CspA) and biochemical analysis of its temperature-dependent interaction with a Y-box ssDNA motif is presented. The Cp-CspA structure contains five ß-strands making up a ß-barrel fold with 11 hydrophobic core residues and two salt bridges that confers it with a melting temperature of ~ 54 °C that is similar to mesophilic Bs-CspB. Chemical shift perturbations analysis revealed that residues in the nucleic acid-binding motifs (RNP 1 and 2) and loop 3 are involved in binding to the Y-box fragment either by direct interaction or by conformational rearrangements remote from the binding region. Fluorescence quenching experiments of Cp-CspA showed that the dissociation constants for Y-box ssDNA binding is nanomolar and the binding affinity decreased as the temperature increased, indicating that the interaction is enthalpically driven and the hydrogen bonds and van der Waals forces are important contributions for complex stabilization. The Y31 of Cp-CspA is a particular occurrence among Csps from mesophilic bacteria that provide a possible explanation for the higher binding affinity to ssDNA than that observed for Bs-CspB. Anisotropy measurements indicated that the reduction in molecular mobility of Cp-CspA upon Y-box binding is characterized by a cooperative process. DATABASE: Resonance assignment and structural data are available in the Biological Magnetic Resonance Data Bank and Protein Data Bank under accession number 26802 and 5O6F, respectively.

A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein-protein interaction networks.

BACKGROUND: Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. RESULTS: Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. CONCLUSIONS: The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

Humoral and cellular immune responses in mice against secreted and somatic antigens from a Corynebacterium pseudotuberculosis attenuated strain: Immune response against a C. pseudotuberculosis strain.

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CL), a chronic disease that affects goats and sheep. CL is characterized by the formation of granulomas in lymph nodes and other organs, such as the lungs and liver. Current knowledge of CL pathogenesis indicates that the induction of humoral and cellular immune responses are fundamental to disease control. The aim of this study was to evaluate the humoral and cellular immune responses in BALB/c mice inoculated with a C. pseudotuberculosis strain isolated in the state of Bahia, Brazil. RESULTS: The lymphocyte proliferation and in vitro production of IFN-γ, IL-4, IL-10, IL-12 and nitric oxide by spleen cells stimulated with secreted and somatic antigens from the studied strain were evaluated. IgG subclasses were also analyzed. Results showed a significant increase of Th1-profile cytokines after 60 days post-inoculation, as well as an important humoral response, represented by high levels of IgG2a and IgG1 against C. pseudotuberculosis. CONCLUSION: The T1 strain of C. pseudotuberculosis was shown to induce humoral and cellular immune responses in BALB/c mice, but, even at a dosage of 1x10(7) CFU, no signs of the disease were observed.

Putative virulence factors of Corynebacterium pseudotuberculosis FRC41: vaccine potential and protein expression.

BACKGROUND: Corynebacterium pseudotuberculosis, a facultative intracellular bacterial pathogen, is the etiological agent of caseous lymphadenitis (CLA), an infectious disease that affects sheep and goats and it is responsible for significant economic losses. The disease is characterized mainly by bacteria-induced caseous necrosis in lymphatic glands. New vaccines are needed for reliable control and management of CLA. Thus, the putative virulence factors SpaC, SodC, NanH, and PknG from C. pseudotuberculosis FRC41 may represent new target proteins for vaccine development and pathogenicity studies. RESULTS: SpaC, PknG and NanH presented better vaccine potential than SodC after in silico analyses. A total of 136 B and T cell epitopes were predicted from the four putative virulence factors. A cluster analysis was performed to evaluate the redundancy degree among the sequences of the predicted epitopes; 57 clusters were formed, most of them (34) were single clusters. Two clusters from PknG and one from SpaC grouped epitopes for B and T-cell (MHC I and II). These epitopes can thus potentially stimulate a complete immune response (humoral and cellular) against C. pseudotuberculosis. Several other clusters, including two from NanH, grouped B-cell epitopes with either MHC I or II epitopes. The four target proteins were expressed in Escherichia coli. A purification protocol was developed for PknG expression. CONCLUSIONS: In silico analyses show that the putative virulence factors SpaC, PknG and NanH present good potential for CLA vaccine development. Target proteins were successfully expressed in E. coli. A protocol for PknG purification is described.

Differential Exoproteome analysis of two corynebacterium pseudotuberculosis biovar ovis strains isolated from goat (1002) and sheep (C231).

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.

Ion Torrent-based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high-performance liquid chromatography a promising rRNA depletion method.

Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.

The core stimulon of Corynebacterium pseudotuberculosis strain 1002 identified using ab initio methodologies.

Corynebacterium pseudotuberculosis is a bacterium which causes diseases such as caseous lymphadenitis in small ruminants, resulting in large-scale economic losses for agribusiness worldwide. Consequently, this bacterium including its transcriptional profile analysis has been the focus of various studies. Identification of the transcripts that appear under conditions that simulate the environment encountered by this bacterial species in the host is of great importance in discovering new targets for the production of more efficient vaccines. We sequenced the cDNA of Corynebacterium pseudotuberculosis strain 1002, using the SOLiD V3 system, under the following conditions: osmotic stress (2 M), acidity (pH), heat shock (50 °C) and control condition (N). To identify the transcripts shared among the stimulons and integrate this information with the results from BLAST and BLAST2GO, we developed the software CoreStImulon (CSI) which allows the user to individually distinguish the genes in terms of their participation in biological processes, their function and cellular location. In the biosynthetic processes, eleven genes represented in the core stimulon and twenty genes in the control were observed. This validates the hypothesis that the organisms strategy for surviving in a hostile environment is through growth reduction. The oxidation reduction process, response to stress process, and cell adhesion are controlled by genes that contribute to bacterial cell maintenance under stress conditions; these could be involved in their pathogenicity. The methodology for identification of transcripts obtained by ab initio assembly and shared among the stimulons permitted candidates selection for vaccine studies. CSI is available at https://sourceforge.net/projects/corestimulon/.

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.

BACKGROUND: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. RESULTS: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. CONCLUSIONS: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

In vivo insertional mutagenesis in Corynebacterium pseudotuberculosis: an efficient means to identify DNA sequences encoding exported proteins.

The reporter transposon-based system TnFuZ was used to identify exported proteins of the animal pathogen Corynebacterium pseudotuberculosis. Thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity. This activity was from 21 C. pseudotuberculosis loci that code for fimbrial and transport subunits and for hypothetical and unknown-function proteins.

Chromatographic and mass spectrometric characterization of 3-O-benzoyl methyl ester derivatives of mycolic acid fractions from Corynebacterium pseudotuberculosis, C. diphtheriae and Rhodococcus rhodochrous.

A benzoyl group was attached to the 3-hydroxyl group of the methyl ester derivative of corynomycolic acid fraction isolated from Corynebacterium pseudotuberculosis. The infrared spectrum of the 3-O-benzoylated compound displayed a series of characteristic absorptions found at 1110, 1267 and 1603 cm-1 that confirmed the presence of a monosubstituted phenyl grouping. The 1H-NMR spectrum showed peaks representing protons of the aromatic ring at 7.4 ppm and 8.0 ppm. The UV spectrum revealed two absorption maxima: at 190 and 228 nm. The mass spectrum of the 3-O-benzoylated material exhibited the following peaks: (1) a prominent peak at m/z 105 of the benzoyl group that constituted the base peak; (2) peaks of methyl esters representing the alpha-hydrocarbon side chain plus carbon atoms C1 and C2 of the mycolic acid molecule; and (3) peaks of molecular ion minus benzoic acid and/or molecular ion minus benzoxyl group. When subjected to liquid chromatography (LC) on an octadecylsilane-silica gel column the 3-O-benzoylated methyl ester derivatives of the corynomycolic acid fraction were separated into their constituent homologous fractions corresponding to underivatized corynomycolic acids with the chain length C30, C32 and C34. Reversed phase HPLC of saturated and monounsaturated species of 3-O-benzoylated derivatives of the mycolic acid fraction from C. diphtheriae and Rhodococcus rhodochrous led to the separation of the corresponding homologous fractions. Mass spectrometry by electron impact mode identified both series of the homologous materials differing in mass by 28 units.