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.

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.

Heat shock stress: Profile of differential expression in Corynebacterium pseudotuberculosis biovar Equi.

Transcriptome studies on Corynebacterium pseudotuberculosis have recently contributed to the understanding about this microorganism's survival mechanisms in various hostile conditions. The gene expression profile of the C. pseudotuberculosis strain 1002 (Ovis biovar), has revealed genes that are possible candidates responsible for its maintenance in adverse environments, such as those found in the host. In another strain of this bacterium, 258 (Equi biovar), a high temperature condition was simulated, in order to verify which genes are responsible for promoting the persistence of the bacterium in these conditions, since it tolerates temperatures higher than 40°C, despite being a mesophilic bacterium. It was possible to generate a list of genes using RNAseq technology that possibly contribute to the survival of the bacteria in this hostile environment. A total of 562 genes were considered as differentially expressed, then, after the fold-change cutoff, 113 were considered induced and 114 repressed, resulting in a total of 227 genes. Therefore, hypothetical proteins presented a fold change above 6, and genes characteristically in control for this type of stress, such as hspR, grpE, and dnaK, presented a fold change above 3. The clpB gene, a chaperone, drew attention due to presenting a fold change above 3 and located in a pathogenicity island. These genes may contribute towards efficient solutions to the effects caused by ulcerative lymphangitis in equines, thus attenuating the damage it causes to agribusiness.

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host.

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.

[Effect of Corynebacterium non diphtheriae on functional activity and apoptosis of macrophages].

AIM: Determine the ability of Corynebacterium non diphtheriae to induce phagocytosis and apoptosis of macrophages and evaluate regulatory effect of nuetrophilokines (NPK) induced by Corynebacterium non diphtheriae on these processes. MATERIALS AND METHODS: The ability of Corynebacterium non diphtheriae, isolated from upper respiratory tract, skin and urogenital tract (UGT) were studied for the ability to induce phagocytosis and apoptosis of mice macrophages (MP; in vitro during staining by May-Grunwald with additional staining by Romanowsky-Giemsa) before and after the addition of NPK induced by Corynebacterium non diphtheriae. RESULTS: Phagocytic index (PI) was the same for all the Corynebacterium non diphtheriae species, phagocytic number (PN) and index of phagocytosis completion (IPC)--were minimal relative to corynebacteria isolated from UGT. All the studied corynebacteria species induced MP apoptosis; the most pronounced apoptogenic effect was detected in Corynebacterium pseudotuberculosis isolated from UGT. NPK increased PN against corynebacteria isolated from the studied biotopes, IPC--only during studies of corynebacteria isolated from skin. The effect of NPK resulted in a reduction of apoptogenic effect for almost all the Corynebacterium non diphtheriae, regardless of the isolation location. CONCLUSION: A pronounced apoptogenic effect and insufficiency of phagocytosis processes induced by corynebacteria are the means of realization of Corynebacterium non diphtheriae pathogenic effect. NPK use is possible for immune correction of immune deficiency conditions developing against the background of diseases determined by Corynebacterium non diphtheriae.

Multiple abscesses caused by Salmonella enterica and Corynebacterium pseudotuberculosis in a dromedary camel.

A rare case of arthritis, peri-arthiritis and pleurits associated with Salmonella enterica and Corynebacterium pseudotuberculosis infection in a dromedary camel is reported. Articular infections caused by Non-typhoidal Salmonella have been exceptionally described in human medicine. To our knowledge, this would be the first description of articular infections associated with Non-thyphoidal Salmonella in other mammals than humans. Possible pathogenesis of the infection is discussed.

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.

Clinical, humoral, and pathologic findings in adult alpacas with experimentally induced Corynebacterium pseudotuberculosis infection.

OBJECTIVE: To experimentally infect adult alpacas by ID inoculation of Corynebacterium pseudotuberculosis, follow the clinical and pathologic course of disease, and study the humoral response to infection. ANIMALS: 13 adult alpacas. PROCEDURES: 9 alpacas were inoculated with 1.1 X 10(6) CFUs of C pseudotuberculosis from llama (n = 4) or alpaca (5) origin, and 4 alpacas were sham inoculated as controls. Alpacas were clinically observed after inoculation and euthanatized on days 16, 58, 93, or 128 after inoculation; necropsy examination and histologic evaluation were performed. An indirect ELISA, which made use of the C pseudotuberculosis cell wall as the antigen, was used to measure antibody titers in serum samples. RESULTS: Alpacas had a persistent febrile response, a local severe inflammatory response, and leucocytosis (> 30 X 10(3) WBCs/microL). Internal abscesses that localized mainly in the renal lymph node were observed. Corynebacterium pseudotuberculosis was recovered from the inoculation site 1 week after inoculation and from internal abscesses at 58 days after inoculation. Initial lesions were typical pyogranulomas with central caseous necrosis, whereas later lesions consisted of connective tissue, mononuclear cells, abundant neutrophils, and liquefactive necrosis. Infected alpacas had detectable serum antibody titers starting on day 16 that persisted until day 93 after inoculation. Shaminoculated alpacas did not develop serum antibody titers, clinical signs of infection, or lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Alpacas inoculated with C pseudotuberculosis developed abscesses at the inoculation site and internally in the renal lymph nodes, without lung lesions. Corynebacterium pseudotuberculosis isolates from llama and alpaca origin were found to be pathogenically indistinct.

An improved protocol for electrotransformation of Corynebacterium pseudotuberculosis.

We developed an improved protocol for the electrotransformation of Corynebacterium pseudotuberculosis, testing variations of parameters in the procedures that are routinely used for the preparation of electrocompetent cells of this species, including (i) culture conditions, (ii) cell growth phase, (iii) electroporation solutions and (iv) quantity of plasmid DNA. We obtained the greatest efficiency of transformation when the cells were grown until the stationary phase and then washed with 10% glycerol electroporation solution. The transformation efficiency was inversely proportional to the quantity of plasmid DNA. The transformation efficiency reached 10(5) colony-forming units (cfu)/mug plasmid DNA. This protocol would be useful for genetic studies of C. pseudotuberculosis.

Comparison of an interferon-gamma to a phospholipase D enzyme-linked immunosorbent assay for diagnosis of Corynebacterium pseudotuberculosis infection in experimentally infected goats.

The optimal method of control of caseous lymphadenitis of goats caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. The objective of this study was to compare detection of C. pseudotuberculosis experimentally infected goats using a commercially available bovine interferon-gamma (IFN-gamma) whole blood enzyme-linked immunosorbent assay (ELISA) to serological response to a recombinant phospholipase D (PLD) ELISA. The tests were assessed repeatedly over 1 year in three infected and three non-infected goats. Using a IFN-gamma optical density cut-off at 0.10 as positive under the conditions used, the test accurately detected C. pseudotuberculosis experimentally infected goats over a 363 day period with a reliability of 89.2% and non-infected goats with a reliability of 97.1%. Using a cut-off value of the mean for negative samples plus two standard deviations, the PLD ELISA detected C. pseudotuberculosis experimentally infected goats over this period with a reliability of 81.0% and non-infected goats with a reliability of 97.0%. The PLD ELISA was however more predictive than the IFN-gamma ELISA of the presence of lesions observed at postmortem examination of infected goats.

An interferon-gamma assay for diagnosis of Corynebacterium pseudotuberculosis infection in adult sheep from a research flock.

The optimal method of control of caseous lymphadenitis of sheep caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. Current serological approaches to identification of infected sheep are generally hampered by low sensitivity and specificity of available tests. The objective of this study was to develop a whole blood assay for detection of C. pseudotuberculosis-infected sheep, based on detection of IFN-gamma response to whole cell C. pseudotuberculosis antigens, and to determine the reliability of the assay. A commercially available bovine interferon-gamma assay enzyme-linked immunosorbent assay was used and the test optimised using experimentally infected sheep. The assay was also tested on known CLA-negative sheep. Setting a IFN-gamma optical density cut-off at 0.100 as positive under the conditions used, the test detected C. pseudotuberculosis experimentally infected sheep over a 450-day period with a reliability of 95.7%. It identified known non-infected sheep with a reliability of 95.5%. Repeated vaccination of three uninfected sheep with a commercially available bacterin-toxoid vaccine did not interfere with the assay. The IFN-gamma response of sheep whole blood to C. pseudotuberculosis antigens offers promise for use in a test-and-removal approach to eradication of CLA in sheep.

Identification and role in virulence of putative iron acquisition genes from Corynebacterium pseudotuberculosis.

Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene. beta-Galactosidase assays on a C. pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media. However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro. Although no defect in iron utilization could be determined for a C. pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis. Thus, expression of the fag genes in the host appears to contribute to virulence.

Caseous lymphadenitis in goats: the pathogenesis, incubation period and serological response after experimental infection.

Twenty goats, in two groups of 10, were injected intradermally with Corynebacterium pseudotuberculosis. The doses of infection were 1 x 10(5) and 5 x 10(4) colony-forming units (cfu) for groups 1 and 2, respectively. Thereafter, a goat from each group was killed every 2-3 days and examined for gross and microscopic caseous lesions in the draining lymph nodes. Bands or zones of macrophages and polymorphonuclear granulocytes were observed microscopically on the second day of infection in both groups. Gross caseous lesions were observed from days 8 and 9 of infection, respectively. Positive bacterial agglutination test and haemolysis inhibition test titres were detected after 15-17 days and 20-25 days of infection, respectively. These results indicated that caseous lymphadenitis is a subacute disease with an incubation period of 8-9 days, but that it is not detectable serologically until after 15 days of infection.

Efficacy of an ovine caseous lymphadenitis vaccine formulated using a genetically inactive form of the Corynebacterium pseudotuberculosis phospholipase D.

Caseous lymphadenitis (CLA) is an economically significant disease of sheep caused by the gram-positive bacterium Corynebacterium pseudotuberculosis. CLA vaccines are currently formulated using formalin inactivated culture supernatants that are rich in the C. pseudotuberculosis phospholipase D (PLD) exotoxin. One alternative to chemical detoxification is to inactivate the PLD genetically. This procedure not only provides a means to remove an onerous chemical treatment step but also the opportunity to increase gene expression, therefore improve protein yields. Using site-specific mutagenesis the C. pseudotuberculosis PLD was inactivated by substituting a serine residue at histidine 20 within the enzyme active site. CLA vaccine formulated using genetically inactivated PLD protected 44% of sheep against C. pseudotuberculosis challenge compared with 95% protection offered by the formalin inactivated preparation. Since there was no apparent difference in immune response mounted by vaccinated sheep the reason for this variation in vaccine efficacy remains unclear. Although genetic inactivation can be a convenient means to produce toxoid vaccines its use to develop a new CLA vaccine provided no net benefit over the conventional formulation.

Role of endogenous tumor necrosis factor alpha and gamma interferon in resistance to Corynebacterium pseudotuberculosis infection in mice.

The production and roles of endogenous tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) in the infection of Corynebacterium (C.) pseudotuberculosis were investigated in mice. The maximum levels of TNF-alpha and IFN-gamma were detected on day 4 after infection. The administration of anti-TNF-alpha monoclonal antibody (mAb) as well as anti-IFN-gamma mAb increased bacterial proliferation in the organs, leading to the death of infected mice, but anti-IFN-gamma mAb showed a less marked effect than anti-TNF-alpha mAb. The suppressive effect of anti-TNF-alpha and anti-IFN-gamma mAbs on anticorynebacterial resistance was augmented by the simultaneous administration of these antibodies. Anti-TNF-alpha mAb was found to be highly effective when administered on day 0 and day 4, suggesting that TNF-alpha produced during the early stage of infection is critical for the generation of resistance. Histologically, many microabscesses, severe follicular swelling and lymphocyte destruction were observed in mice treated with anti-TNF-alpha or anti-IFN-gamma mAb. Injection of anti-CD4 or anti-CD8 mAb also resulted in significantly increased mortality and a marked suppression of IFN-gamma production, but had no effect on TNF-alpha production. Carrageenan also showed a marked effect on the exacerbation of infection. Taken together, these results suggest that endogenously produced TNF-alpha and IFN-gamma are both essential to the host defense against C. pseudotuberculosis infection and that these cytokines may have an additive effect.

Attenuation and vaccine potential of aroQ mutants of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.

Targeted mutagenesis of the phospholipase D gene results in decreased virulence of Corynebacterium pseudotuberculosis.

The chromosomal gene encoding the phospholipase D from Corynebacterium pseudotuberculosis (biovar ovis) isolate Whetten 1 was replaced with an allele containing a nonsense mutation. The virulence of the mutant strain (W1.31r1) and the isogenic parental strain were then compared by inoculation of goats. The wild-type strain caused abscessation at the site of infection, which then spread to the regional lymph node, while W1.31r1 had a reduced ability to establish a primary infection and was incapable of dissemination. Our results confirm that phospholipase D is a virulence determinant of C. pseudotuberculosis that increases the persistence and spread of the bacteria within the host.

Effect of selected cytokines on the replication of Corynebacterium pseudotuberculosis and ovine lentiviruses in pulmonary macrophages.

Opportunistic bacterial pathogens that induce monokine secretion by pulmonary alveolar macrophages (PAM) are frequently encountered complicating factors in lentivirus-associated pneumonias in ungulates and man. We examined the effect of selected cytokines on the replication of Corynebacterium pseudotuberculosis and ovine lentivirus (OvLV) in ovine PAM. Recombinant bovine (rBo) IL 1 beta, rBoIL-2, rBo interferon-gamma (IFN gamma) and rBoTNF alpha, alone and in combination at physiological doses had no apparent effect on the extracellular growth of C. pseudotuberculosis, compared with the growth of the pathogen in medium alone. Untreated ovine PAM, derived from bronchoalveolar lavage, were found to substantially reduce, but not eliminate the growth of C. pseudotuberculosis in culture. This bactericidal effect was neither enhanced nor inhibited by pretreatment of PAM with the recombinant bovine cytokines or low doses of LPS that induce monokines. In contrast, addition of rBoTNF alpha or rBoIL-1 beta, at physiological doses, at the initiation of, or on Day 4, after OvLV infection resulted in a significant increase in viral replication in PAM, as measured in an antigen capture assay for OvLV p25, compared with untreated infected cells. This effect was more pronounced with lower levels of infecting OvLV, and, in the case of TNF alpha, was abrogated by preincubation of the cytokine with specific anti-serum. Conversely, in most instances, inclusion of rBoIFN alpha in OvLV-infected PAM cultures resulted in a significant decrease in viral replication. These results suggest that these soluble mediators that are probably secreted in response to C. pseudotuberculosis infection may have little direct effect on the extra- or intracellular survival of the bacteria in the lung, but may modulate lentiviral replication and, by extension, disease expression, in sheep with dual infection.

Selective medium containing fosfomycin, nalidixic acid, and culture supernatant of Rhodococcus equi for isolation of Corynebacterium pseudotuberculosis.

FNR medium containing fosfomycin, nalidixic acid, bovine blood and culture supernatant of Rhodococcus equi was prepared by the present authors, and the medium did not inhibit growth of Corynebacterium pseudotuberculosis but completely hampered the growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The culture supernatant of R. equi facilitated detection of suspected colonies of C. pseudotuberculosis due to synergistic hemolysis. Rate of isolation of the organisms (from the trachea, larynx and nasal cavity of 16 slaughtered sheep with caseous abscess in the lung) was higher with FNR, the selective medium, than with nonselective medium. The selective medium was thus found to be useful for isolation of C. pseudotuberculosis from sheep.