Cell wall glycolipids from Corynebacterium pseudotuberculosis strains with different virulences differ in terms of composition and immune recognition.

Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.

Critical roles of NLRP3 inflammasome in IL-1ß secretion induced by Corynebacterium pseudotuberculosis in vitro.

Corynebacterium pseudotuberculosis is a prominent human and animal pathogen causing chronic inflammatory diseases. Interleukin-1ß (IL-1ß) is involved in the response to such pathogenic infections. However, the mechanism by which IL-1ß is secreted during C. pseudotuberculosis infection remains unclear. This study aimed to investigate the mechanism underlying IL-1ß secretion by macrophages infected with C. pseudotuberculosis. Herein, we firstly revealed that nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 (Casp1) play critical roles in IL-1ß secretion rather than IL-1ß precursor (pro-IL-1ß) expression in C. pseudotuberculosis-infected macrophages. Toll like receptor 4 (TLR4) is partially involved in IL-1ß secretion, while absent in melanoma 2 (AIM2) is not involved in IL-1ß secretion by C. pseudotuberculosis-infected macrophages. In addition, nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinases (p38 MAPK) inhibitors almost attenuated IL-1ß secretion, implying that NF-κB and p38MAPK pathway are involved in IL-1ß secretion in C. pseudotuberculosis-infected macrophages. Furthermore, C. pseudotuberculosis were significantly more numerous in Nlrp3-/-, Asc-/-, and Casp-1-/- macrophages than in WT macrophages at 24 h after infection (P < 0.05), indicating that NLRP3 inflammasome components limit C. pseudotuberculosis replication in macrophages. Together, these data provide novel insights into the mechanisms underlying IL-1ß secretion in C. pseudotuberculosis-infected macrophages and further the current understanding of the host pro-inflammatory immune response against this pathogen.

Sodium butyrate ameliorates Corynebacterium pseudotuberculosis infection in RAW264.7 macrophages and C57BL/6 mice.

Corynebacterium pseudotuberculosis (CP) infection in livestock has become highly difficult to control. To decrease the incidence of CP infection, the supplementation of feed with non-antibiotic antibacterial substances is a potential approach. The aim of this study was to assess the effects of sodium butyrate (NaB), a potential alternative to antibiotics, on CP infection in RAW264.7 macrophages and C57BL/6 mice. Our data showed that NaB (2 mM) significantly ameliorated CPinfection in RAW264.7 macrophages and decreased the bacterial load in the spleens of infected mice. By real-time PCR, we found that NaB induced significant decreases in zinc-dependent superoxide dismutase (sodC) and tip protein C (spaC) expression in CP from infected-RAW264.7 cells and in phospholipase D (pld) and spaC expression in CP from the spleens of infected mice. NaB treatment significantly up-regulated cathelicidin-related antimicrobial peptide (cramp) expression in spleens of mice infected with CP. Furthermore, NaB alleviated histopathological changes in spleens of CP-infected mice. In conclusion, NaB ameliorated CP infection in RAW264.7 macrophages and C57BL/6 mice, and these effects may be related to the modulation of sodC, spaC, pld, and cramp expression.

Multilocus Sequencing of Corynebacterium pseudotuberculosis Biotype Ovis Strains.

Thirteen Corynebacterium pseudotuberculosis biotype ovis strains isolated from clinical cases of caseous lymphadenitis in Hungary were characterised using multilocus sequencing and their phylogenetic comparison was carried out on the basis of four housekeeping genes (groEL1, infB, dnaK, and leuA). The in silico analysis of the 16 frequently studied housekeeping genes showed that C. pseudotuberculosis strains could be readily distinguished from C. diphtheriae and C. ulcerans strains; however, sequences of the same genes in the two biotypes of the C. pseudotuberculosis were highly similar; the heterogeneity values were low. Genes dnaK, infB, groEL1, and leuA showed marked genetic variation within C. pseudotuberculosis, and strains of the two biotypes of C. pseudotuberculosis could be differentiated. Analysis of the individual genes showed a fairly conservative nature of C. pseudotuberculosis biotype ovis strains. The greatest genetic differentiation was seen in the dnaK and infB genes and concatenations of these two genes were very useful in the genetic separation of the studied strains.

C. pseudotuberculosis Phop confers virulence and may be targeted by natural compounds.

The bacterial two-component system (TCS) regulates genes that are crucial for virulence in several pathogens. One of such TCS, the PhoPR system, consisting of a transmembrane sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP), has been reported to have a major role in mycobacterial pathogenesis. We knocked out the phoP in C. pseudotuberculosis, the causal organism of caseous lymphadenitis (CLA), and using a combination of in vitro and in vivo mouse system, we showed for the first time, that the PhoP of C. pseudotuberculosis plays an important role in the virulence and pathogenicity of this bacterium. Furthermore, we modeled the PhoP of C. pseudotuberculosis and our docking results showed that several natural compounds including Rhein, an anthraquinone from Rheum undulatum, and some drug-like molecules may target PhoP to inhibit the TCS of C. pseudotuberculosis, and therefore may facilitate a remarkable attenuation of bacterial pathogenicity being the CLA. Experiments are currently underway to validate these in silico docking results.

An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge.

Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.

Activity of disinfectants and biofilm production of Corynebacterium pseudotuberculosis / Atividade de desinfetantes e produção de biofilme em Corynebacterium pseudotuberculosis

To verify the occurrence of caseous lymphadenitis in sheep and goats on farms of Pernambuco, Brazil, and in animals slaughtered in two Brazilian cities (Petrolina/PE and Juazeiro/BA), and to characterize the susceptibility profile of Corynebacterium pseudotuberculosis to disinfectants and antimicrobials, and its relationship with biofilm production were the objectives of this study. 398 samples were tested for sensitivity to antimicrobial drugs, disinfectants, and biofilm production. Among the 108 samples collected on the properties, 75% were positive for C. pseudotuberculosis. Slaughterhouse samples indicated an occurrence of caseous lymphadenitis in 15.66% and 6.31% for animals slaughtered in Petrolina and Juazeiro respectively. With respect to antimicrobials, the sensitivity obtained was 100% for florfenicol and tetracycline; 99.25% for enrofloxacin, ciprofloxacin and lincomycin; 98.99% for cephalothin; 98.74% for norfloxacin and sulfazotrim; 97.74% for gentamicin; 94.22% for ampicillin; 91.71% for amoxicillin; 91.21% for penicillin G; 89.19% for neomycin and 0% for novobiocin. In analyzes with disinfectants, the efficiency for chlorhexidine was 100%, 97.20% for quaternary ammonium, 87.40% for chlorine and 84.40% for iodine. 75% of the isolates were weak or non-biofilm producers. For the consolidated biofilm, found that iodine decreased biofilm formation in 13 isolates and quaternary ammonia in 11 isolates. The reduction of the biofilm formation was observed for iodine and quaternary ammonium in consolidated biofilm formation in 33% and 28% of the isolates, respectively. The results of this study highlight the importance of establishing measures to prevent and control the disease.

Verificar a ocorrência de linfadenite caseosa de ovinos e caprinos em fazendas de Pernambuco, no Brasil e em animais abatidos em duas cidades brasileiras (Petrolina/PE e Juazeiro/BA), caracterizar o perfil de suscetibilidade de Corynebacgerium pseudotuberculosis a desinfetantes e a antimicrobianos e analisar sua relação com a produção de biofilme foram os objetivos deste estudo. 398 amostras foram testadas para a sensibilidade aos medicamentos antimicrobianos, desinfetantes e produção de biofilme. Entre as 108 amostras coletadas nas propriedades, 75% foram positivas para C. pseudotuberculosis. Amostras do matadouro indicaram a ocorrência de linfadenite caseosa em 15,66% e 6,31% para animais abatidos em Petrolina (PE) e Juazeiro (BA), respectivamente. Com relação aos antimicrobianos, a sensibilidade obtida foi de 100% para florfenicol e tetraciclina, 99,25% para a enrofloxacina, ciprofloxacina e lincomicina; 98,99% para cefalotina; 98,74% para norfloxacina e sulfazotrim; 97,74% para gentamicina; 94,22% para ampicilina, 91,71% para amoxicilina, 91,21% para a penicilina G, 89,19% para a neomicina e 0% para novobiocina. Nas análises com desinfetantes, a eficiência para a clorexidina foi de 100%, 97,20% para amônia quaternária, 87,40% para o cloro e 84,40% para o iodo. 75% dos isolados foram fracos produtores de biofilme. Para o biofilme consolidado, observou-se que o iodo diminuiu a formação de biofilme em 13 isolados e a amônia quaternária em 11 isolados. A redução da formação de biofilme foi observada no biofilme consolidado, para iodo e amônia quaternária, em 33% e 28% das amostras, respectivamente. Os resultados deste estudo destacam a importância de se estabelecer medidas para prevenir e controlar a doença.

Evaluation of invertebrate infection models for pathogenic corynebacteria.

For several pathogenic bacteria, model systems for host-pathogen interactions were developed, which provide the possibility of quick and cost-effective high throughput screening of mutant bacteria for genes involved in pathogenesis. A number of different model systems, including amoeba, nematodes, insects, and fish, have been introduced, and it was observed that different bacteria respond in different ways to putative surrogate hosts, and distinct model systems might be more or less suitable for a certain pathogen. The aim of this study was to develop a suitable invertebrate model for the human and animal pathogens Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Corynebacterium ulcerans. The results obtained in this study indicate that Acanthamoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis elegans and Galleria larvae seem to offer tractable models for rapid assessment of virulence between strains. Caenorhabtitis elegans gives more differentiated results and might be the best model system for pathogenic corynebacteria, given the tractability of bacteria and the range of mutant nematodes available to investigate the host response in combination with bacterial virulence. Nevertheless, Galleria will also be useful in respect to innate immune responses to pathogens because insects offer a more complex cell-based innate immune system compared with the simple innate immune system of C. elegans.

Ocorrência e fatores de risco associados à infecção por Corynebacterium pseudotuberculosis em caprinos e ovinos do semiárido paraibano / Occurrence and risk factors associated with the Corynebacterium pseudotuberculo sis infection in sheep and goats from the semiarid region of the Paraiba state, Northeastern Brazil

Este trabalho teve como objetivo determinar a ocorrência e os fatores de risco associados à infecção por Corynebacterium pseudotuberculosis em caprinos e ovinos do semiárido da Paraiba. De 640 animais examinados, 7,7% (49/640) apresentavam evidências clínicas de linfadenite caseosa. Em 59,2% (29/49) destes animais havia apenas as cicatrizes de abscessos anteriormente rompidos; em 40,8% (20/49) dos animais, os abscessos estavam intactos. Desses 20 animais, 13 (65%) caprinos apresentaram 14 abscessos, enquanto que sete (35%) ovinos apresentaram oito abscessos. Em ambas as espécies, o linfonodo pré-escapular foi o mais acometido. No exame microbiológico, constatou-se que C. pseudotuberculosis foi o agente mais frequentemente isolado, em 15 (68,2%) amostras; em uma (4,5%) foi isolado Staphylococcus coagulase negativa; uma (4,5%) Enterococcus sp.; uma (4,5%) o Proteus mirabilis e Pseudomonas aeruginosa; e em quatro (18,2%) amostras não houve crescimento bacteriano. O modelo final de regressão logística mostrou que animais provenientes de rebanhos em que seus proprietários deixavam os abscessos romperem naturalmente tiveram maior chance de apresentar linfadenite caseosa (odds ratio =8,19; IC 95% =1,75-38,25; p=0,008). Conclui-se que os caprinovinocultores da região devem adotar medidas profiláticas em seus rebanhos, como abertura e drenagem precoce dos abscessos superficiais e destino adequado do conteúdo. Tais medidas, associadas à inspeção periódica do rebanho, descarte de animais doentes e não introdução de animais infectados, contribuirão significativamente para o controle desta infecção.

This study aimed to determine the occurrence and risk factors associated with Cory nebacterium pseudotuberculosis infection in goats and sheep in semiarid region of Paraiba State, Northeastern Brazil. Of the 640 animals examined, 7.7% (49/640) had clinical evidence of caseous lymphadenitis. In 59.2% (29/49) of these animals there was only the scars of previously ruptured abscesses and in 40.8% (20/49) of the animals abscesses were intact. Of these 20 animals 13 (65%) goats had 14 abscesses, whereas seven (35%) sheep had eight abscesses. In both species, pre-scapular lymph node was the most involved. C. pseudotuberculosis was the agent most frequently isolated, in 15 (68.2%) samples, and in one (4.5%) coagulase-negative Staphylococcus was isolated, one (4.5%) Enterococ cus sp., one (4.5%) Proteus mirabilis and Pseudomonas aeruginosa, and in four (18.2%) samples there was no bacterial growth. The final logistic regression model showed that animals from herds where their owners let the abscesses break naturally presented larger odds of caseous lymphadenitis (odds ratio = 8.19, 95% CI = 1.75 - 38.25, p = 0.008). We conclude that goat/sheep owners of the region should adopt preventive measures in their herds, such as early opening and drainage of superficial abscesses, and appropriate destination of the content. Such measures, in addition to regular inspection of the flock, disposal of diseased animals and introduction of non-infected animals will contribute to the control of this infection.

A description of genes of Corynebacterium pseudotuberculosis useful in diagnostics and vaccine applications

Corynebacterium pseudotuberculosis, a Gram-positive intracellular pathogen, is the etiological agent of caseous lymphadenitis or CLA. This bacterium infects goats and sheep and causes great economic losses worldwide annually, mainly for goat producers. Despite its importance, CLA is still poorly characterized. However, with advances in the genomic field, many C. pseudotuberculosis genes have already been characterized, mainly those related to virulence such as phospholipase D. Here, we examined the use of the several available genes of C. pseudotuberculosis and reviewed their applications in vaccine construction, more efficient diagnostics for CLA, and control of this disease, among other applications.

Expression of phospholipase D, the major virulence factor of Corynebacterium pseudotuberculosis, is regulated by multiple environmental factors and plays a role in macrophage death.

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis, a disease affecting sheep and goats. Phospholipase D (Pld), a major virulence determinant of C. pseudotuberculosis, is believed to play a critical role in dissemination of bacteria from the site of infection to the lymph nodes. Although the pld gene has been studied for some time, it is only recently that it has been identified as being down-regulated following heat shock from 37 to 43 degrees C. To gain insights into the mechanisms of Pld action, this study investigated how it was regulated under varying environmental conditions. Studies measuring pld mRNA levels or utilizing a reporter construct containing the pld promoter upstream of a gfp gene were performed. These showed that pld was upregulated in a cell-density-dependent manner, was regulated by heat shock at all cell-culture densities, and was highly expressed in a tissue-culture macrophage-infection model. Finally, the expression of Pld by intracellular C. pseudotuberculosis was shown to play a small but significant role in the reduction of macrophage viability following infection. This study demonstrates that the regulation of C. pseudotuberculosis pld is complex. This regulatory complexity may play an important role in allowing the pathogen to successfully adapt to the changing host environment during infection, migration, establishment and disease progression.

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.

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.