Association of Corynebacterium pseudotuberculosis recombinant proteins rCP09720 or rCP01850 with rPLD as immunogens in caseous lymphadenitis immunoprophylaxis.

Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLD + rCP09720 (G3), and rPLD + rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21 days after the last immunization. The animals were evaluated daily for 40 days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (p < .05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (p < .05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLD + rCP09720), and 50% (rPLD + rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.

Sphingomyelinase D inhibits store-operated Ca2+ entry in T lymphocytes by suppressing ORAI current.

Infections caused by certain bacteria including Mycobacterium tuberculosis and Corynebacterium pseudotuberculosis provoke inflammatory responses characterized by the formation of granulomas with necrotic foci-so-called caseous necrosis. The granulomas of infected animals show prominent infiltration by T lymphocytes, and T cell depletion increases host mortality. Notorious zoonotic C. pseudotuberculosis secretes sphingomyelinase (SMase) D, a phospholipase that cleaves off the choline moiety of sphingomyelin, a phospholipid found primarily in the outer leaflet of host cell plasma membranes. Experimental C. pseudotuberculosis strains that lack SMase D are markedly less infectious and unable to spread in hosts, indicating that this enzyme is a crucial virulence factor for sustaining the caseous lymphadenitis infections caused by this microbe. However, the molecular mechanism by which SMase D helps bacteria evade the host's immune response remains unknown. Here, we find that SMase D inhibits store-operated Ca(2+) entry (SOCE) in human T cells and lowers the production of the SOCE-dependent cytokines interleukin-2, which is critical for T cell growth, proliferation, and differentiation, and tumor necrosis factor α, which is crucial for the formation and maintenance of granulomas in microbial infections. SMase D inhibits SOCE through a previously unknown mechanism, namely, suppression of Orai1 current, rather than through altering gating of voltage-gated K(+) channels. This finding suggests that, whereas certain genetic mutations abolish Orai1 activity causing severe combined immunodeficiency (SCID), bacteria have the ability to suppress Orai1 activity with SMase D to create an acquired, chronic SCID-like condition that allows persistent infection. Thus, in an example of how virulence factors can disrupt key membrane protein function by targeting phospholipids in host cell membranes, our study has uncovered a novel molecular mechanism that bacteria can use to thwart host immunity.

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

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.

Inhibition of CFTR Cl- channel function caused by enzymatic hydrolysis of sphingomyelin.

Numerous mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR, a Cl(-) channel) disrupt salt and fluid transport and lead to the formation of thick mucus in patients' airways. Obstruction by mucus predisposes CF patients to chronic infections and inflammation, which become gradually harder to control and eventually fatal. Aggressive antibiotic therapy and supportive measures have dramatically lengthened CF patients' lives. Here, we report that sphingomyelinases (SMase) from human respiratory pathogens strongly inhibit CFTR function. The hydrolysis of sphingomyelin by SMase makes it more difficult to activate CFTR by phosphorylation of its regulatory domain. By inhibiting CFTR currents, SMase-producing respiratory tract bacteria may not only aggravate pulmonary infection in some CF patients but may also elicit a condition, analogous to CFTR deficiency, in non-CF patients suffering from bacterial lung infection.

Screening tests for pathogenic corynebacteria.

AIM: To provide simple tests that would help in the identification of corynebacteria that produce diphtheria toxin. METHODS: A collection of 99 freshly isolated corynebacteria was assembled and the cultures identified by conventional tests confirmed by an identification kit. Modifications were made to procedures for preparation of the culture medium for the Elek test and to the test for detection of pyrazinamidase (pyrazine carboxylamidase) activity. These two together with an indicator medium for cystinase activity were applied to the collection of organisms. RESULTS: Cystinase was detected in all 61 members of the toxigenic species and none produced pyrazinamidase. In contrast, all but two of the 38 representatives of non-toxigenic species yielded pyrazinamidase and none formed cystinase. Of the 61 cystinase producing cultures (which were also pyrazinamidase negative), 21 gave a positive Elek test with the modified culture medium. A total of 30 of these 61 were tested for toxigenicity in guinea pigs and the results of the animal and plate tests concorded. At least seven cultures could have been reported as non-toxigenic if Elek tests based on media prepared in the conventional way had been the only test available. CONCLUSION: The three procedures described go some way towards meeting the needs of diagnostic laboratories for efficient procedures for distinguishing pathogenic corynebacteria.