RESUMO
Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (PTB), which is a granulomatous enteritis in ruminants that threatens the dairy industry's healthy development and public health safety worldwide. Because the commercial inactivated vaccines are not completely protective and interfere with bovine tuberculosis diagnostics, we tested four fusion proteins, namely 66NC, 66CN, 90NC, and 90CN, which were constructed with MAP3527, Ag85B, and Hsp70 of MAP in different tandem combinations. Notably, 66NC, which encodes a 66 kDa fusion protein that combines in linear order MAP3527N40-232, Ag85B41-330, and MAP3527C231-361, induced a powerful and specific IFN-γ response. Immunization of C57BL/6 mice with the 66NC fusion protein formulated in Montanide ISA 61 VG adjuvant generated robust Th1, Th2, and Th17 type immune responses and strong antibody responses. The 66NC vaccine protected C57BL/6 mice against virulent MAP K-10 infection. This resulted in a reduction of bacterial load and improvement of pathological damage in the liver and intestine, in addition to a reduction of body weight loss; significantly better protection than the reported 74 F vaccine was also induced. Furthermore, vaccine efficacy correlated with the levels of IFN-γ-, TNF-α-, and IL-17A-secreting antigen-specific CD4+ and CD8+ T lymphocytes as well as with serum IFN-γ and TNF-α levels after vaccination. These results demonstrate that recombinant protein 66NC is an efficient candidate for further development into a protective vaccine in terms of inducing specific protection against MAP.
RESUMO
Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.