Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp paratuberculosis

Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing ou t cross-reactive antigens by using a polyclonal and polyvalent Mycobacteriu m avium subspecies avium antiserum. Two antigens were present in M. avium s ubsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium . They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identifi ed as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of p urified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in so dium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Mo nospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strai ns except for a Mycobacterium gordonae strain, against which a weak cross-r eactive band was produced. Goats experimentally infected with M. avium subs p: paratuberculosis had strong gamma interferon (IFN-gamma) responses towar d both AhpC and AhpD, and they also had antibodies against AhpC. The abilit y of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. Thes e results further show that AhpC and AhpD are immunologically important pro teins which are constitutively and highly expressed in M. avium subsp. para tuberculosis without the bacteria being submitted to oxidative stress and t hat the specificities of antigens can be a matter of different levels of pr otein expression in various species as well as distinct structural differen ces.