Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.

eThe Mycobacterium avium-M. intracellulare complex (MAC) causes a pulmonary disease (PD) similar to tuberculosis (TB). Diagnosis of MAC-PD is complicated and time-consuming. In this study, the serodiagnostic potential of the newly identified MAV2054 and MAV5183 proteins was evaluated in subjects with MAC-PD, pulmonary TB, or latent TB and in noninfected healthy controls (HC), together with HspX and the 38-kDa antigen, well-known serodiagnostic M. tuberculosis antigens. All four antigens evoked significantly higher IgG responses in MAC-PD and active TB than in latent TB and HC subjects. Among the antigens, MAV2054 elicited the highest antibody responses in pulmonary TB and MAC-PD patients. IgG titers against MAV2054 and MAV5183 were significantly higher in MAC-PD than in pulmonary TB subjects. In addition, the levels of IgG against all antigens in the M. intracellulare and fibrocavitary forms were higher than those in the M. avium and nodular bronchiectatic forms, respectively. Based on sensitivity and receiver operator characteristic curve analysis, the best candidates for detection of MAC-PD and pulmonary TB were MAV2054 and the 38-kDa antigen, respectively. In total, 76.0% of MAC-PD and 65.0% of active TB patients were reactive to at least two antigens. In contrast, only 2.8% of HC subjects were reactive with two or more antigens. Our findings suggest that an enzyme-linked immunosorbent assay (ELISA) using the four antigens would be valuable for screening for mycobacterial lung disease, including MAC-PD and pulmonary TB, although it does not provide good discrimination of the disease-causing pathogens.

Antibody responses are useful indicators of Mycobacterium bovis infection in cattle. Many studies have evaluated the ability of immunoglobulin G (IgG) to serodiagnose bovine tuberculosis (TB). In the current study, immunoglobulin A (IgA) and IgG responses against the MPB70 and MPB83 antigens of M. bovis, the 38 kDa phosphate-binding lipoprotein (PstS1) that is a well-known serodiagnostic M. tuberculosis antigen, and a newly identified protein, termed Rv1483c, were compared in M. bovis-infected and noninfected cattle as well as in field samples. The diagnostic utility of the IgA antibody to MPB70 and MPB83 for bovine TB was superior or comparable to that of the IgG antibody, and the sensitivity of serodiagnosis increased when the results of antigen binding by IgA and IgG were combined. The sensitivities of the IgG and IgA antibodies to the Rv1483c and PstS1 proteins were significantly lower than those to MPB70 and MPB83, and no diagnostic utility for Rv1483c was observed in field samples. Importantly, the IgA antibody reacted strongly to the MPB70 and MPB83 antigens and differentiated cattle with TB from healthy cattle in a multiantigen printed immunoassay. The results of this study support the feasibility of using IgA antibody against the MPB70 and MPB83 antigens to detect bovine TB. In addition, approaches using assays for both IgA and IgG antibodies may increase detection accuracy.

Bovine tuberculosis caused by Mycobacterium bovis is a major economic problem in several countries. Antibody responses are useful indicators of M. bovis infection of cattle. To overcome drawback of serological tests with low sensitivity, identification and characterization of multiple serodiagnostic antigens has been required. In this study, the antigens with strong antibody reactivity were searched using fractionation of M. bovis culture filtrate proteins and probing with sera from M. bovis-infected cattle. Twelve proteins which have not previously been described as serologic targets were identified and six proteins among them were expressed in Escherichia coli. The mycobacterial lipoarabinomannan (LAM) with strong seroreactivity in cattle was identified and purified. IgG and IgA responses against the newly identified proteins, the seroreactive proteins with strong antibody reactivity in human tuberculosis, and LAM were compared in M. bovis-infected and non-infected cattle as well as in field samples. In general, sensitivity of the tested antigens was higher in M. bovis-infected cattle than purified protein derivative (PPD) (+) field samples. Although a diverse reactivity and sensitivity according to the antigens were shown, the diagnostic utility of both IgA and IgG antibody to the antigens was similar in M. bovis-infected cattle but utility of IgG antibody was superior to that of IgA in field samples. The antigen with the highest diagnostic value was LAM in both the groups. Other antigens with considerable diagnostic utility were BCG_3488c, BCG_2330, Antigen 85, HspX, and Rv3593 when considered the sensitivity and area under the receiver characteristic curve (AUC) value. These antigens may be valuable candidates to be included in a cocktail test kit for bovine tuberculosis diagnosis.