Recombinant proteins expressed in Mycolicibacterium smegmatis enhance antibody-based detection of bovine tuberculosis

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a zoonosis that threatens public health and causes substantial economic losses in livestock. The suboptimal Escherichia coli-expressed recombinant proteins limit the diagnostic performance of current bTB serological tests. To overcome this limitation, we evaluated Mycolicibacterium smegmatis as an expression host capable of producing recombinant antigens with post-translational modifications comparable to those of M. bovis. Ten antigen candidates were individually expressed in E. coli using the pET-26b( +) vector and in M. smegmatis using the pSOΔBam vector. Their diagnostic performance was evaluated using an enzyme-linked immunosorbent assay (ELISA) with plasma samples from interferon-gamma release assay (IGRA)-negative (n = 30) and -positive (n = 46) cattle in South Korea, followed by receiver operating characteristic (ROC) curve analysis. Among the single antigens, LprA expressed in M. smegmatis demonstrated diagnostic performance comparable to that of the well-established antigen MPB70 (sensitivity: 50.0%, specificity: 96.6%, AUC: 0.791). In addition, several M. smegmatis-derived antigens showed higher concordance with the IGRA results, as assessed by Cohen's kappa and Fisher's exact tests, and a stronger association between age and antigen-specific antibody responses was observed among IGRA-positive cattle. Moreover, a multiple logistic regression model incorporating eight antigens, including those derived from both hosts, achieved high predictive accuracy for IGRA results (sensitivity: 87.0%, specificity: 100%, AUC: 0.991). These findings suggest that M. smegmatis is a promising host for identifying novel antigens and that a multi-host strategy may improve bTB serodiagnosis.