Bovine serum albumin promotes reactivation of viable but non-culturable<i>Mycobacterium tuberculosis</i>via the activation of protein kinase-dependent cell division processes

<jats:title>Abstract</jats:title><jats:sec><jats:title>Objective</jats:title><jats:p><jats:italic>Mycobacterium tuberculosis</jats:italic>(Mtb) H37Ra strain has been reported to rapidly enter the viable but non-culturable (VBNC) state following treatment with an NADH oxidase inhibitor (diphenyleneiodonium [DPI]) and to be reactivated by fetal bovine serum (FBS). However, the mechanism underlying FBS-induced reactivation is unclear. We tried to reveal the mechanism of FBS-induced reactivation using<jats:italic>M. tuberculosis</jats:italic>H37Rv.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>First, we evaluated the effect of DPI on culturability, viability and changes of acid-fastness toward H37Rv. Secondly, we measured the reactivation-promoting effects of human serum albumin, egg-white albumin and antioxidative agents in DPI-induced VBNC cells. We also inhibited adenylyl cyclase and protein kinase which is the downstream of adenylyl cyclase to evaluate the influence to reactivation capacity of bovine serum albumin (BSA).</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>DPI treatment induced VBNC state in H37Rv, resulting in a high proportion of viable cells but a low proportion of culturable cells, loss of acid-fastness and lipid-accumulation. Not only FBS but also BSA alone could reactivate H37Rv. Contrary to our expectation, only human serum albumin had a similar restorative effect to BSA. The inhibition of adenylyl cyclase by SQ22536 did not have a significant effect on reactivation; however, the inhibition of mycobacterial protein kinase by H89 and staurosporine strongly suppressed the BSA-induced reactivation.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>DPI-induced VBNC Mtb cells may be reactivated via the activation of protein kinase-dependent cell division processes through interaction with BSA.</jats:p></jats:sec>