Bovine serum albumin as a resuscitation promoting factor for viable but non-culturable Mycobacterium tuberculosis via the activation of protein kinase A-dependent cellular processes

Objective: Mycobacterium tuberculosis (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 resuscitated by fetal bovine serum (FBS). However, the mechanism underlying FBS-induced resuscitation is currently unclear. We tried to reveal the underlying mechanism of FBS-induced resuscitation using M. tuberculosis H37Rv. Methods: First, we evaluated the effect of DPI on culturability, viability and changes of cellular phenotypes toward H37Rv. Secondly, we measured the resuscitation-promoting effects of human serum albumin, egg-white albumin, N-acetyl-L-cysteine, and D-mannitol in DPI-induced VBNC cells, as antioxidative agents have been reported to be key molecules for resuscitation of other microbes. We also evaluated the effect of inhibition of cAMP production and protein kinase A on BSA-induced resuscitation. Results: DPI treatment successfully induced a VBNC state in H37Rv, resulting in a low proportion of culturable cells, loss of acid-fastness and lipid-accumulation but a high proportion of viable cells. Not only FBS but also bovine serum albumin (BSA) alone could resuscitate H37Rv. Contrary to our expectation, only human serum albumin had a similar resuscitative effect to BSA. The inhibition of adenylyl cyclase by SQ22536 did not have a significant effect on resuscitation; however, the inhibition of protein kinase A by H89 strongly suppressed the BSA-induced resuscitation. Conclusion: DPI-induced VBNC Mtb cells may be resuscitated via the activation of protein kinase A-dependent processes through interaction with BSA.