Bovine and human serum albumins promote recovery of culturability in diphenyleneiodonium-induced viable but non-culturable Mycobacterium tuberculosis , potentially involving protein kinase activity

We investigated the mechanisms underlying the fetal bovine serum (FBS)-induced reactivation of diphenyleneiodonium (DPI)-induced viable but non-culturable (VBNC) state of Mycobacterium tuberculosis (Mtb) using the H37Rv strain. We found that bovine serum albumin (BSA), a major component of FBS, was sufficient to promote recovery of culturability in DPI-induced VBNC Mtb cells under our experimental conditions. Human serum albumin also showed a similar effect, whereas mouse serum albumin and egg-white albumin (ovalbumin) did not induce comparable recovery, suggesting that specific structural or biochemical features shared by bovine and human serum albumin may contribute to this process. In addition, antioxidative agents, including N - acetyl- L -cysteine (NAC), did not induce reactivation, suggesting that the antioxidative property alone is insufficient to explain the effect of BSA. Recovery of culturability was suppressed by protein kinase inhibitors, such as H89 and staurosporine, which are known to target multiple kinases, including serine/threonine protein kinases in Mtb. These findings suggest that protein kinase activity may be involved in BSA-induced recovery of culturability from the DPI-induced VBNC Mtb cells. Overall, our study identifies albumin-dependent recovery of culturability in DPI-induced VBNC model of Mtb and highlights the need to compare recovery mechanisms across distinct dormancy models. IMPORTANCE Mycobacterium tuberculosis (Mtb) cells can enter a viable but non-culturable (VBNC) state under stress, complicating the interpretation of bacterial survival and recovery in experimental dormancy models. However, the factors that restore culturability from defined VBNC populations remain less understood. This study shows that serum albumin promotes recovery of culturability from Mtb cells rendered VBNC state by diphenyleneiodonium (DPI) treatment. The effect was observed with bovine and human serum albumins but not mouse serum albumin or ovalbumin, indicating that albumin-dependent recovery is not a general property of all albumins. The lack of comparable recovery by the antioxidant N-acetyl- L -cysteine (NAC) further suggests that the antioxidative activity alone does not explain this phenomenon. By identifying albumin-dependent recovery in a defined DPI-induced VBNC model, this work provides a basis for testing whether similar or distinct recovery mechanisms operate in other Mtb dormancy models.