Abstract
The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as “hemolysin” which was re-annotated as 2′-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.
Highlights
The causative agent of human tuberculosis disease, Mycobacterium tuberculosis, uses novel mechanisms for evasion of host responses for its intracellular survival (Berry et al, 2013; Cambier et al, 2014)
While the Esx-1 has been shown to be responsible for virulence in M. tuberculosis and M. marinum, the same is responsible for conjugation in M. smegmatis (Gao et al, 2004; Converse and Cox, 2005)
An earlier observation has suggested that expression of tlyA gene of M. tuberculosis (Rv1694; MtbTlyA) in M. smegmatis has resulted in a significant increase in contact dependent hemolysis of red blood cells (King et al, 1993; Wren et al, 1998)
Summary
The causative agent of human tuberculosis disease, Mycobacterium tuberculosis, uses novel mechanisms for evasion of host responses for its intracellular survival (Berry et al, 2013; Cambier et al, 2014). The Tat. TlyA reaches extra cellular milieu and Sec systems recognize amino terminal signal sequences for their transport across the cytoplasmic membrane and the proteins being exported may remain associated with the cell envelope or may get secreted into the extra-cellular milieu depending upon the function of the protein. A comprehensive understanding of protein secretion pathways is essential for delineating the mechanism of translocation and the substrates that utilize these modules for exiting into extra cellular milieu In this regard, an earlier observation has suggested that expression of tlyA gene of M. tuberculosis (Rv1694; MtbTlyA) in M. smegmatis has resulted in a significant increase in contact dependent hemolysis of red blood cells (King et al, 1993; Wren et al, 1998). Our observations reveal that in both native and recombinant hosts, the TlyA can reach the bacterial surface in functional form and such a sequestration, in principle, may aid the intra-cellular survival mechanisms
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