Hemolytic Activity of Mycobacterial Tlya (Rv1694) is Independent of its rRNA Methylation Activity

In the present study, we show that the mycobacterial TlyA (Rv1694) is a 'moonlighting' protein, while the intracellular form methlylates the 2’-O-ribose of ribosomal RNA (conferring susceptibility to the second line of the antibiotic capreomycin); the extracellular form is suspected to be a non-conventional membrane damaging entity. The purified Rv1694 can self-assemble into large oligomers upon contact with phagosomal membranes or red blood cells. Rv1694 has the wellconserved K⁶⁹-D¹⁵⁴-K¹⁸²-E²³⁸ residues that are essential for site-specific methyltransferase activity. Using the Escherichia coli model system, we show that mutation of any of these residues can result in resistance to capreomycin, while the mutated TlyA can reach the cell wall of the bacterium. The E. coli/TlyA system described here would serve as a simple and reliable model for studying the mechanism of export of TlyA and its homologues, their topology on the cell wall and the nature of contacts it must establish with target phagosomal membranes for their susceptibility.