A Novel Zinc Exporter CtpG Enhances Resistance to Zinc Toxicity and Survival in Mycobacterium bovis.

Zinc is a microelement essential for the growth of almost all organisms, but it is toxic at high concentrations and represents an antimicrobial strategy for macrophages. Mycobacterium tuberculosis and Mycobacterium bovis are two well-known intracellular pathogens with strong environmental adaptability, including zinc toxicity. However, the signaling pathway and molecular mechanisms on sensing and resistance to zinc toxicity remains unclear in mycobacteria. Here, we first report that P1B-type ATPase CtpG acts as a zinc efflux transporter and characterize a novel CmtR-CtpG-Zn2+ regulatory pathway that enhances mycobacterial resistance to zinc toxicity. We found that zinc upregulates ctpG expression via transcription factor CmtR and stimulates the ATPase activity of CtpG. The APC residues in TM6 is essential for CtpG to export zinc and enhance M. bovis BCG resistance to zinc toxicity. During infection, CtpG inhibits zinc accumulation in the mycobacteria, and aids bacterial survival in THP-1 macrophage and mice with elevated inflammatory responses. Our findings revealed the existence of a novel regulatory pathway on mycobacteria responding to and adapting to host-mediated zinc toxicity. IMPORTANCE Tuberculosis is caused by the bacillus Mycobacterium tuberculosis and is one of the major sources of mortality. M. tuberculosis has developed unique mechanisms to adapt to host environments, including zinc deficiency and toxicity, during infection. However, the molecular mechanism by which mycobacteria promote detoxification of zinc, and the associated signaling pathways remains largely unclear. In this study, we first report that P1B-type ATPase CtpG acts as a zinc efflux transporter and characterize a novel CmtR-CtpG-Zn2+ regulatory pathway that enhances mycobacterial resistance to zinc toxicity in M. bovis. Our findings reveal the existence of a novel excess zinc-triggered signaling circuit, provide new insights into mycobacterial adaptation to the host environment during infection, and might be useful targets for the treatment of tuberculosis.

The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

P1B-type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P1B-ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu2+, Co2+, Ni2+, Zn2+ Cd2+ and Pb2+ stimulate the ATPase activity of the putative P1B-type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd2+ and Cu2+. As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max/K m ratio 7.4-fold higher for Cd2+ than for Cu2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.