Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Abstract

cCopper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 M copper by 40% and completely suppressed growth at 15 M copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner

“…We found that the susceptibility of the triple-porin mutant to disulfiram in the presence of copper was similar to that of the wild type (Fig. 5B) despite the 15-fold differential in the numbers of outer membrane porin channels (31) and the much greater copper tolerance of ML16 (56). This result strongly suggests that Cu II -(DETC) 2 uptake occurs in a porin-independent fashion.…”

“…To experimentally determine whether the uptake of the disulfiram-copper complex is a porin-independent process, we treated the triple-porin mutant of M. smegmatis, ML16, and the wild type with disulfiram in the presence or absence of copper. The use of ML16 for this purpose was previously validated by several studies (55)(56)(57). We found that the susceptibility of the triple-porin mutant to disulfiram in the presence of copper was similar to that of the wild type (Fig.…”

Copper Oxidation State and Mycobacterial Infection

“…A point of weakness are the porins that are required as entry point for essential nutrients, including copper, but may also allow excess copper to get access to the cytoplasmic membrane ( Figure 2) [24]. Cu homeostasis on the host side proceeds by the components indicated in blue: the high-affinity copper transporter of the plasma membrane, Ctr1, is induced by the activation of the macrophage and transports extra copper into the cytoplasm; Atox1, a cytosolic Cu + chaperone, transfers the copper to ATP7A, which has trafficked from the trans-Golgi network to the phagosome and pumps copper into the phagosomal lumen.…”

MspA-Mycobacterium tuberculosis-transformant with reduced virulence: The “unbirthday paradigm”

“…M. tuberculosis and M. bovis mycobacteria survive in the phagosome of macrophages, a characteristic phenotype in the pathology of tuberculosis [29,30]; and lack of MspA-like porin may facilitate such survival [8,31] as porin deletions increase the survival of M. smegmatis in macrophages [20]. Indeed, MspA porin renders M. smegmatis susceptible to nitric oxide [20]; and M. tuberculosis to copper-killing [32]. However, we observed here that MspA indeed was a non-virulence gene, downregulating the microbicidal program of macrophages by inhibiting the interferon pathway.…”