The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8-to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria.

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

“…These results indicated that the function of the outer membrane as a permeability barrier is not impaired by the loss or overproduction of Rv0888. These results also show that the putative outer membrane channel of full‐length Rv0888 does not increase the susceptibility of Mtb to the tested antibiotics in contrast to the porin MspA of M. smegmatis (Mailaender et al ., ; Stephan et al ., ) or the autotransporter CpnT of Mtb (Danilchanka et al ., ).…”

“…Interestingly, expression of a non‐catalytic Rv0888 mutant was detrimental even for the already impaired Δ rv0888 mutant of Mtb. This observation is consistent with previous findings that the presence of proteins with channel activity in the outer membrane makes Mtb more susceptible to toxic solutes such as nitric oxide produced by macrophages to control growth of Mtb (Fabrino et al ., ; Danilchanka et al ., ). However, mouse infection experiments did not reveal a virulence defect of the Mtb Δ rv0888 mutant.…”

Mechanisms of resistance to aminoglycoside antibiotics: overview and perspectives

“…189 The contribution of CpnT to in vitro resistance of large, hydrophilic drugs including STR was only moderate. 190 …”

Esx Systems and the Mycobacterial Cell Envelope: What's the Connection?

“…The MspA protein in M. smeg-matis is the best characterized mycobacterial porin but has no orthologue in M. tuberculosis (129). More recently, CpnT (Rv3903c) was identified as a mycobacterial toxin and water-filled protein channel that promotes the uptake of glycerol and hydrophilic and hydrophobic antibiotics (131,132).…”

“…In mycobacteria, relatively few proteins that function as channels through the MOM have been characterized (128)(129)(130)(131). The MspA protein in M. smeg-matis is the best characterized mycobacterial porin but has no orthologue in M. tuberculosis (129).…”