SummaryCopper is a required micronutrient that is also toxic at excess concentrations. Currently, little is known about the role of copper in interactions between bacterial pathogens and their human hosts. In this study, we elucidate a mechanism for copper homeostasis in the human pathogen Mycobacterium tuberculosis via characterization of a putative copper exporter, CtpV. CtpV was shown to be required by M. tuberculosis to maintain resistance to copper toxicity. Furthermore, the deletion of ctpV resulted in a 98-gene transcriptional response, which elucidates the increased stress experienced by the bacteria in the absence of this detoxification mechanism. Interestingly, although the DctpV mutant survives close to the wild-type levels in both murine and guinea pig models of tuberculosis, animals infected with the DctpV mutant displayed decreased lung damage, and mutant-infected mice had a reduced immune response to the bacteria as well as a significant increase in survival time relative to mice infected with wild-type M. tuberculosis. Overall, our study provides the first evidence for a connection between bacterial copper response and the virulence of M. tuberculosis, supporting the hypothesis that copper response could be important to intracellular pathogens, in general.
Copper (Cu) is a required micronutrient, but it is highly toxic at high concentrations. Therefore, the levels of Cu must be tightly regulated in all living cells. The phagosome of Mycobacterium tuberculosis has been shown to have variable levels of Cu. Previously, we showed that M. tuberculosis contains a copper-sensitive operon, cso, that is induced during early infection in mice. In this study, we showed that ctpV, a gene in the cso operon, is a copper-responsive gene and most likely encodes an efflux pump for Cu. Furthermore, the transcription of key genes in the cso operon is induced by Cu ions and not by other ions, such as Ni and Zn ions. To elucidate copper-responsive genes other than those in the cso operon, we utilized DNA microarrays to profile mycobacterial responses to physiological levels of Cu. A transcriptome analysis identified a novel set of 30 copperresponsive genes in M. tuberculosis, one-half of which were induced only when toxic levels of Cu were added. Interestingly, several transcriptional regulators, including the furA gene, were induced during toxic Cu exposure, indicating that there was a generalized response to oxidative stressors rather than a Cu-specific response. In general, the Cu-induced transcriptome generated should help elucidate the role of the Cu response in maintaining M. tuberculosis survival during infection and could provide novel targets for controlling this virulent pathogen.
Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the ⌬mosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.
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