Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.

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.

One-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of human tuberculosis, with an annual death rate of more than 2 million (8). Treatment regimens are effective in only 60 to 90% of patients with persistent infection (14). In humans, tuberculosis can be divided into three phases (28): an active phase, characterized by initial replication of M. tuberculosis that triggers host cellmediated immunity; a chronic phase, in which infected individuals are not infectious, with undetectable levels of tuberculous bacilli; and finally, a reactivation phase, which occurs in 10% of patients with an intact immune system. The reactivation rate is even higher for immune-suppressed individuals (19). Both in vitro and in vivo models have been used to investigate tuberculosis during the dormancy and reactivation phases in humans. One of the first in vitro models of tuberculosis dormancy was established by Wayne (49,50) and is based on culturing M. tuberculosis under decreasing concentrations of oxygen levels, creating a microaerophilic environment where mycobacteria transform into a nonreplicating persistent form.More in vitro models were developed to study chronic tuberculosis, and they have shown that the host microenvironment is rich in reactive nitrogen intermediates (29, 48) but deficient in nutrients necessary for bacterial survival (5), suggesting that tuberculous bacilli persist in a metabolically inactive form. For in vivo studies, the mouse model of chronic and reactivation stages of tuberculosis was used to profile the immunological responses activated during both phases (9, 34). Using an artificial-granuloma implant in mice, the DosR regulon was shown to play a role in mycobacterial entry into the dormant phase, with the involvement of relA (20). Unfortunately, the main genetic bases of the mycobacterial conversion from the "dormant" to the "replicating" phase in the lungs has remained largely unknown. In this report, we communicate our efforts to investigate both the chronic and reactivation phases of tuberculosis.A key question relevant to tuberculosis is the physiological status of M. tuberculosis during different stages of infection. The work of different groups (27, 39) using quantitative PCR on both the genomic and transcriptional levels indicated that mycobacterial bacilli persist at a constant level with a very low growth rate. However, many questions related to chronic tuberculosis remain unanswered. Does the low growth rate mean that the bacilli are metabolically inactive? Can the mycobacterial bacilli sense the surrounding microenvironment? Also, do mycobacterial bacilli adapt to the change in their microen-* Corresponding author. Mailing address:

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.