Diisonitrile Lipopeptides Mediate Resistance to Copper Starvation in Pathogenic Mycobacteria

Bacterial pathogens and their hosts engage in intense competition for critical nutrients during infection, including metals such as iron, copper, and zinc. Some metals are limited by the host, and some are deployed by the host as antimicrobials. To counter metal limitation, pathogens deploy high-affinity metal acquisition systems, best exemplified by siderophores to acquire iron. Although pathogen strategies to resist the toxic effects of high Cu have been elucidated, the role of Cu starvation and the existence of Cu acquisition systems are less well characterized. In this study, we examined the role of diisonitrile chalkophores of pathogenic mycobacteria, synthesized by the enzymes encoded by the virulence-associated nrp gene cluster, in metal acquisition. nrp gene cluster expression is strongly induced by starvation or chelation of Cu but not starvation of Zn or excess Cu. Mycobacterium tuberculosis and Mycobacterium marinum strains lacking the nrp-encoded nonribosomal peptide sythetase, the fadD10 adenylate-forming enzyme, or the uncharacterized upstream gene ppe1 are all sensitized to Cu, but not Zn, starvation. This low Cu sensitivity is rescued by genetic complementation or by provision of a synthetic diisonitrile chalkophore. These data demonstrate that diisonitrile lipopeptides in mycobacteria are chalkophores that facilitate survival under Cu-limiting conditions and suggest that Cu starvation is a relevant stress for M. tuberculosis in the host. IMPORTANCE Bacterial pathogens and their hosts engage in intense competition for nutrients, including metals. Mycobacterium tuberculosis, the cause of tuberculosis, lives within host macrophages and is subject to diverse stresses, including metal excess and metal limitation. In this study, we demonstrated that the nrp gene cluster, required for M. tuberculosis virulence and which directs synthesis of diisonitrile lipopeptides, mediates copper acquisition. Copper, but not zinc, deprivation strongly induces diisonitrile biosynthesis, and M. tuberculosis strains lacking the nrp gene, or the associated genes fadD10 or ppe1, are all sensitized to copper chelation or copper deprivation. These results establish a copper binding, or chalkophore, system in M. tuberculosis and indicate that resistance to copper restriction plays an important role in the ability of this global pathogen to cause infection.