Summary Zinc (Zn) is an essential metal that vertebrates sequester from pathogens to protect against infection. Investigating the opportunistic pathogen Acinetobacter baumannii’s response to Zn starvation, we identified a putative Zn metallochaperone, ZigA, which binds Zn, is required for bacterial growth under Zn-limiting conditions, and for disseminated infection in mice. ZigA is encoded adjacent to the histidine (His) utilization (Hut) system. The His ammonia-lyase HutH, binds Zn very tightly only in the presence of high His and makes Zn bioavailable through His catabolism. The released Zn enables A. baumannii to combat host-imposed Zn starvation. These results demonstrate that A. baumannii employs several mechanisms to ensure bioavailability of Zn during infection, with ZigA functioning predominately during Zn starvation, but HutH operating in both Zn deplete and replete conditions to mobilize a labile His-Zn pool.

Copper resistance has emerged as an important virulence determinant of microbial pathogens. In Streptococcus pneumoniae, copper resistance is mediated by the copper-responsive repressor CopY, CupA, and CopA, a copper effluxing P1B-type ATPase. We show here that CupA is a novel cell membrane-anchored Cu(I) chaperone, and that a Cu(I)-binding competent, membrane-localized CupA is obligatory for copper resistance. The crystal structures of the soluble domain of CupA (sCupA) and the N-terminal metal binding domain (MBD) of CopA (CopAMBD) reveal isostructural cupredoxin-like folds each harboring a binuclear Cu(I) cluster unprecedented in bacterial copper trafficking. NMR studies reveal unidirectional Cu(I) transfer from the low-affinity site on sCupA to the high-affinity site of CopAMBD. However, copper binding by CopAMBD is not essential for cellular copper resistance, consistent with a primary role of CupA in cytoplasmic Cu(I) sequestration and/or direct delivery to the transmembrane site of CopA for cellular efflux.

ICP-MS analysis of Streptococcus pneumoniae reveals a high cell-associated Mn(II) concentration that is comparable to that of Zn(II). Stressing these cells with 100–200 μM Zn(II) leads to a slow-growth phenotype and a total Mn(II) concentration that is reduced, with no decrease of other metal ions. Supplementation of the growth media with as little as 10 μM Mn(II) fully restores the growth defect and cell-associated Mn(II) to normal levels. DNA microarray analysis reveals that zinc stress induces the expected upregulation of czcD (encoding a zinc effluxer), but also a pleiotropic transcriptional response suggestive of mild cell wall stress. Genes encoding a nitric oxide (NO) detoxification system (nmlR) and the Mn(II) uptake system (psaBCA) are also induced. We conclude that Zn(II) toxicity results in a cytoplasmic Mn(II) deficiency, possibly caused by competition at the Mn(II) uptake transporter protein PsaA.

Pathogenic bacteria acquire transition metals for cell viability and persistence of infection in competition with host nutritional defenses. The human host employs a variety of mechanisms to stress the invading pathogen with both cytotoxic metal ions and oxidative and nitrosative insults while withholding essential transition metals from the bacterium. For example, the S100 family protein calprotectin (CP) found in neutrophils is a calcium-activated chelator of extracellular Mn and Zn and is found in tissue abscesses at sites of infection by Staphylococcus aureus. In an adaptive response, bacteria have evolved systems to acquire the metals in the face of this competition while effluxing excess or toxic metals to maintain a bioavailability of transition metals that is consistent with a particular inorganic “fingerprint” under the prevailing conditions. This review highlights recent biological, chemical and structural studies focused on manganese (Mn) acquisition and homeostasis and connects this process to oxidative stress resistance and iron (Fe) availability that operates at the human host-pathogen interface.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.