Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response. Dietary zinc deficiency is a global health problem affecting almost two billion people. Infectious diseases associated with zinc deficiency include respiratory infections caused by bacteria, and notably, Streptococcus pneumoniae, which is responsible for more than 1 million deaths annually. The association between zinc and immunity is well known, but the mechanism by which zinc provides protection against infectious diseases has remained a mystery. Previously, we found that manganese was essential for S. pneumoniae growth and its ability to cause disease. Intriguingly, we also observed that zinc could bind to the manganese transport protein. Therefore, we sought to determine if zinc could inhibit manganese transport, and to observe what the effects would be on S. pneumoniae. We found that zinc prevented manganese uptake. This slowed bacterial growth and rendered it hypersensitive to immune cell killing. We also observed that, during S. pneumoniae infection in mice, zinc released by the host increased to concentrations that could compete for manganese uptake. Our study provides direct evidence for how zinc is toxic to bacteria by preventing manganese uptake. Furthermore, we show how this could be harnessed by the immune system, thereby providing a scientific basis for the protective effect of zinc against infectious diseases.

Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

IL-17-producing helper T (Th17) cells are critical for host defense against extracellular pathogens but also drive numerous autoimmune diseases. Th17 cells that differ in their inflammatory potential have been described including IL-10-producing Th17 cells that are weak inducers of inflammation and highly inflammatory, IL-23-driven, GM-CSF/IFNγ-producing Th17 cells. However, their distinct developmental requirements, functions and trafficking mechanisms in vivo remain poorly understood. Here we identify a temporally regulated IL-23-dependent switch from CCR6 to CCR2 usage by developing Th17 cells that is critical for pathogenic Th17 cell-driven inflammation in experimental autoimmune encephalomyelitis (EAE). This switch defines a unique in vivo cell surface signature (CCR6⁻CCR2⁺) of GM-CSF/IFNγ-producing Th17 cells in EAE and experimental persistent extracellular bacterial infection, and in humans. Using this signature, we identify an IL-23/IL-1/IFNγ/TNFα/T-bet/Eomesodermin-driven circuit driving GM-CSF/IFNγ-producing Th17 cell formation in vivo. Thus, our data identify a unique cell surface signature, trafficking mechanism and T-cell intrinsic regulators of GM-CSF/IFNγ-producing Th17 cells.

The importance of Mn ²⁺ for pneumococcal physiology and virulence has been studied extensively. However, the specific cellular role(s) for which Mn ²⁺ is required are yet to be fully elucidated. Here, we analyzed the effect of Mn ²⁺ limitation on the transcriptome and proteome of Streptococcus pneumoniae D39. This was carried out by comparing a deletion mutant lacking the solute binding protein of the high-affinity Mn ²⁺ transporter, pneumococcal surface antigen A (PsaA), with its isogenic wild-type counterpart. We provide clear evidence for the Mn ²⁺ -dependent regulation of the expression of oxidative-stress-response enzymes SpxB and Mn ²⁺ -SodA and virulence-associated genes pcpA and prtA . We also demonstrate the upregulation of at least one oxidative- and nitrosative-stress-response gene cluster, comprising adhC , nmlR , and czcD , in response to Mn ²⁺ stress. A significant increase in 6-phosphogluconate dehydrogenase activity in the psaA mutant grown under Mn ²⁺ -replete conditions and upregulation of an oligopeptide ABC permease (AppDCBA) were also observed. Together, the results of transcriptomic and proteomic analyses provided evidence for Mn ²⁺ having a central role in activating or stimulating enzymes involved in central carbon and general metabolism. Our results also highlight the importance of high-affinity Mn ²⁺ transport by PsaA in pneumococcal competence, physiology, and metabolism and elucidate mechanisms underlying the response to Mn ²⁺ stress.