Characterisation of the Zinc Homeostatic Mechanisms of Streptococcus pneumoniae

Abstract

All living organisms require zinc for essential cellular processes, which necessitates efficient zinc acquisition mechanisms. For the Gram-positive bacterial pathogen Streptococcus pneumoniae, zinc must be scavenged from host tissues in a process akin to piracy. This is facilitated by the high-affinity zinc importer AdcCB, which is paired with two zinc-specific solute-binding proteins (SBPs), AdcA and AdcAII. Although essential for growth, zinc can be toxic in biological systems due to its ability to compete for binding to non-cognate metalloproteins and thereby disrupting their function. To prevent toxic accumulation of zinc ions, S. pneumoniae employs a zinc-efflux pathway known as CzcD. Despite the importance of zinc to pneumococcal biology, these zinc homeostatic mechanisms remain poorly understood. To address this, the mechanisms used by S. pneumoniae to achieve zinc homeostasis were explored in this study at the molecular, cellular and host-pathogen levels. To gain a more detailed understanding of zinc acquisition by S. pneumoniae, AdcA was investigated in detail using a combination of structural and biochemical approaches. AdcA contains additional zinc-recruiting motifs that have been suggested to enhance its ability to recruit zinc. While these zinc-recruiting motifs were found here to bind zinc ions, they were not necessary for pneumococcal growth in zinc-limited media, suggesting that their role in zinc binding may occur under conditions of higher zinc availability. The ligand-binding mechanism of AdcA was also explored, which revealed that zinc ion coordination is facilitated by localised changes in mobility and not significant structural rearrangements. These data indicate a distinct mechanism of ligand binding for a metal-uptake SBP, and provide a plausible explanation for the limited structural rearrangements observed in zincspecific SBPs from other bacteria. The role of zinc was also investigated at the host-pathogen interface using a nutritionally zinc-limited mouse model of infection. During host infection, S. pneumoniae were found to predominantly encounter zinc limitation, even in the zinc-replete mice. This suggested that in the host environment, zinc homeostasis by S. pneumoniae is primarily determined by zinc acquisition, which is facilitated by AdcA and AdcAII, while CzcD permits fine-tuning of zinc accumulation. CzcD was also shown to be important for bacterial resistance to killing by phagocytic cells, which implicates zinc in a direct antimicrobial role against this pathogen during host infection.Collectively, the work presented in this study significantly advances our understanding of the mechanisms of zinc homeostasis by S. pneumoniae, particularly in the context of the host. These findings provide important insights into how this important human pathogen readily colonises and causes disease in humans, which is facilitated, in part, by the tight regulation of its cellular zinc quotient.