Understanding and exploiting zinc toxicity in Streptococcus pneumoniae

Abstract

Antibiotic resistance is associated with increased treatment costs, longer hospital durations and higher rates of mortality. To address this globally significant challenge, novel strategies to control antibiotic resistant pathogens are essential. The world’s foremost bacterial pathogen, Streptococcus pneumoniae, is responsible for more than one million deaths and an estimated 14.5 million cases of serious disease each year. The metal ion zinc has a critical antimicrobial role in innate immune defence and a dietary deficiency in zinc is associated with a marked increase in susceptibility to bacterial infections. Despite this association, precisely how zinc contributes to the control of bacterial infection is poorly defined. The major aim of this thesis was to understand and exploit the antibacterial action of zinc as a novel treatment against S. pneumoniae. A combination of bioinformatic and structural modelling analyses drew on insights from the canonical cation diffusion facilitator family member, YiiP from E. coli, to explore the zinc resistance protein, CzcD, in S. pneumoniae. Further phenotypic analyses showed that S. pneumoniae CzcD directly contributes to pneumococcal zinc efflux and that this protein is important for resistance to zinc stress. Further, this work showed that CzcD contributed to survival within macrophages, indicating that the antimicrobial action of zinc is exploited to aid in the clearance of S. pneumoniae in phagocytic cells. Despite the importance of zinc at the host-pathogen interface and its known antimicrobial role, the basis for zinc toxicity in S. pneumoniae is poorly defined. This work addressed this issue by investigating the pathways affected by zinc stress in S. pneumoniae. Transcriptomic and metabolomic studies showed that zinc altered key pneumococcal pathways, including central carbon metabolism, the pentose phosphate pathway, cell wall biosynthesis and de novo fatty acid biosynthesis. Of these pathways, further activity and phenotypic analyses showed that zinc may impair the activity of a number of putative protein targets, including glycolytic enzyme PfkA, pentose phosphate pathway enzyme Gnd, and the peptidoglycan biosynthesis enzyme GlmU. The capacity for zinc to impair these essential pathways directed studies to assess the influence of zinc on antibiotic action. Abolishment of zinc efflux via CzcD led to enhanced susceptibility to penicillin, indicating that excess intracellular zinc is capable of enhancing susceptibility to antibiotics. Building on these findings, the ionophore activity of the safe for human use drug, PBT2, was repurposed to increase cellular zinc in S. pneumoniae, which rendered antibiotic resistant S. pneumoniae susceptible to a range of antibiotics. Despite in vitro success, preliminary transcriptional studies of S. pneumoniae from murine tissues highlighted the complexity associated with the translation of this treatment strategy. Nevertheless, this work provides a foundation for future studies to further optimise ionophore-induced bacterial zinc intoxication in vivo and therefore enhance pneumococcal susceptibility to antibiotic treatment. Together, the work presented in this thesis significantly expands our understanding of the antimicrobial action of zinc, which may apply to a range of bacterial pathogens. This work describes a novel treatment approach where zinc toxicity may be harnessed to combat antibiotic-resistant S. pneumoniae disease. As antibiotic efficacy continues to decline, insights gained in this study may be applied to the treatment of a range of other clinically significant bacterial pathogens. Together, the improved antibiotic efficacy by zinc may provide a safety net to bridge the gap until the development of novel antimicrobial strategies to combat bacterial disease.