Zinc is an essential nutrient for all forms of life; however, it can also be significantly toxic in excess. Zinc abundance can be manipulated by the innate immune response to bacterial infection, facilitating the clearance of invading pathogens by either restricting or intoxicating bacteria with zinc. Resistance to the antimicrobial activity of zinc occurs via zinc-responsive transcriptional regulators that control intracellular zinc abundance by regulating zinc import and export proteins.
Streptococcus pneumoniae is the causative agent of pneumonia, meningitis and acute otitis media, and is responsible for more than 1 million deaths worldwide each year. In S. pneumoniae, the cation diffusion facilitator (CDF) family protein, CzcD, has been shown to serve in resisting zinc stress, however, a detailed analysis was lacking. Here, we comprehensively investigated the metal ion substrates of CzcD using a combination of microbiological and biochemical approaches in the virulent S. pneumoniae D39 strain. Transcriptional analyses revealed czcD is upregulated under zinc, cobalt and nickel stress. Despite this, phenotypic growth studies and metal ion accumulation analyses revealed that while CzcD provides protection against zinc and cobalt, it does not provide resistance to nickel stress. Recent studies have shown that zinc toxicity is exploited by phagocytic cells, wherein zinc is directed towards bacteria within the phagolysosomes. We examined the contribution of CzcD to S. pneumoniae survival within THP-1 macrophage-like cells, which showed that deletion of czcD impaired survival. Biochemical analyses of recombinant CzcD revealed that the protein binds zinc with an affinity similar to that of YiiP, the archetype of the CDF family. Collectively, these data provide new insight into the regulation of zinc and cobalt stress in S. pneumoniae, highlight the role of CzcD in resisting macrophage-mediated killing, and provide a foundation for structural and biochemical studies of CzcD to elucidate the molecular basis of metal ion transport.