Copper Tolerance and Characterization of a Copper-Responsive Operon, copYAZ, in an M1T1 Clinical Strain of Streptococcus pyogenes

Infection with Streptococcus pyogenes is associated with a breadth of clinical manifestations ranging frommild pharyngitis to severe necrotizing fasciitis. Elevated levels of intracellular copper are highly toxic to this bacterium, and thus, the microbe must tightly regulate the level of this metal ion by one or more mechanisms, which have, to date, not been clearly defined. In this study, we have identified two virulence mechanisms by which S. pyogenes protects itself against copper toxicity. We defined a set of putative genes, copY (for a regulator), copA (for a P1-type ATPase), and copZ (for a copper chaperone), whose expression is regulated by copper. Our results indicate that these genes are highly conserved among a range of clinical S. pyogenes isolates. The copY, copA, and copZ genes are induced by copper and are transcribed as a single unit. Heterologous expression assays revealed that S. pyogenes CopA can confer copper tolerance in a copper-sensitive Escherichia colimutant by preventing the accumulation of toxic levels of copper, a finding that is consistent with a role for CopA in copper export. Evaluation of the effect of copper stress on S. pyogenes in a planktonic or biofilm state revealed that biofilms may aid in protection during initial exposure to cop-per. However, copper stress appears to prevent the shift from the planktonic to the biofilm state. Therefore, our results indicate that S. pyogenesmay use several virulence mechanisms, including altered gene expression and a transition to and from plank-tonic and biofilm states, to promote survival during copper stress. IMPORTANC