Streptococcus pneumoniae (pneumococcus) is a common colonizer of the human upper respiratory tract, and its colonization can lead to sinusitis, pneumonia, otitis media, meningitis, and/or septicemia. Pneumococcal infections result in over one million deaths per year worldwide. Recent work has shown that that a key host defense mechanism against pneumococcus and other respiratory pathogens such as Mycobacterium tuberculosis involves employing to infection sites Zn laden phagocytes, which poison these organisms with Zn. Exposure of these bacteria to excessive Zn concentrations causes intoxication as a consequence of mismetallation of Mn-dependent enzymes. To avoid Zn poisoning, pneumococcus must rapidly respond to changes in the levels of this transition metal in its environment. Small regulatory RNAs (sRNAs) have a pivotal role in mediating, rapid responses to stresses by co- or post-transcriptionally regulating gene expression. However, there is little published information regarding how sRNAs contribute to S. pneumoniae physiology and virulence. The overall objective of this proposal is to understand how the Ccn sRNAs control S. pneumoniae Zn homeostasis. This project is based upon preliminary data showing that the Ccn sRNAs are important for S. pneumoniae virulence and resistance to Zn at concentrations found within the nasopharynx and blood of its host during infection. The central hypothesis for this proposal is that Ccn sRNAs mediate the rapid, adaptive response of S. pneumoniae to excess Zn. In Aim 1, the function of the Ccn sRNAs in regulating gene expression in the presence or absence of Zn stress will be established. In Aim 2, the mechanism by which the Ccn sRNAs prevent Zn intoxication will be elucidated. The significance of the proposed research is that its completion will result in a greater understanding of sRNA-mediated gene regulation and mechanisms of Zn stress response in S. pneumoniae and other Gram-positive bacteria. This proposed research is innovative, because (1) novel approaches will be utilized to identify the targets of S. pneumoniae sRNAs, (2) the proposed work will establish for the first time the function of sRNAs in providing another layer of control of Zn homeostasis, and (3) these studies should reveal the role of a newly identified transporter in maintaining Zn levels.