Although the role of CP against pneumococcal infection has

Although the role of CP against pneumococcal infection has been investigated, the two studies provided opposing findings with varying mechanisms for CP to either inhibit or facilitate pneumococcal virulence. One study reported that CP is an anti-pneumococcal host factor that inhibits bacterial growth during infection (De Filippo, et al., 2014), whereas a second investigation suggested that CP facilitates bacterial infection (Achouiti, et al., 2014). Thus, additional investigations are required to elucidate the precise role of CP against pneumococcal infection. In this regard, results from this study provided significant evidence that GAS encounters CP-mediated Zn limitation during invasive infection and indicated similar susceptibility of other streptococcal pathogens to CP. The observations that CP is abundant at GAS infection sites, CP imposes Zn limitation on GAS, and adaptive responses to Zn starvation are highly upregulated during GAS invasive infections lend support to our findings (Brenot et al., 2007). However, it is likely that GAS may encounter additional nutritional immune mechanisms such as zinc toxicity in other host compartments during infection (Ong et al., 2014). The GAS arsenals to combat CP-mediated zinc withholding include the high-affinity zinc importer, AdcABC and the zinc sensor, AdcR. Although several studies demonstrated the importance of metal importers to bacterial pathogenesis (Ammendola et al., 2007; Campoy et al., 2002; Davis et al., 2009; Bayle, et al., 2011; Sabri, et al., 2009), this study delineated the direct role of a zinc transporter in evading host nutritional immune defenses and highlighted its significant contribution to GAS survival during infection (Fig. 2). The adcCB-inactivated strain is unable to compete against CP due to defective zinc acquisition, which likely results in growth retardation and premature bacterial clearance from the site of infection. Consistent with this, ∆ mutant exhibited reduced in vivo survival and significantly attenuated virulence compared to WT and trans-complemented strains. Furthermore, GAS actively monitors alterations in zinc availability during infection and mounts a measured induction of adaptive responses that correlates with the degree of buy Dorsomorphin in zinc concentration. AdcR employs two metal-sensing sites to monitor zinc availability, and each site has defined biological roles in gene regulation and GAS pathogenesis. Metallation of the primary zinc-sensing site is essential for AdcR-promoter interactions and transcription repression, and vacant site 1 results in full derepression of regulated genes. The secondary zinc-sensing site has a lesser regulatory role as zinc binding at this site causes tighter repression, and loss of zinc at site 2 leads to lower levels of target gene derepression. Such gradual gene regulation by AdcR may afford significant growth advantage as it allows the pathogen to sense and respond to broader range of alterations in zinc concentrations. CP levels are relatively low during early stages of infection, which likely imposes milder zinc limitation on the pathogen and requires lower level induction of GAS adaptive responses. As CP concentration increases during later stages of infection, a further drop in zinc concentration may necessitate robust induction of adaptive responses. Varied target gene regulation in response to altering zinc concentration has been observed in zinc metalloregulators, Zur from B. subtilis and Streptomyces coelicolor, and Zap1 from Saccharomyces cerevisiae (Ma et al., 2011; North et al., 2012; Shin et al., 2011; Shin and Helmann, 2016; Wu et al., 2008). However, this is the first study demonstrating the functional relevance of gradual gene regulation by a metalloregulator to the virulence of a human pathogen. Since the GAS-host confrontation for zinc occurs during early stages of infection and zinc acquisition systems are upregulated to disarm host defenses (Brenot et al., 2007), we hypothesized that bacterial zinc uptake machinery could be targeted as a protective vaccine against GAS infection. To probe this, we demonstrated that the extracellular component of zinc uptake system, AdcA, is expressed during infection, accessible on the bacterial surface, antigenic, and immunization with recombinant AdcA-NTD confers protection against systemic GAS infection (Fig. 4). Importantly, as a vaccine candidate, AdcA has the potential to overcome longstanding barriers in GAS vaccine research. AdcA is highly conserved and elicited protection against different tested GAS serotypes, thus increasing the likelihood of conferring protection across multiple GAS serotypes (Figs. 4, and S6). Structural modeling indicated the lack of coiled-coil structures in AdcA, as observed in M protein structures (Kirvan et al., 2014; McNamara et al., 2008), which minimizes the possibility for elicitation of autoimmune reactions against human cardiac tissues, the etiology behind rheumatic heart disease (Figs. S7 and S8). Finally, all the tested representatives of different groups of streptococci exhibited susceptibility to CP and encode structurally similar zinc acquisition machinery in their genomes (Figs. 5, and S8), suggesting that other streptococcal pathogens as well as L. monocytogenes may engage in a nutrient battle against the host during infection. Thus, a vaccine developed against AdcA would likely be protective against many pathogenic streptococci. In conclusion, we have identified and delineated the roles of the major players in the arms race between GAS and the host for zinc, and demonstrated the translational potential of this signaling pathway for development of an effective GAS vaccine.