Successful pathogens use complex signaling mechanisms to monitor their environment and reprogram global gene expression during specific stages of infection. Group A (GAS) is a major human pathogen that causes significant disease burden worldwide. A secreted cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB) is a key virulence factor that is produced abundantly during infection and is critical for GAS pathogenesis. Although identified nearly a century ago, the molecular basis for growth phase control of gene expression remains unknown. We have discovered that GAS uses a previously unknown peptide-mediated intercellular signaling system to control SpeB production, alter global gene expression, and enhance virulence. GAS produces an eight-amino acid leaderless peptide [SpeB-inducing peptide (SIP)] during high cell density and uses the secreted peptide for cell-to-cell signaling to induce population-wide expression. The SIP signaling pathway includes peptide secretion, reimportation into the cytosol, and interaction with the intracellular global gene regulator Regulator of Protease B (RopB), resulting in SIP-dependent modulation of DNA binding and regulatory activity of RopB. Notably, SIP signaling causes differential expression of ∼14% of GAS core genes. Several genes that encode toxins and other virulence genes that enhance pathogen dissemination and infection are significantly up-regulated. Using three mouse infection models, we show that the SIP signaling pathway is active during infection and contributes significantly to GAS pathogenesis at multiple host anatomic sites. Together, our results delineate the molecular mechanisms involved in a previously undescribed virulence regulatory pathway of an important human pathogen and suggest new therapeutic strategies.

Bacteria control gene expression in concert with their population density by a process called quorum sensing, which is modulated by bacterial chemical signals and environmental factors. In the human pathogen Streptococcus pyogenes , production of secreted virulence factor SpeB is controlled by a quorum-sensing pathway and environmental pH. The quorum-sensing pathway consists of a secreted leaderless peptide signal (SIP), and its cognate receptor RopB. Here, we report that the SIP quorum-sensing pathway has a pH-sensing mechanism operative through a pH-sensitive histidine switch located at the base of the SIP-binding pocket of RopB. Environmental acidification induces protonation of His144 and reorganization of hydrogen bonding networks in RopB, which facilitates SIP recognition. The convergence of two disparate signals in the SIP signaling pathway results in induction of SpeB production and increased bacterial virulence. Our findings provide a model for investigating analogous crosstalk in other microorganisms.

Group A Streptococcus (GAS) is a human-only pathogen that causes a spectrum of disease conditions. Given its survival in inflamed lesions, the ability to sense and overcome oxidative stress is critical for GAS pathogenesis. PerR senses oxidative stress and coordinates the regulation of genes involved in GAS antioxidant defenses. In this study, we investigated the role of PerR-controlled metal transporter A (PmtA) in GAS pathogenesis. Previously, PmtA was implicated in GAS antioxidant defenses and suggested to protect against zinc toxicity. Here, we report that PmtA is a P 1B4 -type ATPase that functions as an Fe(II) exporter and aids GAS defenses against iron intoxication and oxidative stress. The expression of pmtA is specifically induced by excess iron, and this induction requires PerR. Furthermore, a pmtA mutant exhibited increased sensitivity to iron toxicity and oxidative stress due to an elevated intracellular accumulation of iron. RNA-sequencing analysis revealed that GAS undergoes significant alterations in gene expression to adapt to iron toxicity. Finally, using two mouse models of invasive infection, we demonstrated that iron efflux by PmtA is critical for bacterial survival during infection and GAS virulence. Together, these data demonstrate that PmtA is a key component of GAS antioxidant defenses and contributes significantly to GAS virulence.KEYWORDS bacterial pathogenesis, gene regulation, iron efflux, metal homeostasis, oxidative stress I ron is a critical micronutrient required for bacterial survival and proliferation due to its role as a cofactor in cellular macromolecules involved in metabolism and electron transport. Given the bacterial requirement for iron, hosts limit iron availability to pathogens by using a variety of extracellular and intracellular mechanisms (1, 2). The eukaryotic host recruits an array of extracellular antimicrobial factors to chelate iron present at the microbial colonization surfaces to retard bacterial growth (2-4). As a countermeasure, pathogens employ high-affinity iron importers and iron-scavenging siderophores to acquire metal ions from nutrient-sparse infection sites (5). Consistent with this, the inactivation of iron importers resulted in an attenuated virulence of several pathogens, underscoring their importance to bacterial pathogenesis (5-7). Emerging evidence indicates that the host also employs metal toxicity at microbial colonization surfaces, predominantly within phagosomes, to mediate microbial killing and control bacterial infection (8,9). Host innate immune cells, such as neutrophils and macrophages, accumulate copper and zinc around phagocytosed bacteria and impose metal toxicity (8-12). Conversely, bacteria employ metal efflux pumps to reduce the intracellular metal concentration and negate host-induced metal toxicity (8,11,12). In

Type VI CRISPR-Cas systems use the RNA-guided RNase Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. Here we show that Csx28, of Type VI-B2 systems, forms membrane pore structures to slow cellular metabolism upon viral infection, and this activity drastically increases anti-viral defense. High- resolution cryo-EM reveals that Csx28 exists unexpectedly as a detergent-encapsulated octameric pore, and we then show these Csx28 pores are membrane localized in vivo. Activation of Csx28 in vivo strictly requires sequence-specific recognition of viral mRNAs by Cas13b, and this activation results in Csx28-mediated membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Together, our work reveals an unprecedented mechanism by which Csx28 acts as a downstream, Cas13b-activated, effector protein that uses membrane perturbation as an anti-viral defense strategy.

Bacterial virulence factor production is a highly coordinated process. The temporal pattern of bacterial gene expression varies in different host anatomic sites to overcome niche-specific challenges. The human pathogen group A streptococcus (GAS) produces a potent secreted protease, SpeB, that is crucial for pathogenesis. Recently, we discovered that a quorum sensing pathway comprised of a leaderless short peptide, SpeB-inducing peptide (SIP), and a cytosolic global regulator, RopB, controls expression in concert with bacterial population density. The SIP signaling pathway is active and contributes significantly to GAS invasive infections. In the current study, we investigated the role of the SIP signaling pathway in GAS-host interactions during oropharyngeal colonization. The SIP signaling pathway is functional during growth in human saliva. SIP-mediated expression plays a crucial role in GAS colonization of the mouse oropharynx. GAS employs a distinct pattern of SpeB production during growth in saliva that includes a transient burst of expression during early stages of growth coupled with sustained levels of secreted SpeB protein. SpeB production aids GAS survival by degrading LL37, an abundant human antimicrobial peptide. We found that SIP signaling occurs during growth in human blood o. Moreover, the SIP signaling pathway is critical for GAS survival in blood. SIP-dependent regulation is functional in strains of diverse types, indicating that SIP signaling is a conserved virulence regulatory mechanism. Our discoveries have implications for future translational studies.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo–electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28’s antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.

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