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Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phospho t ransferase s ystem (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes , has an EIIB Gat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes , harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Streptococcus pyogenes is an obligate human pathobiont associated with many disease states. Here, we present a model of S. pyogenes infection using intact murine epithelium. We were able to perform RNA sequencing to evaluate genetic changes undertaken by both the bacterium and host at 5 and 24 h post-infection. Analysis of these genomic data demonstrate that S. pyogenes undergoes genetic adaptation to successfully infect the murine epithelium, including changes to metabolism and activation of the Rgg2/Rgg3 quorum-sensing (QS) system. Subsequent experiments demonstrate that an intact Rgg2/Rgg3 QS cascade is necessary to establish a stable superficial skin infection. QS cascade activation results in increased murine morbidity and bacterial burden on the skin. This phenotype is associated with gross changes to the murine skin and with evidence of inflammation. These experiments offer a method to investigate S. pyogenes-epithelial interactions and demonstrate that a well-studied QS pathway is critical to a persistent infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Quorum sensing (QS) is a means of bacterial communication accomplished by microbe-produced signals and sensory systems. QS systems regulate important population-wide behaviors in bacteria, including secondary metabolite production, swarming motility, and bioluminescence. The human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) utilizes Rgg-SHP QS systems to regulate biofilm formation, protease production, and activation of cryptic competence pathways. Given their reliance on small-molecule signals, QS systems are attractive targets for small-molecule modulators that would then affect gene expression. In this study, a high-throughput luciferase assay was employed to screen an Actinobacteria -derived secondary metabolite (SM) fraction library to identify small molecule inhibitors of Rgg regulation. A metabolite produced by Streptomyces tendae D051 was found to be a general inhibitor of GAS Rgg-mediated QS. Herein, we describe the biological activity of this metabolite as a QS inhibitor. IMPORTANCE Streptococcus pyogenes, a human pathogen known for causing infections such as pharyngitis and necrotizing fasciitis, uses quorum sensing (QS) to regulate social responses in its environment. Previous studies have focused on disrupting QS as a means to control specific bacterial signaling outcomes. In this work, we identified and described the activity of a naturally derived S. pyogenes QS inhibitor. This study demonstrates that the inhibitor affects three separate but similar QS signaling pathways., Competing Interests: The authors declare no conflict of interest.

Streptococcus pneumoniae is an agent of otitis media, septicemia, and meningitis and remains the leading cause of community-acquired pneumonia regardless of vaccine use. Of the various strategies that S. pneumoniae takes to enhance its potential to colonize the human host, quorum sensing (QS) is an intercellular communication process that provides coordination of gene expression at a community level. Numerous putative QS systems are identifiable in the S. pneumoniae genome, but their gene-regulatory activities and contributions to fitness have yet to be fully evaluated. To contribute to assessing regulatory activities of rgg paralogs present in the D39 genome, we conducted transcriptomic analysis of mutants of six QS regulators. Our results find evidence that at least four QS regulators impact the expression of a polycistronic operon (encompassing genes spd_1517 to spd_1513 ) that is directly controlled by the Rgg/SHP1518 QS system. As an approach to unravel the convergent regulation placed on the spd_1513-1517 operon, we deployed transposon mutagenesis screening in search of upstream regulators of the Rgg/SHP1518 QS system. The screen identified two types of insertion mutants that result in increased activity of Rgg1518-dependent transcription, one type being where the transposon inserted into pepO , an annotated endopeptidase, and the other type being insertions in spxB , a pyruvate oxidase. We demonstrate that pneumococcal PepO degrades SHP1518 to prevent activation of Rgg/SHP1518 QS. Moreover, the glutamic acid residue in the conserved "HExxH" domain is indispensable for the catalytic function of PepO. Finally, we confirmed the metalloendopeptidase property of PepO, which requires zinc ions, but not other ions, to facilitate peptidyl hydrolysis. IMPORTANCE Streptococcus pneumoniae uses quorum sensing to communicate and regulate virulence. In our study, we focused on one Rgg quorum sensing system (Rgg/SHP1518) and found that multiple other Rgg regulators also control it. We further identified two enzymes that inhibit Rgg/SHP1518 signaling and revealed and validated one enzyme's mechanisms for breaking down quorum sensing signaling molecules. Our findings shed light on the complex regulatory network of quorum sensing in Streptococcus pneumoniae., Competing Interests: The authors declare no conflict of interest.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX ), whose expression is induced by two types of peptide signals: CSP ( c ompetence s timulating p eptide, encoded by comC ) and XIP ( sig X - i nducing p eptide, encoded by comS ). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus , but not homologs of S. mutans blpRH (also known as comDE ). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism., Competing Interests: The authors declare no conflict of interest.

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1β (IL-1β). However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1β production. A Δ sagB mutant fails to promote IL-1β production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1β production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a Δ sagB mutant leads to reduced caspase-independent cleavage of pro-IL-1β, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1β production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1β production by innate immune cells through a previously undescribed mechanism related to IL-1β maturation.

Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GAS's ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and tandem mass tag (TMT)-LC-MS/MS analysis on cells in which this system was induced with short hydrophobic peptide (SHP) pheromone or disrupted. Primary findings confirmed that pheromone stimulation in wild-type cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, a deletion mutant of rgg3 , a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions, a connection was identified between Rgg2/3 and the stringent response. Mutation of relA , the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB , impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. IMPORTANCE Streptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogenes's gene expression and, in extreme conditions, its physiology. Using transcriptomic and proteomic approaches, the results define the pheromone-dependent regulon of the Rgg2/3 quorum sensing system. In addition, we find that excessive pheromone stimulation, generated by genetic disruption of the Rgg2/3 system, leads to stress responses that are associated with the stringent response. Disruption of stringent response affects the ability of the cell-cell communication system to respond under normally inducing conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to nontraditional sources of stress.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes , including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Streptococcus pneumoniae, an opportunistic human pathogen, is the leading cause of community-acquired pneumonia and an agent of otitis media, septicemia, and meningitis. Although genomic and transcriptomic studies of S. pneumoniae have provided detailed perspectives on gene content and expression programs, they have lacked information pertaining to the translational landscape, particularly at a resolution that identifies commonly overlooked small open reading frames (sORFs), whose importance is increasingly realized in metabolism, regulation, and virulence. To identify protein-coding sORFs in S. pneumoniae, antibiotic-enhanced ribosome profiling was conducted. Using translation inhibitors, 114 novel sORFs were detected, and the expression of a subset of them was experimentally validated. Two loci associated with virulence and quorum sensing were examined in deeper detail. One such sORF, rio3 , overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio3 from srf-02 revealed that rio3 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in the genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome. IMPORTANCE This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae. We identified 114 unannotated intergenic small open reading frames (sORFs). The described procedures and data sets provide a model for microbiologists seeking to explore the translational landscape of bacteria. The biological roles of four sORF examples are characterized: two control the regulation of a cell-cell communication (quorum sensing) system, one contributes to the ability of S. pneumoniae to colonize the upper respiratory tract of mice, and a fourth governs the translation of PrfB, a protein enabling ribosome release at stop codons. We propose that Ribo-RET is a valuable approach to identifying unstudied microproteins and difficult-to-find pheromone genes used by Gram-positive organisms, whose genomes are replete with pheromone receptors.

Atypical antipsychotic (AAP) medication is a critical tool for treating symptoms of psychiatric disorders. While AAPs primarily target dopamine (D2) and serotonin (5HT2A and 5HT1A) receptors, they also exhibit intrinsic antimicrobial activity as an off-target effect. Because AAPs are often prescribed to patients for many years, a potential risk associated with long-term AAP use is the unintended emergence of bacteria with antimicrobial resistance (AMR). Here, we show that exposure to the AAP quetiapine at estimated gut concentrations promotes AMR in Escherichia coli after 6 weeks. Quetiapine-exposed isolates exhibited an increase in MICs for ampicillin, tetracycline, ceftriaxone, and levofloxacin. By whole-genome sequencing analysis, we identified mutations in genes that confer AMR, including the repressor for the multiple antibiotic resistance mar operon ( marR ), and real-time reverse transcription-quantitative PCR (RT-qPCR) analysis showed increased levels of marA , acrA , and tolC mRNAs and reduced levels of ompF mRNA in the isolates carrying marR mutations. To determine the contribution of each marR mutation to AMR, we constructed isogenic strains carrying individual mutant marR alleles in the parent background and reevaluated their resistance phenotypes using MIC and RT-qPCR assays. While marR mutations induced robust activity of the mar operon, they resulted in only modest increases in MICs. Interestingly, although these marR mutations did not fully recapitulate the AMR phenotype of the quetiapine-exposed isolates, we show that marR mutations promote growth fitness in the presence of quetiapine. Our findings revealed an important link between the use of AAPs and AMR development in E. coli. IMPORTANCE AAP medication is a cornerstone in the treatment of serious psychiatric disease. The AAPs are known to exhibit antimicrobial activity; therefore, a potential unintended risk of long-term AAP use may be the emergence of AMR, although such risk has received little attention. In this study, we describe the development of multidrug antibiotic resistance in Escherichia coli after 6 weeks of exposure to the AAP quetiapine. Investigation of mutations in the marR gene, which encodes a repressor for the multiple antibiotic resistance ( mar ) operon, reveals a potential mechanism that increases the fitness of E. coli in the presence of quetiapine. Our findings establish a link between the use of AAPs and AMR development in bacteria.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins., (© 2022. The Author(s).)

Streptococcus pyogenes, also known as group A Streptococcus or GAS, is a human-restricted pathogen causing a diverse array of infections. The ability to adapt to different niches requires GAS to adjust gene expression in response to environmental cues. We previously identified the abundance of biometals and carbohydrates led to natural induction of the Rgg2/3 cell-cell communication system (quorum sensing, QS). Here we determined the mechanism by which the Rgg2/3 QS system is stimulated exclusively by mannose and repressed by glucose, a phenomenon known as carbon catabolite repression (CCR). Instead of carbon catabolite protein A, the primary mediator of CCR in Gram-positive bacteria; CCR of Rgg2/3 requires the PTS regulatory domain (PRD)-containing transcriptional regulator Mga. Deletion of Mga led to carbohydrate-independent activation of Rgg2/3 by down-regulating rgg3, the QS repressor. Through phosphoablative and phosphomimetic substitutions within Mga PRDs, we demonstrated that selective phosphorylation of PRD1 conferred repression of the Rgg2/3 system. Moreover, given the carbohydrate specificity mediating Mga-dependent governance over Rgg2/3, we tested mannose-specific PTS components and found the EIIA/B subunit ManL was required for Mga-dependent repression. These findings provide newfound connections between PTS Man , Mga, and QS, and further demonstrate that Mga is a central regulatory nexus for integrating nutritional status and virulence., (© 2021 John Wiley & Sons Ltd.)

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-β) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. IMPORTANCE Streptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes , on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response., (Copyright © 2021 Rahbari et al.)

The genus Streptococcus encompasses a large bacterial taxon that commonly colonizes mucosal surfaces of vertebrates and is capable of disease etiologies originating from diverse body sites, including the respiratory, digestive, and reproductive tracts. Identifying new modes of treating infections is of increasing importance, as antibiotic resistance has escalated. Streptococcus mutans is an important opportunistic pathogen that is an agent of dental caries and is capable of systemic diseases such as endocarditis. As such, understanding how it regulates virulence and competes in the oral niche is a priority in developing strategies to defend from these pathogens. We determined that S. mutans UA159 possesses a bona fide short hydrophobic peptide (SHP)/Rgg quorum-sensing system that regulates a specialized biosynthetic operon featuring a radical-SAM ( S -adenosyl-l-methionine) (RaS) enzyme and produces a ribosomally synthesized and posttranslationally modified peptide (RiPP). The pairing of SHP/Rgg regulatory systems with RaS biosynthetic operons is conserved across streptococci, and a locus similar to that in S. mutans is found in Streptococcus ferus , an oral streptococcus isolated from wild rats. We identified the RaS-RiPP product from this operon and solved its structure using a combination of analytical methods; we term these RiPPs tryglysin A and B for the unusual Trp-Gly-Lys linkage. We report that tryglysins specifically inhibit the growth of other streptococci, but not other Gram-positive bacteria such as Enterococcus faecalis or Lactococcus lactis We predict that tryglysin is produced by S. mutans in its oral niche, thus inhibiting the growth of competing species, including several medically relevant streptococci. IMPORTANCE Bacteria interact and compete with a large community of organisms in their natural environment. Streptococcus mutans is one such organism, and it is an important member of the oral microbiota. We found that S. mutans uses a quorum-sensing system to regulate production of a novel posttranslationally modified peptide capable of inhibiting growth of several streptococcal species. We find inhibitory properties of a similar peptide produced by S. ferus and predict that these peptides play a role in interspecies competition in the oral niche., (Copyright © 2021 Rued et al.)

Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci., Competing Interests: The authors declare no competing interest.

Mammalian microbiomes encode thousands of biosynthetic gene clusters (BGCs) and represent a new frontier in natural product research. We recently found an abundance of quorum sensing-regulated BGCs in mammalian microbiome streptococci that code for ribosomally synthesized and post-translationally modified peptides (RiPPs) and contain one or more radical S-adenosylmethionine (RaS) enzymes, a versatile superfamily known to catalyze some of the most unusual reactions in biology. In the current work, we target a widespread group of streptococcal RiPP BGCs and elucidate both the reaction carried out by its encoded RaS enzyme and identify its peptide natural product, which we name streptosactin. Streptosactin is the first sactipeptide identified from Streptococcus spp.; it contains two sequential four amino acid sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays reveal potent but narrow-spectrum activity against the producing strain and its closest relatives that carry the same BGC, suggesting streptosactin may be a long-suspected fratricidal agent of Streptococcus thermophilus . Our results highlight mammalian streptococci as a rich source of unusual enzymatic chemistries and bioactive natural products.

Streptococcus pyogenes is a human-restricted pathogen most often found in the human nasopharynx. Multiple bacterial factors are known to contribute to persistent colonization of this niche, and many are important in mucosal immunity and vaccine development. In this work, mice were infected intranasally with transcriptional regulator mutants of the Rgg2/3 quorum sensing (QS) system-a peptide-based signaling system conserved in sequenced isolates of S. pyogenes Deletion of the QS system's transcriptional activator (Δ rgg2 ) dramatically diminished the percentage of colonized mice, while deletion of the transcriptional repressor (Δ rgg3 ) increased the percentage of colonized mice compared to that of the wild type (WT). Stimulation of the QS system using synthetic pheromones prior to inoculation did not significantly increase the percentage of animals colonized, indicating that QS-dependent colonization is responsive to the intrinsic conditions within the host upper respiratory tract. Bacterial RNA extracted directly from oropharyngeal swabs and evaluated by quantitative reverse transcription-PCR (qRT-PCR) subsequently confirmed QS upregulation within 1 h of inoculation. In the nasal-associated lymphoid tissue (NALT), a muted inflammatory response to the Δ rgg2 bacteria suggests that their rapid elimination failed to elicit the previously characterized response to intranasal inoculation of GAS. This work identifies a new transcriptional regulatory system governing the ability of S. pyogenes to colonize the nasopharynx and provides knowledge that could help lead to decolonization therapeutics., (Copyright © 2020 American Society for Microbiology.)

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro , affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS. IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae , binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization., (Copyright © 2019 Cook et al.)

Streptococcus pyogenes , or Group A Streptococcus (GAS), is a human-restricted pathogen most commonly found in the posterior oropharynx of the human host. The bacterium is responsible for 600 million annual cases of pharyngitis globally and has been found to asymptomatically colonize the pharynxes of 4-30% of the population. As such, many studies have utilized animals as models in order to decipher bacterial and host elements that contribute to the bacterial-pharyngeal interaction and determine differences between acute infection and asymptomatic colonization. The aim of this review is to first describe both bacterial and host factors that are important for the pharyngeal persistence of GAS in humans, then to detail the bacterial and host factors that are important for colonization in murine model, and finally to compare the two in order to evaluate the strength of murine pharyngeal colonization as a model for the human-GAS pharyngeal interaction.

Both cervical and throat cancers are associated with human papillomavirus (HPV). HPV infection requires cleavage of the minor capsid protein L2 by furin. While furin is present in the vaginal epithelium, it is absent in oral epithelial basal cells where HPV infection occurs. The objective of this study was to investigate whether common oral bacteria express furin-like peptidases. By screening strains representing 12 oral Streptococcus and Enterococcus species, we identified that eight Streptococcus strains displayed high levels of furin-like peptidase activity, with S. gordonii V2016 the highest. We constructed null mutations for 14 genes encoding putative endopeptidases in S. gordonii V2016. Results showed that three endopeptidases, PepO, PulO, and SepM, had furin-like activities. All three mutants showed decreased natural transformation by chromosomal DNA, while the pepO mutant also showed reduced transformation by plasmid DNA, indicating involvement of these endopeptidases in competence development. The purified S. gordonii PepO protein promoted infection of epithelial 293TT cells in vitro by HPV16 pseudovirus. In conclusion, oral bacteria might promote HPV infection and contribute to HPV tissue tropism and subsequent carcinogenesis in the oral cavity and throat by providing furin-like endopeptidases., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become "competent" and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus.

The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A Streptococcus (GAS; Streptococcus pyogenes ). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, spy49_0414c , abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides. IMPORTANCE This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface., (Copyright © 2018 American Society for Microbiology.)

Streptococcus agalactiae (group B streptococcus [GBS]) can colonize the human vaginal tract, leading to both superficial and serious infections in adults and neonates. To study bacterial colonization of the reproductive tract in a mammalian system, we employed a murine vaginal carriage model. Using transcriptome sequencing (RNA-Seq), the transcriptome of GBS growing in vivo during vaginal carriage was determined. Over one-quarter of the genes in GBS were found to be differentially regulated during in vivo colonization compared to laboratory cultures. A two-component system (TCS) homologous to the staphylococcal virulence regulator SaeRS was identified as being upregulated in vivo One of the SaeRS targets, pbsP , a proposed GBS vaccine candidate, is shown to be important for colonization of the vaginal tract. A component of vaginal lavage fluid acts as a signal to turn on pbsP expression via SaeRS. These data demonstrate the ability to quantify RNA expression directly from the murine vaginal tract and identify novel genes involved in vaginal colonization by GBS. They also provide more information about the regulation of an important virulence and colonization factor of GBS, pbsP , by the TCS SaeRS., (Copyright © 2018 American Society for Microbiology.)

Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2 Sd ). Here, we compared two Rgg2 Sd (C45S) X-ray crystal structures with that of wild-type Rgg2 Sd and found that in the absence of the intermolecular disulfide, the Rgg2 Sd dimer interface is destabilized and Rgg2 Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2 Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes ( rgg2 Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2 Sp *, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2 Sd (C45S) crystal structures. The Rgg2 Sp * mutations increased Rgg2 Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2 Sp * mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2 Sp ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Research on the Gram-positive human-restricted pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) has long focused on invasive illness, the most severe manifestations of GAS infection. Recent advances in descriptions of molecular mechanisms of GAS virulence, coupled with massive sequencing efforts to isolate genomes, have allowed the field to better understand the molecular and evolutionary changes leading to pandemic strains. These findings suggest that it is necessary to rethink the dogma involving GAS pathogenesis, and that the most productive avenues for research going forward may be investigations into GAS in its 'normal' habitat, the nasopharynx, and its ability to either live with its host in an asymptomatic lifestyle or as an agent of superficial infections. This review will consider these advances, focusing on the natural history of GAS, the evolution of pandemic strains, and novel roles for several key virulence factors that may allow the field to better understand their physiological role., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The human pathogens Streptococcus pneumoniae and Streptococcus mutans have both evolved complex quorum sensing (QS) systems that regulate the production of bacteriocins and the entry into the competent state, a requirement for natural transformation. Natural transformation provides bacteria with a mechanism to repair damaged genes or as a source of new advantageous traits. In S. pneumoniae , the competence pathway is controlled by the two-component signal transduction pathway ComCDE, which directly regulates SigX, the alternative sigma factor required for the initiation into competence. Over the past two decades, effectors of cellular killing (i.e., fratricides) have been recognized as important targets of the pneumococcal competence QS pathway. Recently, direct interactions between the ComCDE and the paralogous BlpRH pathway, regulating bacteriocin production, were identified, further strengthening the interconnections between these two QS systems. Interestingly, a similar theme is being revealed in S. mutans , the primary etiological agent of dental caries. This review compares the relationship between the bacteriocin and the competence QS pathways in both S. pneumoniae and S. mutans , and hopes to provide clues to regulatory pathways across the genus Streptococcus as a potential tool to efficiently investigate putative competence pathways in nontransformable streptococci., Competing Interests: The authors declare no conflict of interest or any role of the funding sponsors in the composition of this manuscript. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

A transposon mutagenesis screen designed to identify mutants that were defective in peptide-pheromone signaling of the Rgg2/Rgg3 pathway in Streptococcus pyogenes generated insertions in sixteen loci displaying diminished reporter activity. Fourteen unique transposon insertions were mapped to pptAB, an ABC-type transporter recently described to export sex pheromones of Enterococcus faecalis. Consistent with an idea that PptAB exports signaling peptides, the pheromones known as SHPs (short hydrophobic peptides) were no longer detected in cell-free culture supernatants in a generated deletion mutant of pptAB. PptAB exporters are conserved among the Firmicutes, but their function and substrates remain unclear. Therefore, we tested a pptAB mutant generated in Streptococcus mutans and found that while secretion of heterologously expressed SHP peptides required PptAB, secretion of the S. mutans endogenous pheromone XIP (sigX inducing peptide) was only partially disrupted, indicating that a secondary secretion pathway for XIP exists., Competing Interests: The authors have declared that no competing interests exist.

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a 'test-bed' assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity., Competing Interests: The authors have declared that no competing interests exist.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci., Competing Interests: The authors have declared that no competing interests exist.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg(2+) and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles., (© 2015 John Wiley & Sons Ltd.)

The human-restricted pathogen Streptococcus pyogenes (Group A Streptococcus, GAS) is responsible for wide-ranging pathologies at numerous sites in the body but has the proclivity to proliferate in individuals asymptomatically. The ability to survive in diverse tissues is undoubtedly benefited by sensory pathways that recognize environmental cues corresponding to stress and nutrient availability and thereby trigger adaptive responses. We investigated the impact that environmental signals contribute to cell-to-cell chemical communication [quorum sensing (QS)] by monitoring activity of the Rgg2/Rgg3 and SHP-pheromone system in GAS. We identified metal limitation and the alternate carbon source mannose as two environmental indicators likely to be encountered by GAS in the host that significantly induced the Rgg-SHP system. Disruption of the metal regulator MtsR partially accounted for the response to metal depletion, whereas ptsABCD was primarily responsible for QS induction due to mannose, but each sensory system induced Rgg-SHP signaling apparently by different mechanisms. Significantly, we found that induction of QS, regardless of the GAS serotype tested, led to enhanced resistance to the antimicrobial agent lysozyme. These results indicate the benefits for GAS to integrate environmental signals with intercellular communication pathways in protection from host defenses., (© 2015 John Wiley & Sons Ltd.)

Purpose: To analyze the bacterial microbiota colonizing the ocular surface of patients with Boston type 1 keratoprostheses (K-Pros) for antibacterial resistance patterns and capacity to form biofilms., Methods: Twenty-seven eyes with a Boston type 1 K-Pro and 16 fellow control eyes from 26 patients were enrolled. The surface of the K-Pro optic and/or the inferior conjunctival fornix was swabbed and plated separately on culture media. Positive cultures were processed to assess for biofilm-forming capability. Microtiter plate adherence assay and polymerase chain reaction for ica and atlE genes were used. An in vitro assay of vancomycin tolerance was performed on isolated strains and compared to standard controls with and without biofilm-forming capability., Results: Eighty-five percent of K-Pro eyes and 69% of control eyes had positive cultures (P = 0.20). All Gram-positive strains exhibited susceptibility to vancomycin by standard testing. Biofilm-forming bacterial isolates were detected in 57.7% of K-Pro eyes and 53.3% of control eyes. A vancomycin tolerance assay showed that the antibiotic susceptibility of coagulase-negative staphylococcus (CNS) within biofilms was significant in only three of five biofilm-forming strains (P < 0.05). In all strains, bacterial cells in planktonic form were more susceptible to vancomycin than in biofilm form (P < 0.001)., Conclusions: Coagulase-negative staphylococcus can be isolated from K-Pro surfaces despite the use of vancomycin prophylaxis. In this study, the majority of isolated strains had biofilm-forming capability. In vitro vancomycin tolerance assays suggest that biofilm formation decreases susceptibility to vancomycin. This may contribute to higher rates of infectious complications observed in these patients.

The 5 th American Society for Microbiology Conference on Cell-Cell Communication in Bacteria (CCCB-5), which convened from October 18 - 21, 2014 in San Antonio, Texas, highlighted recent advances in our understanding of microbial intercellular signaling. While the CCCB meetings arose from interests in pheromone signaling and quorum sensing, it was evident at CCCB-5 that the cell-cell communication field is continuing to mature, expanding into new areas and integrating cutting-edge technologies. In this review, we recap some of the research discussed at CCCB-5 as well as the questions that have arisen from it., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Bacteria coordinate a variety of social behaviors, important for both environmental and pathogenic bacteria, through a process of intercellular chemical signaling known as quorum sensing (QS). As microbial resistance to antibiotics grows more common, a critical need has emerged to develop novel anti-infective therapies, such as an ability to attenuate bacterial pathogens by means of QS interference. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development in response to the short hydrophobic peptides SHP2 and SHP3. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that could specifically inhibit Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt complexes of Rgg and FITC (fluorescein isothiocyanate)-conjugated SHP using a fluorescence polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with submicromolar affinities, to specifically abolish transcription of Rgg2/3-controlled genes, and to prevent biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit, cyclosporine A, as well as its nonimmunosuppressive analog, valspodar, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The Rgg-FITC-peptide-based screen provides a platform to identify inhibitors specific for each Rgg type. Discovery of Rgg inhibitors constitutes a step toward the goal of manipulating bacterial behavior for purposes of improving health., Importance: The global emergence of antibiotic-resistant bacterial infections necessitates discovery not only of new antimicrobials but also of novel drug targets. Since antibiotics restrict microbial growth, strong selective pressures to develop resistance emerge quickly in bacteria. A new strategy to fight microbial infections has been proposed, namely, development of therapies that decrease pathogenicity of invading organisms while not directly inhibiting their growth, thus decreasing selective pressure to establish resistance. One possible means to this goal is to interfere with chemical communication networks used by bacteria to coordinate group behaviors, which can include the synchronized expression of genes that lead to disease. In this study, we identified chemical compounds that disrupt communication pathways regulated by Rgg proteins in species of Streptococcus. Treatment of cultures of S. pyogenes with the inhibitors diminished the development of biofilms, demonstrating an ability to control bacterial behavior with chemicals that do not inhibit growth., (Copyright © 2015 Aggarwal et al.)

Peptide pheromone cell-cell signaling (quorum sensing) regulates the expression of diverse developmental phenotypes (including virulence) in Firmicutes, which includes common human pathogens, e.g., Streptococcus pyogenes and Streptococcus pneumoniae. Cytoplasmic transcription factors known as "Rgg proteins" are peptide pheromone receptors ubiquitous in Firmicutes. Here we present X-ray crystal structures of a Streptococcus Rgg protein alone and in complex with a tight-binding signaling antagonist, the cyclic undecapeptide cyclosporin A. To our knowledge, these represent the first Rgg protein X-ray crystal structures. Based on the results of extensive structure-function analysis, we reveal the peptide pheromone-binding site and the mechanism by which cyclosporin A inhibits activation of the peptide pheromone receptor. Guided by the Rgg-cyclosporin A complex structure, we predicted that the nonimmunosuppressive cyclosporin A analog valspodar would inhibit Rgg activation. Indeed, we found that, like cyclosporin A, valspodar inhibits peptide pheromone activation of conserved Rgg proteins in medically relevant Streptococcus species. Finally, the crystal structures presented here revealed that the Rgg protein DNA-binding domains are covalently linked across their dimerization interface by a disulfide bond formed by a highly conserved cysteine. The DNA-binding domain dimerization interface observed in our structures is essentially identical to the interfaces previously described for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface appears to be unique. We hypothesize that this disulfide bond may, under the right conditions, affect Rgg monomer-dimer equilibrium, stabilize Rgg conformation, or serve as a redox-sensitive switch.

Quorum sensing (QS) is a widespread phenomenon in the microbial world that has important implications in the coordination of population-wide responses in several bacterial pathogens. In Group A Streptococcus (GAS), many questions surrounding QS systems remain to be solved pertaining to their function and their contribution to the GAS lifestyle in the host. The QS systems of GAS described to date can be categorized into four groups: regulator gene of glucosyltransferase (Rgg), Sil, lantibiotic systems, and LuxS/AI-2. The Rgg family of proteins, a conserved group of transcription factors that modify their activity in response to signaling peptides, has been shown to regulate genes involved in virulence, biofilm formation and competence. The sil locus, whose expression is regulated by the activity of signaling peptides and a putative two-component system (TCS), has been implicated on regulating genes involved with invasive disease in GAS isolates. Lantibiotic regulatory systems are involved in the production of bacteriocins and their autoregulation, and some of these genes have been shown to target both bacterial organisms as well as processes of survival inside the infected host. Finally AI-2 (dihydroxy pentanedione, DPD), synthesized by the LuxS enzyme in several bacteria including GAS, has been proposed to be a universal bacterial communication molecule. In this review we discuss the mechanisms of these four systems, the putative functions of their targets, and pose critical questions for future studies.

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Group A streptococcus (GAS) commonly colonizes the oropharynx and nonintact skin. However, colonization has been little studied and the role of biofilm formation is unclear, as biofilm experiments to date have not been conducted under conditions that mimic the host environment., Methods: In this study we grew GAS biofilms on human keratinocytes under various environmental conditions and used this model to evaluate colonization, invasive disease and natural transformation., Results: GAS grown on epithelial cells, but not biofilms grown on abiotic surfaces, produced biofilms with characteristics similar to in vivo colonization. These biofilm bacteria showed a 100-fold higher bacterial burden of nasal-associated lymphoid tissue in mice than broth-grown bacteria, and were not virulent during septic infection, which was attributed in part to down-regulation of genes typically involved in localized and invasive disease. We also showed for the first time that GAS were naturally transformable when grown in biofilms and during colonization of NALT in vivo., Conclusions: These findings provide novel model systems to study biofilm formation of GAS in vitro and in vivo, suggest an important role for biofilm formation during GAS colonization, and provide an explanation for the known genome diversity within the GAS population.

Intercellular chemical signaling in bacteria, commonly referred to as quorum sensing (QS), relies on the production and detection of compounds known as pheromones to elicit coordinated responses among members of a community. Pheromones produced by Gram-positive bacteria are comprised of small peptides. Based on both peptide structure and sensory system architectures, Gram-positive bacterial signaling pathways may be classified into one of four groups with a defining hallmark: cyclical peptides of the Agr type, peptides that contain Gly-Gly processing motifs, sensory systems of the RNPP family, or the recently characterized Rgg-like regulatory family. The recent discovery that Rgg family members respond to peptide pheromones increases substantially the number of species in which QS is likely a key regulatory component. These pathways control a variety of fundamental behaviors including conjugation, natural competence for transformation, biofilm development, and virulence factor regulation. Overlapping QS pathways found in multiple species and pathways that utilize conserved peptide pheromones provide opportunities for interspecies communication. Here we review pheromone signaling identified in the genera Enterococcus and Streptococcus, providing examples of all four types of pathways., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released., (© 2013.)

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ∼ 0.5-1 mg/L of E. coli culture., (© 2013 American Institute of Chemical Engineers.)

All bacterial quorum sensing (QS) systems are based on the production, secretion, and detection of small signaling molecules. Gram-positive bacteria typically use small peptides as QS effectors, and each QS circuit generally requires the interaction of a single signaling molecule with a single receptor protein. The recently described Rgg2 and Rgg3 (Rgg2/3) regulatory circuit of Streptococcus pyogenes (group A streptococcus [GAS]) is one of only a few QS circuits known to utilize multiple signaling peptides. In this system, two distinct, endogenously produced peptide pheromones (SHP2 and SHP3) both function to activate the QS circuit. The aim of this study was to further define the roles of SHP2 and SHP3 in activation of the Rgg2/3 QS system, specifically with regard to shp gene identity and dosage. Results from our studies using transcriptional reporters and isogenic GAS mutants demonstrate that shp gene dosage does contribute to Rgg2/3 system induction, as decreased gene dosage results in decreased or absent induction. Beyond this, however, data indicate that the shp genes possess distinct potentials for supporting system activation, with shp3 more readily able to support system activation than shp2. Studies using synthetic peptides and shp gene mutants indicate that the disparate activities of endogenous SHPs are due to production, rather than signaling, differences and are conferred by the N-terminal regions rather than the C-terminal signaling regions of the peptides. These data provide evidence that the N-terminal, noneffector sequences of SHP pheromones influence their production efficiencies and thereby the relative activation potentials of endogenous SHPs.

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Unlabelled: Quorum sensing (QS) regulates diverse and coordinated behaviors in bacteria, including the production of virulence factors, biofilm formation, sporulation, and competence development. It is now established that some streptococci utilize Rgg-type proteins in concert with short hydrophobic peptides (SHPs) to mediate QS, and sequence analysis reveals that several streptococcal species contain highly homologous Rgg/SHP pairs. In group A streptococcus (GAS), two SHPs (SHP2 and SHP3 [SHP2/3]) were previously identified to be important in GAS biofilm formation. SHP2/3 are detected by two antagonistic regulators, Rgg2 and Rgg3, which control expression of the shp genes. In group B streptococcus (GBS), RovS is a known virulence gene regulator and ortholog of Rgg2, whereas no apparent Rgg3 homolog exists. Adjacent to rovS is a gene (shp1520) encoding a peptide nearly identical to SHP2. Using isogenic mutant strains and transcriptional reporters, we confirmed that RovS/SHP1520 comprise a QS circuit in GBS. More important, we performed experiments demonstrating that production and secretion of SHP1520 by GBS can modulate Rgg2/3-regulated gene expression in GAS in trans; likewise, SHP2/3 production by GAS can stimulate RovS-mediated gene regulation in GBS. An isolate of Streptococcus dysgalactiae subsp. equisimilis also produced a secreted factor capable of simulating the QS circuits of both GAS and GBS, and sequencing confirms the presence of an orthologous Rgg2/SHP2 pair in this species as well. To our knowledge, this is the first documented case of bidirectional signaling between streptococcal species in coculture and suggests a role for orthologous Rgg/SHP systems in interspecies communication between important human pathogens., Importance: Pathogenic streptococci, such as group A (GAS) and group B (GBS) streptococcus, are able to persist in the human body without causing disease but become pathogenic under certain conditions that are not fully characterized. Environmental cues and interspecies signaling between members of the human flora likely play an important role in the transition to a disease state. Since quorum-sensing (QS) peptides have been consistently shown to regulate virulence factor production in pathogenic species, the ability of bacteria to signal via these peptides may prove to be an important link between the carrier and pathogenic states. Here we provide evidence of a bidirectional QS system between GAS, GBS, and Streptococcus dysgalactiae subsp. equisimilis, demonstrating the possibility of evolved communication systems between human pathogens.