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The trend of marine non-indigenous species in India has been increasing, with more than half of the species probably being introduced by shipping. A live specimen of buckler crab Cryptopodia angulata was found along the west coast of India at 40 m depth. The recent new records at different Indian coastal locations suggest that the crab is widening its distribution. Shipping is thought to be the possible introduction vector (via ballast) for the spread of C. angulata in the coastal waters of India. Further, the favorable environmental conditions prevalent in the Indian coastal waters may facilitate the establishment and subsequent spread of C. angulata. The invasion of this buckler crab may have negative impact on the native species. Although not present in detectable numbers, C. angulata may pose a major threat to the native species, if it establishes. Information on the establishment and distribution of C. angulata from other locations along the Indian coast would be essential to comprehensively and effectively address the threat. [ABSTRACT FROM AUTHOR]

The interplay between host nutritional immune mechanisms and bacterial nutrient uptake systems has a major impact on the disease outcome. The host immune factor calprotectin (CP) limits the availability of essential transition metals, such as manganese (Mn) and zinc (Zn), to control the growth of invading pathogens. We previously demonstrated that the competition between CP and the human pathogen group A streptococcus (GAS) for Zn impacts GAS pathogenesis. However, the contribution of Mn sequestration by CP in GAS infection control and the role of GAS Mn acquisition systems in overcoming host-imposed Mn limitation remain unknown. Using a combination of in vitro and in vivo studies, we show that GAS-encoded mtsABC is a Mn uptake system that aids bacterial evasion of CP-imposed Mn scarcity and promotes GAS virulence. Mn deficiency caused by either the inactivation of mtsC or CP also impaired the protective function of GAS-encoded Mn-dependent superoxide dismutase. Our ex vivo studies using human saliva show that saliva is a Mn-scant body fluid, and Mn acquisition by MtsABC is critical for GAS survival in human saliva. Finally, animal infection studies using wild-type (WT) and CP-/ - mice showed that MtsABC is critical for GAS virulence in WT mice but dispensable in mice lacking CP, indicating the direct interplay between MtsABC and CP in vivo . Together, our studies elucidate the role of the Mn import system in GAS evasion of host-imposed metal sequestration and underscore the translational potential of MtsABC as a therapeutic or prophylactic target., Competing Interests: The authors declare no conflict of interest.

Probiotic supplements are suggested to promote human health by preventing pathogen colonization. However, the mechanistic bases for their efficacy in vivo are largely uncharacterized. Here using metabolomics and bacterial genetics, we show that the human oral probiotic Streptococcus salivarius K12 (SAL) produces salivabactin, an antibiotic that effectively inhibits pathogenic Streptococcus pyogenes (GAS) in vitro and in mice. However, prophylactic dosing with SAL enhanced GAS colonization in mice and ex vivo in human saliva. We showed that, on co-colonization, GAS responds to a SAL intercellular peptide signal that controls SAL salivabactin production. GAS produces a secreted protease, SpeB, that targets SAL-derived salivaricins and enhances GAS survival. Using this knowledge, we re-engineered probiotic SAL to prevent signal eavesdropping by GAS and potentiate SAL antimicrobials. This engineered probiotic demonstrated superior efficacy in preventing GAS colonization in vivo. Our findings show that knowledge of interspecies interactions can identify antibiotic- and probiotic-based strategies to combat infection., (© 2024. The Author(s).)

The human pathogen Streptococcus pyogenes secretes a short peptide (leaderless communication peptide, LCP) that mediates intercellular communication and controls bacterial virulence through interaction with its receptor, RopB. Here, we show that LCP and RopB homologues are present in other Firmicutes. We experimentally validate that LCPs with distinct peptide communication codes act as bacterial intercellular signals and regulate gene expression in Streptococcus salivarius, Streptococcus porcinus, Enterococcus malodoratus and Limosilactobacillus reuteri. Our results indicate that LCPs are more widespread than previously thought, and their characterization may uncover new signaling mechanisms and roles in coordinating diverse bacterial traits., (© 2023. Springer Nature Limited.)

Pathogenic streptococci require manganese for survival in the host. In response to invading pathogens, the host recruits nutritional immune effectors at infection sites to withhold manganese from the pathogens and control bacterial growth. The manganese scarcity impairs several streptococcal processes including oxidative stress defenses, de novo DNA synthesis, bacterial survival, and virulence. Emerging evidence suggests that pathogens also encounter manganese toxicity during infection and manganese excess impacts streptococcal virulence by manganese mismetallation of non-cognate molecular targets involved in bacterial antioxidant defenses and cell division. To counter host-imposed manganese stress, the streptococcal species employ a sophisticated sensory system that tightly coordinates manganese stress-specific molecular strategies to negate host induced manganese stress and proliferate in the host. Here we review the molecular details of host-streptococcal interactions in the battle for manganese during infection and the significance of streptococcal effectors involved to bacterial pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aggarwal and Kumaraswami.)

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster larvae, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure (WC), another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveal that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal WC or hemocyte spreading., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The influence of distinct tidal characteristics and nutrient status on mesozooplankton community was studied in six major estuaries along the west coast of India during the late-monsoon (MS) and post-monsoon (PM) periods. The macro-tidal estuaries in the north (Amba and Thane) exhibited higher nutrient concentration compared to the micro- and meso-tidal estuaries located in the south (Cochin and Nethravati) and central (Zuari and Mandovi) west coast of India. The markedly higher nitrate and phosphate levels in the macro-tidal estuaries during PM indicated anthropogenic contributions from domestic and industrial effluents, which significantly impacted the mesozooplankton community structure. Nutrient enrichments favored higher phytoplankton standing stock leading to low DO levels. In the micro- and meso-tidal estuaries, meso- and euryhaline copepods dominated whereas in the macro-tidal estuaries, the copepod community was dominated by euryhaline and coastal species. Furthermore, the high-saline eutrophic environment of macro-tidal estuaries formed congenial for the increased jellyfish preponderance during PM. The predation pressure exerted by the jellyfish population on the crustacean zooplankton and ichthyoplankton exerted an adverse impact on the potential fishery stock in the macro-tidal estuaries. Thus, the study reveals that the nutrient enrichment favoring a shift in the mesozooplankton community structure from nutritionally superior crustacean plankton to less desirable jellyfishes, which in turn, may lead to a threat on the estuarine pelagic energy transfer and ecosystem deliverables., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves., (Copyright © 2020 Long et al.)

Colonization by pathogenic bacteria depends on their ability to overcome host nutritional defenses and acquire nutrients. The human pathogen group A streptococcus (GAS) encounters the host defense factor calprotectin (CP) during infection. CP inhibits GAS growth in vitro by imposing zinc (Zn) limitation. However, GAS counterstrategies to combat CP-mediated Zn limitation and the in vivo relevance of CP-GAS interactions to bacterial pathogenesis remain unknown. Here, we report that GAS upregulates the AdcR regulon in response to CP-mediated Zn limitation. The AdcR regulon includes genes encoding Zn import ( adcABC ), Zn sparing ( rpsN.2 ), and Zn scavenging systems ( adcAII , phtD , and phtY ). Each gene in the AdcR regulon contributes to GAS Zn acquisition and CP resistance. The Δ adcC and Δ rpsN.2 mutant strains were the most susceptible to CP, whereas the Δ adcA , Δ adcAII , and Δ phtD mutant strains displayed less CP sensitivity during growth in vitro However, the Δ phtY mutant strain did not display an increased CP sensitivity. The varied sensitivity of the mutant strains to CP-mediated Zn limitation suggests distinct roles for individual AdcR regulon genes in GAS Zn acquisition. GAS upregulates the AdcR regulon during necrotizing fasciitis infection in WT mice but not in S100a9 -/- mice lacking CP. This suggests that CP induces Zn deficiency in the host. Finally, consistent with the in vitro results, several of the AdcR regulon genes are critical for GAS virulence in WT mice, whereas they are dispensable for virulence in S100a9 -/- mice, indicating the direct competition for Zn between CP and proteins encoded by the GAS AdcR regulon during infection., (Copyright © 2020 American Society for Microbiology.)

Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE -encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N -cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo , revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials., (Copyright © 2019 Beggs et al.)

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.

Streptococcus pyogenes causes 700 million human infections annually worldwide, yet, despite a century of intensive effort, there is no licensed vaccine against this bacterium. Although a number of large-scale genomic studies of bacterial pathogens have been published, the relationships among the genome, transcriptome, and virulence in large bacterial populations remain poorly understood. We sequenced the genomes of 2,101 emm28 S. pyogenes invasive strains, from which we selected 492 phylogenetically diverse strains for transcriptome analysis and 50 strains for virulence assessment. Data integration provided a novel understanding of the virulence mechanisms of this model organism. Genome-wide association study, expression quantitative trait loci analysis, machine learning, and isogenic mutant strains identified and confirmed a one-nucleotide indel in an intergenic region that significantly alters global transcript profiles and ultimately virulence. The integrative strategy that we used is generally applicable to any microbe and may lead to new therapeutics for many human pathogens.

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains., Competing Interests: The authors have declared that no competing interests exist.

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 speB expression in concert with bacterial population density. The SIP signaling pathway is active in vivo 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 ex vivo in human saliva. SIP-mediated speB expression plays a crucial role in GAS colonization of the mouse oropharynx. GAS employs a distinct pattern of SpeB production during growth ex vivo in saliva that includes a transient burst of speB 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 ex viv o. Moreover, the SIP signaling pathway is critical for GAS survival in blood. SIP-dependent speB regulation is functional in strains of diverse emm types, indicating that SIP signaling is a conserved virulence regulatory mechanism. Our discoveries have implications for future translational studies., (Copyright © 2018 American Society for Microbiology.)

Group A Streptococccus (GAS) is a major human pathogen that causes significant morbidity and mortality. Zinc is an essential trace element required for GAS growth, however, zinc can be toxic at excess concentrations. The bacterial strategies to maintain zinc sufficiency without incurring zinc toxicity play a crucial role in host-GAS interactions and have a significant impact on GAS pathogenesis. The host deploys nutritional immune mechanisms to retard GAS growth by causing either zinc deprivation or zinc poisoning. However, GAS overcomes the zinc-dependent host defenses and survives in the hostile environment by employing complex adaptive strategies. In this review, we describe the different host immune strategies that employ either zinc limitation or zinc toxicity in different host environments to control GAS infection. We also discuss the molecular mechanisms and machineries used by GAS to evade host nutritional defenses and establish successful infection. Emerging evidence suggests that the metal transporters are major GAS virulence factors as they compete against host nutritional immune mechanisms to acquire or expel metals and promote bacterial survival in the host. Thus, identification of GAS molecules and elucidation of the mechanisms by which GAS combats host-mediated alterations in zinc availability may lead to novel interference strategies targeting GAS metal acquisition systems.

Successful pathogens use complex signaling mechanisms to monitor their environment and reprogram global gene expression during specific stages of infection. Group A Streptococcus (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 speB 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 speB 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., Competing Interests: The authors declare no conflict of interest.

Bacterial pathogens must overcome host immune mechanisms to acquire micronutrients for successful replication and infection. Streptococcus pyogenes, also known as group A streptococcus (GAS), is a human pathogen that causes a variety of clinical manifestations, and disease prevention is hampered by lack of a human GAS vaccine. Herein, we report that the mammalian host recruits calprotectin (CP) to GAS infection sites and retards bacterial growth by zinc limitation. However, a GAS-encoded zinc importer and a nuanced zinc sensor aid bacterial defense against CP-mediated growth inhibition and contribute to GAS virulence. Immunization of mice with the extracellular component of the zinc importer confers protection against systemic GAS challenge. Together, we identified a key early stage host-GAS interaction and translated that knowledge into a novel vaccine strategy against GAS infection. Furthermore, we provided evidence that a similar struggle for zinc may occur during other streptococcal infections, which raises the possibility of a broad-spectrum prophylactic strategy against multiple streptococcal pathogens., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 American Society for Microbiology.)

Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism., Importance: The modulation of gene transcription is key to the ability of bacterial pathogens to infect hosts to cause disease. Here, we discovered that the group A Streptococcus transcription factor RivR negatively regulates the ability of this pathogen to survive in human blood, and we also began biochemical characterization of this protein. We determined that, in order for RivR to function, it must self-associate, forming both dimers (consisting of two RivR proteins) and higher-order complexes (consisting of more than two RivR proteins). This functional requirement for RivR is shared by other regulators in the same family of proteins, suggesting a common regulatory mechanism. Insight into how these transcription factors function may facilitate the development of novel therapeutic agents targeting their activity., (Copyright © 2016 American Society for Microbiology.)

The members of RRNPP family of bacterial regulators sense population density-specific secreted oligopeptides and modulate the expression of genes involved in cellular processes, such as sporulation, competence, virulence, biofilm formation, conjugative plasmid transfer and antibiotic resistance. Signaling by RRNPP regulators include several steps: generation and secretion of the signaling oligopeptides, re-internalization of the signaling molecules into the cytoplasm, signal sensing by the cytosolic RRNPP regulators, signal-specific allosteric structural changes in the regulators, and interaction of the regulators with their respective regulatory target and gene regulation. The recently determined structures of the RRNPP regulators provide insight into the mechanistic aspects for several steps in this signaling circuit. In this review, we discuss the structural principles underlying peptide specificity, regulatory target recognition, and ligand-induced allostery in RRNPP regulators and its impact on gene regulation. Despite the conserved tertiary structure of these regulators, structural analyses revealed unexpected diversity in the mechanism of activation and molecular strategies that couple the peptide-induced allostery to gene regulation. Although these structural studies provide a sophisticated understanding of gene regulation by RRNPP regulators, much needs to be learned regarding the target DNA binding by yet-to-be characterized RNPP regulators and the several aspects of signaling by Rgg regulators., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Group A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB-dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C-terminal domain of RopB. RopB-CTD has the TPR motif, a signature motif involved in protein-peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density-specific cell-free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB-dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure-guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB-dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Unlabelled: The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection., Importance: More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We used a colorimetric assay to determine the presence of chlorhexidine on skin, and we identified deficiencies in preoperative bathing and daily bathing in the intensive care unit. Both types of bathing improved with an intervention that included feedback to nursing staff. The assay provides a simple and rapid method of monitoring the performance of chlorhexidine bathing.

Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Altering zinc bioavailability to bacterial pathogens is a key component of host innate immunity. Thus, the ability to sense and adapt to the alterations in zinc concentrations is critical for bacterial survival and pathogenesis. To understand the adaptive responses of group A Streptococcus (GAS) to zinc limitation and its regulation by AdcR, we characterized gene regulation by AdcR. AdcR regulates the expression of 70 genes involved in zinc acquisition and virulence. Zinc-bound AdcR interacts with operator sequences in the negatively regulated promoters and mediates differential regulation of target genes in response to zinc deficiency. Genes involved in zinc mobilization and conservation are derepressed during mild zinc deficiency, whereas the energy-dependent zinc importers are upregulated during severe zinc deficiency. Further, we demonstrated that transcription activation by AdcR occurs by direct binding to the promoter. However, the repression and activation by AdcR is mediated by its interactions with two distinct operator sequences. Finally, mutational analysis of the metal ligands of AdcR caused impaired DNA binding and attenuated virulence, indicating that zinc sensing by AdcR is critical for GAS pathogenesis. Together, we demonstrate that AdcR regulates GAS adaptive responses to zinc limitation and identify molecular components required for GAS survival during zinc deficiency., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

Humans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group A Streptococcus (GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator. Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in the mga promoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange, we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier strain. Compared to strains with the wild-type mga promoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewer mga and Mga-regulated gene transcripts. Consistent with decreased mga transcripts, strains containing the variant mga promoter were also significantly less virulent in in vivo and ex vivo models of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to the mga promoter and that the 12-bp deletion in the mga promoter reduces CodY-mediated mga transcription. We conclude that the naturally occurring 12-bp deletion in the mga promoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new insight into the molecular basis of the carrier state of an important human pathogen.

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.

Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.