RESUMEN
Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.
Asunto(s)
Metaloendopeptidasas/metabolismo , Zinc , Animales , GTP Fosfohidrolasas/metabolismo , Homeostasis , Metalochaperonas/metabolismo , Metaloproteínas/genética , Ratones , Pez Cebra/metabolismo , Zinc/metabolismoRESUMEN
Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.
Asunto(s)
Calcio , Proteómica , Animales , Ratones , Proteómica/métodos , Biología Computacional/métodos , Análisis Multivariante , Proteoma/metabolismoRESUMEN
Characterization of transcriptional networks is one of the main strategies used to understand how bacteria interact with their environment. To reveal novel regulatory elements in the human pathogen Staphylococcus aureus, we adapted a traditional transduction protocol to be used in a high-throughput format in combination with the publicly available S. aureus Nebraska Transposon Mutant Library. Specifically, plasmid transductions are performed in 96-well format, so that a single plasmid can be simultaneously transferred into numerous recipient strains. When used in conjunction with bioluminescent reporter constructs, this strategy enables parallel and continuous monitoring of downstream transcriptional effects of hundreds of defined mutations. Here, we use this workflow in a proof-of-concept study to identify novel regulators of the staphylococcal metalloprotease aureolysin. Importantly, this strategy can be utilized with any other bacterium where plasmid transduction is possible, making it a versatile and efficient tool to probe transcriptional regulatory connections.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Biblioteca de Genes , Humanos , Plásmidos/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genéticaRESUMEN
The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Metionina Sulfóxido Reductasas/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , Staphylococcus aureus/inmunología , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Peróxido de Hidrógeno/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/inmunología , Ratones , Viabilidad Microbiana/inmunología , Mutación , Oxidación-Reducción , Estrés Oxidativo , Staphylococcus aureus/genéticaRESUMEN
Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.
Asunto(s)
Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/microbiología , Hemo/metabolismo , Virulencia/fisiología , Animales , Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Infecciones por Clostridium/metabolismo , Genes Bacterianos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Operón/genéticaRESUMEN
The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of â¼110 µm in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (â¼250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.
Asunto(s)
Microscopía Fluorescente/métodos , Péptidos/metabolismo , Proteómica/métodos , Animales , Colorantes Fluorescentes/química , Riñón/metabolismo , Riñón/patología , Extracción Líquido-Líquido/métodos , Ratones , Péptidos/química , Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus aureus/metabolismo , Tripsina/metabolismoRESUMEN
Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering ZnII to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for ZnII metallochaperone activity. ZnII chelator competition experiments reveal one high-affinity (KZn1 ≈ 1010-1011 M-1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. CoII titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with ZnII more effective than CoII. Both GTPases bind the product, GDP, more tightly in the apoprotein than the ZnII-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.
Asunto(s)
Metalochaperonas/metabolismo , Zinc/metabolismo , Sitios de Unión , Metalochaperonas/química , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Zinc/químicaRESUMEN
Heme is a cofactor that is essential for cellular respiration and for the function of many enzymes. If heme levels become too low within the cell, S. aureus switches from producing energy via respiration to producing energy by fermentation. S. aureus encodes two heme oxygenases, IsdI and IsdG, which cleave the porphyrin heme ring releasing iron for use as a nutrient source. Both isdI and isdG are only expressed under low iron conditions and are regulated by the canonical Ferric Uptake Regulator (Fur). Here we demonstrate that unregulated expression of isdI and isdG within S. aureus leads to reduced growth under low iron conditions. Additionally, the constitutive expression of these enzymes leads to decreased heme abundance in S. aureus, an increase in the fermentation product lactate, and increased resistance to gentamicin. This work demonstrates that S. aureus has developed tuning mechanisms, such as Fur regulation, to ensure that the cell has sufficient quantities of heme for efficient ATP production through aerobic respiration.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/fisiología , Homeostasis , Proteínas Represoras/metabolismo , Staphylococcus aureus/enzimología , Aerobiosis , Proteínas Bacterianas/genética , Hemo Oxigenasa (Desciclizante)/genética , Hierro/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas/genética , Proteínas Represoras/genética , Staphylococcus aureus/genéticaRESUMEN
Staphylococcus aureus is a major human pathogen that causes infection in a wide variety of sites within the human body. Its ability to adapt to the human host and to produce a successful infection requires precise orchestration of gene expression. While DNA-dependent RNA polymerase (RNAP) is generally well characterized, the roles of several small accessory subunits within the complex have yet to be fully explored. This is particularly true for the omega (ω or RpoZ) subunit, which has been extensively studied in Gram-negative bacteria but largely neglected in Gram-positive counterparts. In Escherichia coli, it has been shown that ppGpp binding, and thus control of the stringent response, is facilitated by ω. Interestingly, key residues that facilitate ppGpp binding by ω are not conserved in S. aureus, and consequently, survival under starvation conditions is unaffected by rpoZ deletion. Further to this, ω-lacking strains of S. aureus display structural changes in the RNAP complex, which result from increased degradation and misfolding of the ß' subunit, alterations in δ and σ factor abundance, and a general dissociation of RNAP in the absence of ω. Through RNA sequencing analysis we detected a variety of transcriptional changes in the rpoZ-deficient strain, presumably as a response to the negative effects of ω depletion on the transcription machinery. These transcriptional changes translated to an impaired ability of the rpoZ mutant to resist stress and to fully form a biofilm. Collectively, our data underline, for the first time, the importance of ω for RNAP stability, function, and cellular physiology in S. aureus IMPORTANCE: In order for bacteria to adjust to changing environments, such as within the host, the transcriptional process must be tightly controlled. Transcription is carried out by DNA-dependent RNA polymerase (RNAP). In addition to its major subunits (α2ßß') a fifth, smaller subunit, ω, is present in all forms of life. Although this small subunit is well studied in eukaryotes and Gram-negative bacteria, only limited information is available for Gram-positive and pathogenic species. In this study, we investigated the structural and functional importance of ω, revealing key roles in subunit folding/stability, complex assembly, and maintenance of transcriptional integrity. Collectively, our data underline, for the first time, the importance of ω for RNAP function and cellular harmony in S. aureus.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Staphylococcus aureus/enzimología , Proteínas Bacterianas/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Guanosina Tetrafosfato , Estabilidad Proteica , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transcripción GenéticaRESUMEN
Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Staphylococcus aureus/enzimología , Factores de Virulencia , Proteínas Bacterianas/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Mutación , Isomerasa de Peptidilprolil/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
UNLABELLED: The ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NOË) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NOË typically results in growth inhibition and induction of stress regulons. S. aureus, however, induces a metabolic state in response to NOË that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NOË resistance in S. aureus: three virulence regulons not formerly associated with NOË resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NOË stress and show that the S. aureus ΔsarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NOË exposure. Altogether, our results broadly characterize the regulatory requirements for NOË resistance in S. aureus and suggest an intriguing overlap between the regulation of NOË resistance and virulence in this well-adapted human pathogen. IMPORTANCE: The prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NOË), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NOË is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NOË response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NOË resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NOË is highly pleiotropic and intrinsically tied to metabolism and virulence regulation.
Asunto(s)
Proteínas Bacterianas/genética , Óxido Nítrico/farmacología , Staphylococcus aureus/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Inmunidad Innata , Hierro/metabolismo , Quelantes del Hierro , Mutación , Peróxidos , Staphylococcus aureus/inmunología , Staphylococcus aureus/fisiologíaRESUMEN
Prenylation is the addition of prenyl groups to peptide chains or metabolites via the condensation of geranyl- or isopentenyl-diphosphate moieties by geranyltranstransferases. Although this process is extensively studied in eukaryotes, little is known about the influence of prenylation in prokaryotic species. To explore the role of this modification in bacteria, we generated a mutation in the geranyltranstransferase (IspA) of Staphylococcus aureus. Quite strikingly, the ispA mutant completely lacked pigment and exhibited a previously undescribed small colony variant-like phenotype. Further pleiotropic defects in cellular behavior were noted, including impaired growth, decreased ATP production, increased sensitivity to oxidative stress, increased resistance to aminoglycosides and cationic antimicrobial peptides, and decreased resistance to cell wall-targeting antibiotics. These latter effects appear to result from differences in envelope composition as ispA mutants have highly diffuse cell walls (particularly at the septum), marked alterations in fatty acid composition and increased membrane fluidity. Taken together, these data present an important characterization of prokaryotic prenylation and demonstrate that this process is central to a wealth of pathways involved in mediating cellular homeostasis in S. aureus.
Asunto(s)
Pared Celular/metabolismo , Geraniltranstransferasa/genética , Prenilación de Proteína , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Farmacorresistencia Bacteriana , Ácidos Grasos/análisis , Perfilación de la Expresión Génica , Geraniltranstransferasa/metabolismo , Fluidez de la Membrana , Pruebas de Sensibilidad Microbiana , Mutación , Fenotipo , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Proteínas de la Membrana/metabolismo , Factor sigma/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Animales , Proteínas Bacterianas/genética , Daño del ADN/efectos de los fármacos , Farmacorresistencia Bacteriana , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Humanos , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Mutación , Proteómica , Staphylococcus aureus/genética , Estrés Fisiológico , Porcinos , Sitio de Iniciación de la TranscripciónRESUMEN
In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the ß and/or ß' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.
Asunto(s)
ADN Polimerasa Dirigida por ADN/fisiología , Regiones Promotoras Genéticas/fisiología , Subunidades de Proteína/fisiología , Factor sigma/fisiología , Staphylococcus aureus/patogenicidad , Factores de Transcripción/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Fagocitosis , Factor sigma/genética , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Factores de Transcripción/genética , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
SSR42 is the longest noncoding RNA in the S. aureus cell and the second-most abundant transcript in the stationary phase transcriptome, second only to RNAIII. It is highly conserved across strains and exhibits pronounced stability in stationary phase, however the mechanism behind its regulatory role has yet to be fully elucidated. Herein, we used transcriptomic and proteomic approaches to probe the role of SSR42, revealing that it is a powerful, novel activator of the primary leukocidin LukAB. SSR42 is required for cytotoxicity towards, and escape from within, human neutrophils, and also mediates survival within human blood. We show that SSR42 wields this role via derepression by the peroxide repressor PerR in response to the presence of human neutrophils and governs lukAB induction in this niche. Importantly, this regulation is driven by direct RNA-RNA interaction, as we show binding of the 5' UTR of the lukAB transcript with the 3' end of SSR42, which ultimately modulates transcript stability as well as translational activity. Finally, we demonstrate that this behavior is absolutely required for full virulence of S. aureus in murine models of both pneumonia and sepsis. Collectively, we present SSR42 as a pleiotropic regulatory RNA that acts as a nexus between environmental sensing and the regulation of pathogenesis, responding to environmental stimuli and host immune factors to bolster cytotoxic behavior and facilitate infection in S. aureus . Importance: S. aureus is a master pathogen due to its formidable collection of virulence factors. These are tightly controlled by a diverse group of regulators that titrate their abundance to adapt to unique infectious niches. The role of regulatory RNAs in stress adaptation and pathogenesis is becoming increasingly more relevant in S. aureus . In this study, we provide the most comprehensive global analysis to date of just such a factor, SSR42. Specifically, we uncover that SSR42 is required for mediating cytotoxicity - one of the pillars of infection - in response to phagocytosis by human neutrophils. We find that SSR42 is induced by components of the host immune system and facilitates downstream activation of cytotoxic factors via RNA-RNA interactions. This illustrates that SSR42 forms a pivotal link between sensing the external environment and mediating resistance to oxidative stress while promoting virulence, solidifying it as a major global regulator in S. aureus .
RESUMEN
The U.S. Centers for Disease Control and Prevention (CDC) received surveillance data on how many people tested positive for SARS-CoV-2, but there was little information about what individuals did to mitigate transmission. To fill the information gap, we conducted an online, probability-based survey among a nationally representative panel of adults living in the United States to better understand the behaviors of individuals following a positive SARS-CoV-2 test result. Given the low response rates commonly associated with panel surveys, we assessed how well the survey data aligned with CDC surveillance data from March, 2020 to March, 2022. We used CDC surveillance data to calculate monthly aggregated COVID-19 case counts and compared these to monthly COVID-19 case counts captured by our survey during the same period. We found high correlation between our overall survey data estimates and monthly case counts reported to the CDC during the analytic period (r: +0.94; p < 0.05). When stratified according to demographic characteristics, correlations remained high. These correlations strengthened our confidence that the panel survey participants were reflective of the cases reported to CDC and demonstrated the potential value of panel surveys to inform decision making.
RESUMEN
The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals. IMPORTANCE: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.
Asunto(s)
Complejo de Antígeno L1 de Leucocito , Infecciones Estafilocócicas , Staphylococcus aureus , Complejo de Antígeno L1 de Leucocito/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Infecciones Estafilocócicas/microbiología , Metales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Animales , Regulación Bacteriana de la Expresión Génica , Endopeptidasa Clp/metabolismo , Endopeptidasa Clp/genética , Ratones , Adaptación FisiológicaRESUMEN
CONTEXT.: The COVID-19 pandemic has triggered a worldwide crisis that created unprecedented challenges for the health care system, including diagnostic laboratories that faced an ever-increasing demand for SARS-CoV-2 testing. OBJECTIVE.: To share our experiences mobilizing a large-scale volunteer operation within a diagnostic laboratory in response to the COVID-19 crisis. In particular, during the early stages of the pandemic, research scientists at Vanderbilt University Medical Center were called upon to address challenges put forth by the rapid increase in testing demands. Volunteer scientists became a valuable resource to the clinical laboratory team after stay-at-home orders were in place and rapid diagnostic capabilities for COVID-19 were not yet widespread, thus necessitating significant manual laboratory analysis to support patient care. However, these volunteer efforts were not without challenges, including considerations around the licensure of clinical laboratory workers. Requirements can differ significantly between states and, in our case, were alleviated by an emergency gubernatorial decree. DATA SOURCES.: We summarize these experiences here as an operational roadmap for other institutions that wish to leverage biomedical research staff in response to future emergencies. We include recruitment and organizational schemes, as well as results of a survey that details participant experiences and identifies strategies for optimization. Lastly, we present considerations around long-term hosting of clinical laboratory volunteers, beyond just the initial stages of an emergency. CONCLUSIONS.: Through strategic implementation, scientists can provide diagnostic laboratories with invaluable support in times of need, while maintaining high clinical quality and regulatory compliance.
RESUMEN
Previous microarray analyses revealed that in Bradyrhizobium japonicum, about 100 genes are induced by genistein, an isoflavonoid secreted by soybean. This includes the three genes freC, freA, and freB (systematic designations bll4319, bll4320, and bll4321), which are likely to form a genistein-, daidzein-, and coumestrol-inducible operon and to encode a multidrug efflux system. Upstream of freCAB and in the opposite orientation, FrrA (systematic designation Blr4322), which has similarity to TetR-type regulators, is encoded. A deletion of frrA leads to increased expression of freB in the absence of an inducer. We identified the correct translational start codon of frrA and showed that the gene is inducible by genistein and daidzein. The protein, which was heterologously expressed and purified from Escherichia coli, binds to two palindrome-like DNA elements (operator A and operator B), which are located in the intergenic region between freC and frrA. The replacement of several nucleotides or the insertion of additional spacer nucleotides prevented binding. Binding of FrrA was also affected by the addition of genistein. By mapping the transcription start sites, we found that operator A covers the transcriptional start site of freC and operator B is probably located between the -35 regions of the two divergently oriented genes. Operator A seems to be conserved in a few similar gene constellations in other proteobacteria. Our data indicate that in B. japonicum, besides NodD1 (the LysR family) and NodVW (a two-component response regulator), a third regulator type (a TetR family member) which responds to the plant signal molecules genistein and daidzein exists.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bradyrhizobium/metabolismo , Flavonoides/farmacología , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Bacterianas/genética , Bradyrhizobium/efectos de los fármacos , Bradyrhizobium/genética , Codón Iniciador , Mutación , Nodulación de la Raíz de la Planta , Glycine max/microbiología , Glycine max/fisiologíaRESUMEN
HitRS is a two-component system that responds to cell envelope damage in the human pathogen Bacillus anthracis. Here we identify an RNA-binding protein, KrrA, that regulates HitRS function by modulating the stability of the hitRS mRNA. In addition to hitRS, KrrA binds to over 70 RNAs and, directly or indirectly, affects the expression of over 150 genes involved in multiple processes, including genetic competence, sporulation, RNA turnover, DNA repair, transport, and cellular metabolism. KrrA does not exhibit detectable nuclease activity in vitro, and thus the mechanism by which it modulates mRNA stability remains unclear.