RESUMEN
Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.
Asunto(s)
Antibacterianos , Clostridioides difficile , Resistencia a la Vancomicina , Vancomicina , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Antibacterianos/farmacología , Aptitud Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Transducción de Señal , Mutación , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/genéticaRESUMEN
Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.
Asunto(s)
Proteínas Bacterianas , Clostridioides difficile , Peptidoglicano , Peptidil Transferasas , Proteínas Bacterianas/química , Resistencia betalactámica , beta-Lactamas/farmacología , Catálisis , Clostridioides difficile/enzimología , Clostridioides difficile/genética , Peptidoglicano/química , Peptidil Transferasas/química , Peptidil Transferasas/genéticaRESUMEN
Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well understood. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer variants, following infection with the S-layer-null strain, FM2.5. These variants carry either correction of the original point mutation, or sequence modifications which restored the reading frame, and translation of slpA. Selection of these variant clones was rapid in vivo, and independent of toxin production, with up to 90% of the recovered C. difficile population encoding modified slpA sequence within 24 h post infection. Two variants, subsequently named FM2.5varA and FM2.5varB, were selected for study in greater detail. Structural determination of SlpA from FM2.5varB indicated an alteration in the orientation of protein domains, resulting in a reorganisation of the lattice assembly, and changes in interacting interfaces, which might alter function. Interestingly, variant FM2.5varB displayed an attenuated, FM2.5-like phenotype in vivo compared to FM2.5varA, which caused disease severity more comparable to that of R20291. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates revealed large changes in gene expression between R20291 and FM2.5. Downregulation of tcdA/tcdB and several genes associated with sporulation and cell wall integrity may account for the reported attenuated phenotype of FM2.5 in vivo. RNA-seq data correlated well with disease severity with the more virulent variant, FM2.5varA, showing s similar profile of gene expression to R20291 in vitro, while the attenuated FM2.5varB showed downregulation of many of the same virulence associated traits as FM2.5. Cumulatively, these data add to a growing body of evidence that the S-layer contributes to C. difficile pathogenesis and disease severity.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Clostridioides , Clostridioides difficile/genética , Pared Celular , Células ClonalesRESUMEN
The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.
Asunto(s)
Clostridioides difficile/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , ARN Bacteriano/metabolismo , Regiones no Traducidas 5'/genética , Clostridioides difficile/genética , Ambiente , Etanolamina/metabolismo , Genoma Bacteriano , Ligandos , Chaperonas Moleculares/metabolismo , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Operón/genética , Regiones Promotoras Genéticas/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Sitio de Iniciación de la Transcripción , Terminación de la Transcripción Genética , Transcriptoma/genéticaRESUMEN
BACKGROUND: Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile. METHODS: Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. RESULTS: Deletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. CONCLUSIONS: We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , Esporas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Pared Celular/química , Pared Celular/genética , Pared Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , Clostridioides difficile/crecimiento & desarrollo , Calor , Proteínas de Unión a las Penicilinas/genética , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
OBJECTIVES: The vascular endothelium is a major target of sepsis-induced events, and endothelial activation accounts for much of the pathology of sepsis. Urinary tract infections and pneumonia caused by Escherichia coli are among of the most common infections causing sepsis in both community and hospital settings. Currently, there are no approved drugs on the market to treat the underlying pathophysiology of sepsis. The aim of this study is to elucidate the molecular mechanism by which E. coli induces endothelial injury as a result of attachment. DESIGN: Laboratory research using a hemodynamic perfusion ex vivo model. SETTING: Research Laboratories of Royal College of Surgeons in Ireland and Beaumont Hospital. PATIENTS: Ex vivo human vascular endothelial cells. INTERVENTIONS: Addition of αVß3 antagonist, cilengitide. MEASUREMENTS AND MAIN RESULTS: Clinical strains of E. coli isolated from patients with sepsis bound to sheared human endothelial cells under static and hemodynamic shear conditions. Binding was dependent on E. coli cell membrane protein outer membrane protein A attaching directly to endothelial cell integrin αVß3. Attachment resulted in disturbances in endothelial barrier integrity, as determined by loss of tight junction protein staining, permeability changes, and ultimately cell death by apoptosis. Using a low concentration of the αVß3 antagonist cilengitide or using a strain deficient in outer membrane protein A resulted in a significant reduction in endothelial dysfunction following infection. CONCLUSIONS: Inhibition of E. coli binding to endothelial cell αVß3 by cilengitide prevents endothelial dysfunction and may, therefore, present as a novel early therapeutic for the treatment of sepsis.
Asunto(s)
Células Endoteliales/microbiología , Escherichia coli/crecimiento & desarrollo , Integrina alfaVbeta3/antagonistas & inhibidores , Sepsis/microbiología , Venenos de Serpiente/farmacología , Permeabilidad Capilar , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.
Asunto(s)
Secuencias de Aminoácidos , Pared Celular/metabolismo , Clostridioides difficile/metabolismo , Proteínas de la Membrana/metabolismo , Polisacáridos Bacterianos/metabolismo , Técnicas de Silenciamiento del Gen , Unión Proteica , Secuencias Repetitivas de AminoácidoRESUMEN
Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.
Asunto(s)
Proteínas Bacterianas/química , Clostridium botulinum/química , Clostridium/química , Esporas Bacterianas/química , Electroforesis en Gel de Poliacrilamida , Homología de Secuencia de Aminoácido , Esporas Bacterianas/metabolismo , Esporas Bacterianas/ultraestructura , Espectrometría de Masas en TándemRESUMEN
Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with.
Asunto(s)
Cromosomas Bacterianos/metabolismo , Clostridioides difficile/genética , Conjugación Genética , Recombinación Homóloga , Plásmidos/metabolismo , Cromosomas Bacterianos/química , Clostridioides difficile/aislamiento & purificación , Clostridioides difficile/metabolismo , Infecciones por Clostridium/microbiología , Medios de Cultivo/farmacología , Calor , Humanos , Plásmidos/química , Ribotipificación , Transformación Bacteriana/efectos de los fármacosRESUMEN
BACKGROUND: Clostridium difficile is an anaerobic, Gram-positive bacterium that can reside as a commensal within the intestinal microbiota of healthy individuals or cause life-threatening antibiotic-associated diarrhea in immunocompromised hosts. C. difficile can also form highly resistant spores that are excreted facilitating host-to-host transmission. The C. difficile spo0A gene encodes a highly conserved transcriptional regulator of sporulation that is required for relapsing disease and transmission in mice. RESULTS: Here we describe a genome-wide approach using a combined transcriptomic and proteomic analysis to identify Spo0A regulated genes. Our results validate Spo0A as a positive regulator of putative and novel sporulation genes as well as components of the mature spore proteome. We also show that Spo0A regulates a number of virulence-associated factors such as flagella and metabolic pathways including glucose fermentation leading to butyrate production. CONCLUSIONS: The C. difficile spo0A gene is a global transcriptional regulator that controls diverse sporulation, virulence and metabolic phenotypes coordinating pathogen adaptation to a wide range of host interactions. Additionally, the rich breadth of functional data allowed us to significantly update the annotation of the C. difficile 630 reference genome which will facilitate basic and applied research on this emerging pathogen.
Asunto(s)
Clostridioides difficile/fisiología , Clostridioides difficile/patogenicidad , Redes y Vías Metabólicas , Proteoma , Transcriptoma , Butiratos/metabolismo , Clostridioides difficile/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Glucosa/metabolismo , Humanos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Mutación , Proteómica , Esporas Bacterianas , Virulencia/genéticaRESUMEN
Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of mechanisms used in colonization of the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family and is expressed in a phase variable manner. Here we show CwpV promotes C. difficile aggregation, mediated by the C-terminal repetitive domain. This domain varies markedly between strains; five distinct repeat types were identified and were shown to be antigenically distinct. Other aspects of CwpV are, however, conserved. All CwpV types are expressed in a phase variable manner. Using targeted gene knock-out, we show that a single site-specific recombinase RecV is required for CwpV phase variation. CwpV is post-translationally cleaved at a conserved site leading to formation of a complex of cleavage products. The highly conserved N-terminus anchors the CwpV complex to the cell surface. Therefore CwpV function, regulation and processing are highly conserved across C. difficile strains, whilst the functional domain exists in at least five antigenically distinct forms. This hints at a complex evolutionary history for CwpV.
Asunto(s)
Pared Celular/metabolismo , Clostridioides difficile/metabolismo , Evolución Molecular , Glicoproteínas de Membrana/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Pared Celular/genética , Pared Celular/inmunología , Clostridioides difficile/genética , Clostridioides difficile/inmunología , Técnicas de Silenciamiento del Gen , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Estructura Terciaria de Proteína , Recombinasas/genética , Recombinasas/inmunología , Recombinasas/metabolismoRESUMEN
Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Humanos , Virulencia , Clostridioides difficile/genética , Clostridioides , Factores de Virulencia , Diarrea , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ÏCD38-2, ÏCD111, and ÏCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ÏCD111 and SLCT-11 enabled infection by ÏCD38-2 and ÏCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ÏCD38-2 and ÏCD146 but not ÏCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.
RESUMEN
Protein translocation across the cytoplasmic membrane is an essential process in all bacteria. The Sec system, comprising at its core an ATPase, SecA, and a membrane channel, SecYEG, is responsible for the majority of this protein transport. Recently, a second parallel Sec system has been described in a number of gram-positive species. This accessory Sec system is characterized by the presence of a second copy of the energizing ATPase, SecA2; where it has been studied, SecA2 is responsible for the translocation of a subset of Sec substrates. In common with many pathogenic gram-positive species, Clostridium difficile possesses two copies of SecA. Here, we describe the first characterization of the C. difficile accessory Sec system and the identification of its major substrates. Using inducible antisense RNA expression and dominant-negative alleles of secA1 and secA2, we demonstrate that export of the S-layer proteins (SLPs) and an additional cell wall protein (CwpV) is dependent on SecA2. Accumulation of the cytoplasmic precursor of the SLPs SlpA and other cell wall proteins was observed in cells expressing dominant-negative secA1 or secA2 alleles, concomitant with a decrease in the levels of mature SLPs in the cell wall. Furthermore, expression of either dominant-negative allele or antisense RNA knockdown of SecA1 or SecA2 dramatically impaired growth, indicating that both Sec systems are essential in C. difficile.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/genética , Mutación , Plásmidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Clostridium difficile is a major cause of chronic antibiotic-associated diarrhea and a significant health care-associated pathogen that forms highly resistant and infectious spores. Spo0A is a highly conserved transcriptional regulator that plays a key role in initiating sporulation in Bacillus and Clostridium species. Here, we use a murine model to study the role of the C. difficile spo0A gene during infection and transmission. We demonstrate that C. difficile spo0A mutant derivatives can cause intestinal disease but are unable to persist within and effectively transmit between mice. Thus, the C. difficile Spo0A protein plays a key role in persistent infection, including recurrence and host-to-host transmission in mice.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Infecciones por Clostridium/microbiología , Regulación Bacteriana de la Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/transmisión , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Mutación , Organismos Libres de Patógenos Específicos , Factores de Transcripción/genética , VirulenciaRESUMEN
Clostridium difficile, a leading cause of hospital-acquired bacterial infection, is coated in a dense surface layer (S-layer) that is thought to provide both physicochemical protection and a scaffold for host-pathogen interactions. The key structural components of the S-layer are two proteins derived from a polypeptide precursor, SlpA, via proteolytic cleavage by the protease Cwp84. Here, we report the design, synthesis and in vivo characterization of a panel of protease inhibitors and activity-based probes (ABPs) designed to target S-layer processing in live C. difficile cells. Inhibitors based on substrate-mimetic peptides bearing a C-terminal Michael acceptor warhead were found to be promising candidates for further development.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/metabolismo , Cisteína Endopeptidasas/química , Inhibidores de Proteasas/química , Proteínas Bacterianas/metabolismo , Clostridioides difficile/enzimología , Cisteína Endopeptidasas/metabolismo , Interacciones Huésped-Patógeno , Péptidos/síntesis química , Péptidos/química , Inhibidores de Proteasas/síntesis química , Relación Estructura-ActividadRESUMEN
Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.
Asunto(s)
Bacteriófagos , Clostridioides difficile , Bacteriófagos/genética , Pared Celular/metabolismo , Clostridioides , Endopeptidasas/química , Endopeptidasas/genética , Endopeptidasas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/análisis , N-Acetil Muramoil-L-Alanina Amidasa/genética , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Polisacáridos Bacterianos/metabolismoRESUMEN
Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, the main S-layer protein of the bacterial pathogen Clostridioides difficile, and use electron microscopy to study S-layer organisation and assembly. The SlpA crystal lattice mimics S-layer assembly in the cell, through tiling of triangular prisms above the cell wall, interlocked by distinct ridges facing the environment. Strikingly, the array is very compact, with pores of only ~10 Å in diameter, compared to other S-layers (30-100 Å). The surface-exposed flexible ridges are partially dispensable for overall structure and assembly, although a mutant lacking this region becomes susceptible to lysozyme, an important molecule in host defence. Thus, our work gives insights into S-layer organisation and provides a basis for development of C. difficile-specific therapeutics.
Asunto(s)
Clostridioides difficile , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Clostridioides difficile/genéticaRESUMEN
Clostridium difficile expresses a surface layer (S-layer) which coats the surface of the bacterium and acts as an adhesin facilitating interaction of the bacterium with host enteric cells. The S-layer contains a high-molecular-weight S-layer protein (HMW SLP) and its low-molecular-weight partner protein (LMW SLP). We show that these proteins form a tightly associated non-covalent complex, the H/L complex, and we identify the regions of both proteins responsible for complex formation. The 2.4 A X-ray crystal structure of a truncated derivative of the LMW SLP reveals two domains. Domain 1 has a two-layer sandwich architecture while domain 2, predicted to orientate towards the external environment, contains a novel fold. Small-angle X-ray scattering analysis of the H/L complex shows an elongated molecule, with the two SLPs arranged 'end-to-end' interacting with each other through a small contact area. Alignment of LMW SLPs, which exhibit high sequence diversity, reveals a core of conserved residues that could reflect functional conservation, while allowing for immune evasion through sequence variation. These structures are the first described for the S-layer of a bacterial pathogen, and provide insights into the assembly and biogenesis of the S-layer.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Pared Celular/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.