RESUMEN
During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris. This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris. Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism. IMPORTANCE: Gram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae, SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance.
Asunto(s)
Actinomyces , Proteínas Bacterianas , Cisteína Endopeptidasas , Proteínas Fimbrias , Fimbrias Bacterianas , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , Actinomyces/genética , Actinomyces/enzimología , Actinomyces/metabolismo , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/genética , Pared Celular/metabolismo , Señales de Clasificación de ProteínaRESUMEN
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
Asunto(s)
Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Proteínas Fimbrias , Fimbrias Bacterianas , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/química , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Cristalografía por Rayos X , Actinomyces/metabolismo , Actinomyces/enzimología , Especificidad por Sustrato , Modelos MolecularesRESUMEN
IMPORTANCE: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly.
Asunto(s)
Fimbrias Bacterianas , Streptococcus sanguis , Streptococcus sanguis/genética , Streptococcus sanguis/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Bacterias/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , ADN/metabolismoRESUMEN
Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.
Asunto(s)
Escherichia coli Enteropatógena , Humanos , Escherichia coli Enteropatógena/metabolismo , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , Adenilil Imidodifosfato/análisis , Adenilil Imidodifosfato/metabolismo , Fimbrias Bacterianas/metabolismo , Proteínas Fimbrias/metabolismoRESUMEN
Background: Acinetobacter baumannii is an important nosocomial pathogen causing high morbidity and mortality in immunocompromised patients with prolonged hospitalization. Multidrug-resistant A. baumannii infections are on the rise worldwide. Therefore, the discovery of an effective vaccine against this bacterium seems necessary as a cost-effective and preventive strategy. Methods: In this present study, 35 genomes of A. baumannii strains were considered, and the extracellular proteins were selected, maximally having one transmembrane helix with high adhesion probability and no similarity to host proteins, as immunogenic candidates using the web tool Vaxign. Subsequently, the role of these selected proteins in bacterial pathogenesis was investigated using VICMpred. Then, the major histocompatibility complex class II, linear B-cell epitopes, and conservation of epitopes were identified using the Immune Epitope Database, BepiPred, and Epitope Conservancy Analysis, respectively. Finally, the B-cell discontinuous epitopes of each protein were predicted using ElliPro and plotted on the three-dimensional structure (3D) of the proteins. The role of the unknown proteins was predicted using the STRING database. Results: In this study, eight acceptable immunogenic candidates, including FilF, FimA, putative acid phosphatase, putative exported protein, subtilisin-like serine protease, and three uncharacterized proteins, were identified in A. baumannii. Conclusions: The results of the STRING database showed that these three uncharacterized proteins play a role in nutrition (heme utilization), peptide bond cleavage (serine peptidases), and cellular processes (MlaD protein). Extracellular proteins might play a catalyst role in the outer membrane protein-based vaccine of A. baumannii. Furthermore, this study proposed a list of potent immunogenic candidates of extracellular proteins.
RESUMEN
Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.
Asunto(s)
Actinobacteria/metabolismo , Aminoaciltransferasas , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Homeostasis , Proteínas de la Membrana , Morfogénesis , Serina EndopeptidasasRESUMEN
Acinetobacter baumannii, a prominent pathogen responsible for chronic infections in the blood, urinary tract, and lungs, has a high mortality due to its virulence and limited preventive methods. The present study aims to characterize the pilus assembly protein of A. baumannii to offer leads for epitope-based vaccine development. FilF is the putative pilus assembly protein that reportedly plays a supreme character in the virulence of this WHO-listed ESKAPE bacterium. Implementing various bioinformatics tools, led to the recognition of many antigenic B and T cell epitopes. Most promising B and T-cell epitopes were selected based on their binding efficiency with commonly occurring MHC alleles. Finally, we stepped down to fourteen protective antigenic peptides. These epitopes were also revealed to be non-allergenic and non-toxic. As a result, a vaccine chimera was created by linking these epitopes with appropriate linkers and adjuvant such as ß-defensins. Furthermore, homology modeling and validation were carried out, with the modeled structure being employed for molecular docking with the immunological receptor (TLR-4) found on lymphocyte cells. As a result of the molecular dynamics simulation, the interaction between human TLR-4 and the multi-epitope vaccine sequence was stable. Finally, in silico cloning and immune simulation were carried out to see the efficacy of the construct vaccine. This is the first study targeting the pilus assembly protein from A. baumannii to identify novel epitopes that hold potential for further experimental design of multi-peptide vaccine construct against the pathogen.
Asunto(s)
Acinetobacter baumannii , Acinetobacter baumannii/genética , Biología Computacional/métodos , Epítopos de Linfocito B , Epítopos de Linfocito T/genética , Humanos , Simulación del Acoplamiento Molecular , Receptor Toll-Like 4 , Vacunas de Subunidad/químicaRESUMEN
Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as "hypothetical," is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery.IMPORTANCE Cyanobacteria, environmentally prevalent photosynthetic prokaryotes, contribute â¼25% of global primary production. Cyanobacterial biofilms elicit biofouling, thus leading to substantial economic losses; however, these microbial assemblages can also be beneficial, e.g., in wastewater purification processes and for biofuel production. Mechanistic aspects of cyanobacterial biofilm development were long overlooked, and genetic and molecular information emerged only in recent years. The importance of this study is 2-fold. First, it identifies novel components of cyanobacterial biofilm regulation, thus contributing to the knowledge of these processes and paving the way for inhibiting detrimental biofilms or promoting beneficial ones. Second, the data suggest that cyanobacteria may employ the same complex for the assembly of the motility appendages, type 4 pili, and protein secretion. A shared pathway was previously shown in only a few cases of heterotrophic bacteria, whereas numerous studies demonstrated distinct systems for these functions. Thus, our study broadens the understanding of pilus assembly/secretion in diverse bacteria and furthers the aim of controlling the formation of cyanobacterial biofilms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Fimbrias Bacterianas/fisiología , Proteoma , Synechococcus/química , Synechococcus/fisiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Biogénesis de Organelos , Transporte de Proteínas , Vías Secretoras/genética , Vías Secretoras/fisiología , Synechococcus/genéticaRESUMEN
Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain.IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.
Asunto(s)
Proteínas Bacterianas/genética , Fimbrias Bacterianas/fisiología , Neisseria gonorrhoeae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Fimbrias Bacterianas/clasificación , Regulación Bacteriana de la Expresión Génica , Neisseria gonorrhoeae/metabolismo , Fenotipo , Dominios Proteicos , VirulenciaRESUMEN
Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.
Asunto(s)
Actinobacteria/metabolismo , Proteínas Fimbrias/metabolismo , Bacterias Grampositivas/metabolismo , Actinobacteria/genética , Actinomyces , Pared Celular/metabolismo , Corynebacterium diphtheriae/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas , Humanos , Peptidoglicano/metabolismo , VirulenciaRESUMEN
Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.
Asunto(s)
Actinomyces , Adhesinas Bacterianas , Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Fimbrias Bacterianas , Actinomyces/química , Actinomyces/genética , Actinomyces/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismoRESUMEN
Natural competence is the term used to describe the uptake of "naked" extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large ß-solenoid domain inserted into the ß-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and ß-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.IMPORTANCEThermus thermophilus is a thermophilic bacterium which is capable of natural transformation, the uptake of external DNA with high efficiency. DNA uptake is thought to be mediated by a competence-associated pilus, which binds the DNA substrate and mediates its transfer across the outer membrane and periplasm. Here, we describe the structural and functional analysis of two pilins which are known to be essential for DNA uptake, ComZ and PilA2. ComZ adopts an unusual structure, incorporating a large ß-solenoid domain into the pilin structural framework. We argue on structural grounds that this structure cannot readily be accommodated into the competence pilus fiber unless it is at the tip. We also show that ComZ binds DNA and identify two lysine residues which appear to be important for DNA binding. These results suggest a model in which ComZ and PilA2 form a tip-associated DNA receptor which mediates DNA uptake.
Asunto(s)
Competencia de la Transformación por ADN , Proteínas Fimbrias/química , Fimbrias Bacterianas/química , Receptores de Superficie Celular/química , Thermus thermophilus/genética , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Dominios Proteicos , Receptores de Superficie Celular/genéticaRESUMEN
Bacterial type 4a pili are dynamic surface filaments that promote bacterial adherence, motility, and macromolecular transport. Their genes are highly conserved among enterobacteria and their expression in enterohemorrhagic Escherichia coli (EHEC) promotes adhesion to intestinal epithelia and pro-inflammatory signaling. To define the molecular basis of EHEC pilus assembly, we determined the structure of the periplasmic domain of its major subunit PpdD (PpdDp), a prototype of an enterobacterial pilin subfamily containing two disulfide bonds. The structure of PpdDp, determined by NMR, was then docked into the density envelope of purified EHEC pili obtained by cryoelectron microscopy (cryo-EM). Cryo-EM reconstruction of EHEC pili at â¼8 Å resolution revealed extremely high pilus flexibility correlating with a large extended region of the pilin stem. Systematic mutagenesis combined with functional and interaction analyses identified charged residues essential for pilus assembly. Structural information on exposed regions and interfaces between EHEC pilins is relevant for vaccine and drug discovery.
Asunto(s)
Escherichia coli Enterohemorrágica/química , Proteínas de Escherichia coli/química , Proteínas Fimbrias/química , Fimbrias Bacterianas/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática , TermodinámicaRESUMEN
Initial attachment and subsequent colonization of the intestinal epithelium comprise critical events allowing enteric pathogens to survive and express their pathogenesis. In enterotoxigenic Escherichia coli (ETEC), these are mediated by a long proteinaceous fiber termed type IVb pilus (T4bP). We have reported that the colonization factor antigen/III (CFA/III), an operon-encoded T4bP of ETEC, possesses a minor pilin, CofB, that carries an H-type lectin domain at its tip. Although CofB is critical for pilus assembly by forming a trimeric initiator complex, its importance for bacterial attachment remains undefined. Here, we show that T4bP is not sufficient for bacterial attachment, which also requires a secreted protein CofJ, encoded within the same CFA/III operon. The crystal structure of CofB complexed with a peptide encompassing the binding region of CofJ showed that CofJ interacts with CofB by anchoring its flexible N-terminal extension to be embedded deeply into the expected carbohydrate recognition site of the CofB H-type lectin domain. By combining this structure and physicochemical data in solution, we built a plausible model of the CofJ-CFA/III pilus complex, which suggested that CofJ acts as a molecular bridge by binding both T4bP and the host cell membrane. The Fab fragments of a polyclonal antibody against CofJ significantly inhibited bacterial attachment by preventing the adherence of secreted CofJ proteins. These findings signify the interplay between T4bP and a secreted protein for attaching to and colonizing the host cell surface, potentially constituting a therapeutic target against ETEC infection.
Asunto(s)
Adhesión Bacteriana , Escherichia coli Enterotoxigénica/química , Proteínas de Escherichia coli/química , Fimbrias Bacterianas/química , Cristalografía por Rayos X , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Escherichia coli Enterotoxigénica/patogenicidad , Escherichia coli K12/química , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Operón , Dominios ProteicosRESUMEN
Bacterial surface appendages of the type 4 pilus superfamily play diverse roles in adherence, aggregation, motility, signaling, and macromolecular transport. Here we describe two analytical approaches to study assembly of type 4 pili and of pseudopili produced by type 2 protein secretion systems: the shearing assay and immunofluorescence microscopy. These complementary antibody-based methods allow for semiquantitative analysis of fiber assembly. The shearing assay can be scaled up to yield crude extracts of pili that can be further analyzed by electron and atomic force microscopy or by mass spectrometry.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Fimbrias Bacterianas/metabolismo , Klebsiella oxytoca/metabolismo , Sustancias Macromoleculares/metabolismo , Microscopía Fluorescente/métodos , Adhesión Bacteriana , Fimbrias Bacterianas/químicaRESUMEN
The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbACd). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbACm) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro, we demonstrated that MdbACm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbACm in the C. diphtheriae ΔmdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbACm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in ActinobacteriaIMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide bond formation in vitro Furthermore, a new gene deletion method revealed that deletion of mdbA is lethal in C. matruchotii Remarkably, C. matruchotii MdbA can replace C. diphtheriae MdbA to maintain normal cell growth and morphology, toxin production, and pilus assembly. Overall, our studies support the hypothesis that C. matruchotii utilizes MdbA as a major oxidoreductase to catalyze oxidative protein folding.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium/enzimología , Corynebacterium/genética , Proteína Disulfuro Reductasa (Glutatión)/química , Proteínas Bacterianas/genética , Biopelículas , Catálisis , Corynebacterium diphtheriae/enzimología , Corynebacterium diphtheriae/genética , Disulfuros/química , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Eliminación de Gen , Genoma Bacteriano , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Proteína Disulfuro Reductasa (Glutatión)/genéticaRESUMEN
Acinetobacter baumannii is emerging as a multidrug-resistant nosocomial pathogen of increasing threat to human health worldwide. Pili are important bacterial virulence factors, playing a role in attachment to host cells and biofilm formation. The Csu pilus, which is assembled via the chaperone-usher secretion system, has been studied in A. baumannii ATCC 19606. Here we show that, in opposition to previous reports, the common laboratory strain ATCC 17978 produces Csu pili. We found that, although ATCC 17978 was resistant to sulfamethoxazole (Smx) and trimethoprim (Tmp), subinhibitory concentrations of these antibiotics abolished the expression of Csu and consequently produced a dramatic reduction in biofilm formation by ATCC 17978. Smx and Tmp acted synergistically to inhibit the enzymatic systems involved in the bacterial synthesis of tetrahydrofolate (THF), which is required for the synthesis of nucleotides. The effects of these antibiotics were partially relieved by exogenous THF addition, indicating that Smx and Tmp turn off Csu assembly by inducing folate stress. We propose that, for Acinetobacter, nanomolar concentrations of Smx and Tmp represent a "danger signal." In response to this signal, Csu expression is repressed, allowing biofilm dispersal and escape from potentially inhibitory concentrations of antibiotics. The roles of antibiotics as signaling molecules are being increasingly acknowledged, with clear implications for both the treatment of bacterial diseases and the understanding of complex microbial interactions in the environment.
Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Sulfametoxazol/farmacología , Trimetoprim/farmacología , Infecciones por Acinetobacter/metabolismo , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Factores de Virulencia/metabolismoRESUMEN
The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors.
Asunto(s)
Actinomyces/fisiología , Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Transporte de Electrón , Fimbrias Bacterianas/metabolismo , Biogénesis de Organelos , Streptococcus oralis/fisiología , Actinomyces/genética , Actinomyces/crecimiento & desarrollo , Actinomyces/metabolismo , Elementos Transponibles de ADN , Pruebas Genéticas , Mutagénesis InsercionalRESUMEN
Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacterium Actinomyces oris is mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterial vitamin K epoxide reductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of the A. oris membrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent ß-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of the Mycobacterium tuberculosis VKOR analog in the A. oris VKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression of M. tuberculosis VKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA in Escherichia coli Altogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the class ActinobacteriaIMPORTANCE It has recently been shown in the high-GC Gram-positive bacteria (or Actinobacteria) Actinomyces oris and Corynebacterium diphtheriae that oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. In A. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that the A. oris membrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the class Actinobacteria, including Mycobacterium tuberculosis.
Asunto(s)
Actinomyces/enzimología , Actinomyces/metabolismo , Biopelículas/crecimiento & desarrollo , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Vitamina K Epóxido Reductasas/metabolismo , Actinomyces/genética , Actinomyces/fisiología , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Cisteína/genética , Cisteína/metabolismo , Análisis Mutacional de ADN , Fimbrias Bacterianas/metabolismo , Microscopía Electrónica de Transmisión , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Biogénesis de Organelos , Oxidación-Reducción , Vitamina K Epóxido Reductasas/genéticaRESUMEN
In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural "tip"-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal ß-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly.