RESUMO
Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.
Assuntos
Fímbrias Bacterianas , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/fisiologia , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Bactérias/genética , Bactérias/metabolismo , Aderência Bacteriana/genética , Transferência Genética Horizontal , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Myxococcus xanthus/genética , Myxococcus xanthus/fisiologia , Myxococcus xanthus/metabolismoRESUMO
The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.
Assuntos
Proteínas de Escherichia coli , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Escherichia coli/metabolismo , GMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Biofilmes , Regulação Bacteriana da Expressão GênicaRESUMO
Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.
Assuntos
Alginatos , Pseudomonas aeruginosa , Acetilação , Alginatos/química , Proteínas de Bactérias/metabolismo , Biofilmes , Periplasma/metabolismo , Processamento de Proteína Pós-Traducional , Pseudomonas aeruginosa/metabolismoRESUMO
Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the pelABCDEFG operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P. aeruginosa PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in Bacillus cereus ATCC 10987, which has a predicted extracellular PelA deacetylase in its pel operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets. IMPORTANCE Present in more than 500 diverse Gram-negative and 900 Gram-positive organisms, the Pel polysaccharide is one of the most phylogenetically widespread biofilm matrix determinants found to date. Partial de-N-acetylation of this α-1,4 linked N-acetylgalactosamine polymer by the carbohydrate modification enzyme PelA is required for Pel-dependent biofilm formation in Pseudomonas aeruginosa and Bacillus cereus. Given this and our observation that extracellular Pel is not produced by a P. aeruginosa PelA deactylase mutant, we developed an enzyme-based high-throughput screen and identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) and its phenyl derivative as specific Pel-dependent biofilm inhibitors. Michaelis-Menten kinetics revealed SK-017154-O is a noncompetitive inhibitor and that its noncytotoxic, phenyl derivative does not directly inhibit P. aeruginosa PelA esterase activity. We provide proof of concept that exopolysaccharide modification enzymes can be targeted with small molecule inhibitors to block Pel-dependent biofilm development in both Gram-negative and Gram-positive bacteria.
Assuntos
Polissacarídeos Bacterianos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Biofilmes , Periplasma , Esterases , Proteínas de Bactérias/genéticaRESUMO
Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence.
Assuntos
Biofilmes , Glicosídeo Hidrolases , Polissacarídeos Bacterianos , Pseudomonas aeruginosa , Animais , Fenômenos Biomecânicos , Drosophila melanogaster/microbiologia , Glicosídeo Hidrolases/genética , Pseudomonas aeruginosa/fisiologia , Virulência , Caenorhabditis elegans/microbiologiaRESUMO
Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.
Assuntos
Alginatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Alginatos/metabolismo , Ácidos Hexurônicos/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Glucurônico/metabolismo , Biofilmes , Polímeros/metabolismoRESUMO
Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.
Assuntos
Alginatos , Periplasma , Polissacarídeo-Liases , Pseudomonas aeruginosa , Alginatos/química , Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/genética , Ácidos Hexurônicos/química , Homeostase , Humanos , Periplasma/enzimologia , Periplasma/metabolismo , Polímeros/metabolismo , Polissacarídeo-Liases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismoRESUMO
Bacteria and archaea rely on appendages called type IV pili (T4P) to participate in diverse behaviors including surface sensing, biofilm formation, virulence, protein secretion and motility across surfaces. T4P are broadly distributed fibers that dynamically extend and retract, and this dynamic activity is essential for their function in broad processes. Despite the essentiality of dynamics in T4P function, little is known about the role of these dynamics and molecular mechanisms controlling them. Recent advances in microscopy have yielded insight into the role of T4P dynamics in their diverse functions and recent structural work has expanded what is known about the inner workings of the T4P motor. This review discusses recent progress in understanding the function, regulation, and mechanisms of T4P dynamics.
Assuntos
Proteínas de Fímbrias , Fímbrias Bacterianas , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/análise , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , VirulênciaRESUMO
Bacteria synthesize and export adhesive macromolecules to enable biofilm formation. These macromolecules, collectively called the biofilm matrix, are structurally varied and often unique to specific bacterial species or subspecies. This heterogeneity in matrix utilization makes it difficult to facilitate direct comparison between biofilm formation mechanisms of different bacterial species. Despite this, some matrix components, in particular the polysaccharides poly-ß-1,6-N-acetyl-glucosamine (PNAG) and bacterial cellulose, are utilized by many Gram-negative species for biofilm formation. However, there is a very narrow distribution of these components across Gram-positive organisms, whose biofilm matrix determinants remain largely undiscovered. We found that a genetic locus required for the production of a biofilm matrix component of P. aeruginosa, the Pel polysaccharide, is widespread in Gram-negative bacteria and that there is a variant form of this cluster present in many Gram-positive bacterial species. We demonstrated that this locus is required for biofilm formation by Bacillus cereus ATCC 10987, produces a polysaccharide that is similar to Pel, and is post-translationally regulated by cyclic-3',5'-dimeric-guanosine monophosphate (c-di-GMP) in a manner identical to P. aeruginosa. However, while the proposed mechanism for Pel production appears remarkably similar between B. cereus and P. aeruginosa, we identified several key differences between Gram-negative and Gram-positive Pel biosynthetic components in other monoderms. In particular, 4 different architectural subtypes of the c-di-GMP-binding component PelD were identified, including 1 found only in Streptococci that has entirely lost the c-di-GMP recognition domain. These observations highlight how existing multi-component bacterial machines can be subtly tweaked to adapt to the unique physiology and regulatory mechanisms of Gram-positive organisms. Collectively, our analyses suggest that the Pel biosynthetic locus is one of the most phylogenetically widespread biofilm matrix determinants in bacteria, and that its mechanism of production and regulation is extraordinarily conserved across the majority of organisms that possess it.
RESUMO
Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.
Assuntos
Aderência Bacteriana/fisiologia , Fenômenos Fisiológicos Bacterianos , Biofilmes , Percepção de Quorum/fisiologia , Biofilmes/crescimento & desenvolvimentoRESUMO
Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of pel gene clusters has helped us identify an additional gene, pelX, that is present adjacent to pelABCDEFG in >100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to elucidate PelX function as Pseudomonas aeruginosa lacks a pelX homologue in its pel gene cluster. We found that P. protegens forms Pel-dependent biofilms; however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led us to identify a pelX paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to pelX In line with this, a ΔpelX ΔpgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 Å resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Carboidratos Epimerases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/fisiologia , Pseudomonas/fisiologia , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética , Polissacarídeos Bacterianos/genética , Uridina Difosfato N-Acetilglicosamina/genética , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.
Assuntos
Fenômenos Fisiológicos Bacterianos , Caulobacter crescentus/fisiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Ciclo Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Replicação do DNA , Fímbrias Bacterianas/fisiologia , Modelos Biológicos , MutaçãoRESUMO
Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.
Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Óperon , Polissacarídeos/metabolismo , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Filogenia , Conformação ProteicaRESUMO
In bacteria functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated cellulose, poly-ß-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).
Assuntos
Biologia Computacional/métodos , Óperon/genética , Óperon/fisiologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Evolução Biológica , Evolução Molecular , Duplicação Gênica , Filogenia , Fatores de VirulênciaRESUMO
The Pel polysaccharide is a structural component of the extracellular matrix of Pseudomonas aeruginosa biofilms. Recent analyses suggest that Pel production proceeds via a synthase-dependent polysaccharide secretion pathway, which in Gram-negative bacteria is defined by an outer membrane ß-barrel porin, a periplasmic tetratricopeptide repeat-containing scaffold protein, and an inner membrane-embedded synthase. Polymerization is catalyzed by the glycosyltransferase domain of the synthase component of these systems, which is allosterically regulated by cyclic 3',5'-dimeric GMP (c-di-GMP). However, while the outer membrane and periplasmic components of the Pel system have been characterized, the inner membrane complex required for Pel polymerization has yet to be defined. To address this, we examined over 500 pel gene clusters from diverse species of Proteobacteria This analysis identified an invariant set of four syntenic genes, three of which, pelD, pelE, and pelG, are predicted to reside within the inner membrane, while the fourth, pelF, encodes a glycosyltransferase domain. Using a combination of gene deletion analysis, subcellular fractionation, coimmunoprecipitation, and bacterial two-hybrid assays, we provide evidence for the existence of an inner membrane complex of PelD, PelE, and PelG. Furthermore, we show that this complex interacts with PelF in order to facilitate its localization to the inner membrane. Mutations that abolish c-di-GMP binding to the known receptor domain of PelD had no effect on complex formation, suggesting that c-di-GMP binding stimulates Pel production through quaternary structural rearrangements. Together, these data provide the first experimental evidence of an inner membrane complex involved in Pel polysaccharide production.IMPORTANCE The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant pelDEFG gene clusters in over 500 diverse proteobacterial species. Using Pseudomonas aeruginosa, we demonstrate that PelD, PelE, PelF, and PelG form a complex at the inner membrane and propose that this complex represents the previously unidentified Pel polysaccharide synthase, which is responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of cyclic 3',5'-dimeric GMP (c-di-GMP) binding to the receptor PelD. Collectively, these data establish the widespread Pel apparatus as a member of the synthase-dependent pathway of polysaccharide biosynthetic systems and broaden the architectural diversity of already-established bacterial polysaccharide synthases.
Assuntos
Proteínas de Bactérias/metabolismo , Polissacarídeos Bacterianos/biossíntese , Pseudomonas aeruginosa/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genéticaRESUMO
The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Matriz Extracelular/metabolismo , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal ß-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.
Assuntos
Biologia Computacional , Polissacarídeo-Liases/química , Polissacarídeos Bacterianos/química , Pseudomonas aeruginosa/química , Biofilmes/crescimento & desenvolvimento , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Lipoproteínas/química , Lipoproteínas/genética , Periplasma/química , Periplasma/genética , Periplasma/metabolismo , Polissacarídeo-Liases/genética , Polissacarídeos Bacterianos/genética , Pseudomonas aeruginosa/patogenicidadeRESUMO
While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example, Pseudomonas aeruginosa and Staphylococcus aureus are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that P. aeruginosa and/or S. aureus adopts phenotypes that allow coexistence during infection. We compared competitive interactions of P. aeruginosa and S. aureus isolates from mono- or coinfected CF patients employing in vitro coculture models. P. aeruginosa isolates from monoinfected patients were more competitive toward S. aureus than P. aeruginosa isolates from coinfected patients. We also observed that the least competitive P. aeruginosa isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill S. aureus Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureus activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids-each required for efficient killing of S. aureus These studies demonstrate alginate overproduction may be an important factor driving P. aeruginosa coinfection with S. aureusIMPORTANCE Numerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains of P. aeruginosa is sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompete S. aureus, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoid P. aeruginosa can coexist with S. aureus Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.
Assuntos
Alginatos/metabolismo , Coinfecção/microbiologia , Interações Microbianas , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/crescimento & desenvolvimento , Infecções Estafilocócicas/complicações , Staphylococcus aureus/crescimento & desenvolvimento , Fibrose Cística/complicações , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Humanos , Modelos Teóricos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Infecções Respiratórias , Infecções Estafilocócicas/microbiologiaRESUMO
A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose, and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo-structure of the periplasmic domain of PslG (PslG(31-442)) and its complex with mannose were determined to 2.0 and 1.9 Å resolution, respectively. Despite a domain architecture and positioning of catalytic residues similar to those of other family 39 glycoside hydrolases, PslG(31-442) exhibits a unique 32-Å-long active site groove that is distinct from other structurally characterized family members. PslG formed a complex with two mannose monosaccharides in this groove, consistent with binding data obtained from intrinsic tryptophan fluorescence. PslG was able to catalyze the hydrolysis of surface-associated Psl, and this activity was abolished in a E165Q/E276Q double catalytic variant. Surprisingly, P. aeruginosa variants with these chromosomal mutations as well as a pslG deletion mutant were still capable of forming Psl biofilms. However, overexpression of PslG in a pslG deletion background impaired biofilm formation and resulted in less surface-associated Psl, suggesting that regulation of this enzyme is important during polysaccharide biosynthesis.
Assuntos
Biofilmes , Glicosídeo Hidrolases/metabolismo , Polissacarídeos/biossíntese , Pseudomonas aeruginosa/enzimologia , Sequência de Carboidratos , Glicosídeo Hidrolases/química , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-Atividade , Frações Subcelulares/enzimologiaRESUMO
Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes.