RESUMEN
Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat due to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Further, sera from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.
RESUMEN
Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium. Some of these bacteria, including Gardnerella, produce glycosyl hydrolase enzymes. However, glycans of the human vaginal epithelial surface have not been studied in detail. Here, we elucidate key characteristics of the "normal" vaginal epithelial glycan landscape and analyze the impact of resident microbes on the surface glycocalyx. In human BV, glycocalyx staining was visibly diminished in electron micrographs compared to controls. Biochemical and mass spectrometric analysis showed that, compared to normal vaginal epithelial cells, BV cells were depleted of sialylated N- and O-glycans, with underlying galactose residues exposed on the surface. Treatment of primary epithelial cells from BV-negative women with recombinant Gardnerella sialidases generated BV-like glycan phenotypes. Exposure of cultured VK2 vaginal epithelial cells to recombinant Gardnerella sialidase led to desialylation of glycans and induction of pathways regulating cell death, differentiation, and inflammatory responses. These data provide evidence that vaginal epithelial cells exhibit an altered glycan landscape in BV and suggest that BV-associated glycosidic enzymes may lead to changes in epithelial gene transcription that promote cell turnover and regulate responses toward the resident microbiome.
Asunto(s)
Gardnerella vaginalis , Vaginosis Bacteriana , Femenino , Humanos , Gardnerella vaginalis/genética , Gardnerella vaginalis/metabolismo , Vagina , Vaginosis Bacteriana/genética , Vaginosis Bacteriana/microbiología , Bacterias/metabolismo , Polisacáridos , Neuraminidasa/genética , Neuraminidasa/metabolismoRESUMEN
Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.
Asunto(s)
Infecciones por Klebsiella , Vacunas , Ratones , Animales , Virulencia , Antígenos O , Klebsiella pneumoniae , Lipopolisacáridos/metabolismo , Antibacterianos/farmacología , Infecciones por Klebsiella/prevención & controlRESUMEN
Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.
Asunto(s)
Hexosiltransferasas , Moraxellaceae , Animales , Ratones , Moraxellaceae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Filogenia , Hexosiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Fimbrias , Polisacáridos/metabolismo , Bacterias/metabolismoRESUMEN
Klebsiella pneumoniae is a concerning pathogen that is now the leading cause of neonatal sepsis and is increasingly difficult to treat due to heightened antibiotic resistance. Thus, there is an urgent need for preventive and effective immunotherapies targeting K. pneumoniae. Vaccination represents a tractable approach to combat this resistant bacterium in some settings; however, there is currently not a licensed K. pneumoniae vaccine available. K. pneumoniae surface polysaccharides, including the terminal O-antigen polysaccharides of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven of the predominant O-antigen subtypes in K. pneumoniae. Each of the seven bioconjugates were immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains, including suspected hypervirulent strains, all expressing different O-antigen and capsular polysaccharide combinations. Further, sera from vaccinated mice induced complement-mediated killing of many of these K. pneumoniae strains. Finally, we found that increased quantity of capsule interferes with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains, including those carrying hypervirulence-associated genes. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits promising efficacy against some, but not all, K. pneumoniae isolates.
RESUMEN
Capsular polysaccharides (CPSs), with which most pathogenic bacterial surfaces are decorated, have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide. Pneumococcal conjugate vaccines (PCVs) are administered globally to prevent invasive pneumococcal disease (IPD). While PCVs have played important roles in controlling IPD in all age groups, their empirical, and labor-intensive chemical conjugation yield poorly characterized, heterogeneous, and variably immunogenic vaccines, with poor immune responses in high-risk populations such as the elderly and patients with weak immune systems. We previously developed a method that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems to produce pneumococcal conjugate vaccines. The bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to an engineered carrier protein all within the lab safe bacterium E. coli. In these studies, we demonstrate that a serotype 8 pneumococcal bioconjugate vaccine is highly immunogenic and elicits functionally protective anti-serotype 8 antibody responses. Specifically, using multiple models we show that mice immunized with multiple doses of a serotype 8 bioconjugate vaccine elicit antibody responses that mediate opsonophagocytic killing, protect mice from systemic infection, and decrease the ability of serotype 8 pneumococci to colonize the nasopharynx and disseminate. Collectively, these studies demonstrate the utility of bioconjugation to produce efficacious pneumococcal conjugate vaccines.
Asunto(s)
Infecciones Neumocócicas , Vacunas Neumococicas , Animales , Anticuerpos Antibacterianos , Proteínas Portadoras , Escherichia coli , Ratones , Polisacáridos Bacterianos , Vacunas ConjugadasRESUMEN
Group B Streptococcus (GBS) is a leading cause of neonatal infections and invasive diseases in nonpregnant adults worldwide. Developing a protective conjugate vaccine targeting the capsule of GBS has been pursued for more than 30 years; however, it has yet to yield a licensed product. In this study, we present a novel bioconjugation platform for producing a prototype multivalent GBS conjugate vaccine and its subsequent analytical and immunological characterizations. Using a glycoengineering strategy, we generated strains of Escherichia coli that recombinantly express the type Ia, type Ib, and type III GBS capsular polysaccharides. We then combined the type Ia-, Ib-, and III-capsule-expressing E. coli strains with an engineered Pseudomonas aeruginosa exotoxin A (EPA) carrier protein and the PglS oligosaccharyltransferase. Coexpression of a GBS capsule, the engineered EPA protein, and PglS enabled the covalent attachment of the target GBS capsule to an engineered serine residue on EPA, all within the periplasm of E. coli. GBS bioconjugates were purified, analytically characterized, and evaluated for immunogenicity and functional antibody responses. This proof-of-concept study signifies the first step in the development of a next-generation multivalent GBS bioconjugate vaccine, which was validated by the production of conjugates that are able to elicit functional antibodies directed against the GBS capsule.
Asunto(s)
Escherichia coli , Infecciones Estreptocócicas , Adulto , Anticuerpos Antibacterianos , Escherichia coli/genética , Humanos , Recién Nacido , Infecciones Estreptocócicas/prevención & control , Streptococcus agalactiae/genética , Vacunas CombinadasRESUMEN
Bioconjugate vaccines, consisting of polysaccharides attached to carrier proteins, are enzymatically generated using prokaryotic glycosylation systems in a process termed bioconjugation. Key to bioconjugation are a group of enzymes known as oligosaccharyltransferases (OTases) that transfer polysaccharides to engineered carrier proteins containing conserved amino acid sequences known as sequons. The most recently discovered OTase, PglS, has been shown to have the broadest substrate scope, transferring many different types of bacterial glycans including those with glucose at the reducing end. However, PglS is currently the least understood in terms of the sequon it recognizes. PglS is a pilin-specific O-linking OTase that naturally glycosylates a single protein, ComP. In addition to ComP, we previously demonstrated that an engineered carrier protein containing a large fragment of ComP is also glycosylated by PglS. Here we sought to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested >100 different ComP fragments individually fused to Pseudomonas aeruginosa exotoxin A (EPA), leading to the identification of an 11-amino acid sequence sufficient for robust glycosylation by PglS. We also demonstrate that the placement of the ComP sequon on the carrier protein is critical for stability and subsequent glycosylation. Moreover, we identify novel sites on the surface of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive Material 197 fused to a ComP fragment is also glycosylated. These results represent a significant expansion of the glycoengineering toolbox as well as our understanding of bacterial O-linking sequons.
Asunto(s)
Hexosiltransferasas , Secuencia de Aminoácidos , Proteínas Fimbrias/metabolismo , Glicosilación , Hexosiltransferasas/metabolismo , Proteínas de la MembranaRESUMEN
Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.
Asunto(s)
Proteínas Bacterianas/genética , Disbiosis/microbiología , Fusobacterium/metabolismo , Gardnerella vaginalis/metabolismo , Neuraminidasa/genética , Polisacáridos/metabolismo , Vaginosis Bacteriana/microbiología , Animales , Proteínas Bacterianas/metabolismo , Técnicas de Tipificación Bacteriana , Disbiosis/patología , Femenino , Fusobacterium/genética , Fusobacterium/aislamiento & purificación , Fusobacterium/patogenicidad , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Gardnerella vaginalis/patogenicidad , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Microbiota/genética , Neuraminidasa/metabolismo , ARN Ribosómico 16S/genética , Ácidos Siálicos/metabolismo , Simbiosis/genética , Vagina/microbiología , Vaginosis Bacteriana/patologíaRESUMEN
Gardnerella vaginalis is abundant in bacterial vaginosis (BV), a condition associated with adverse reproductive health. Sialidase activity is a diagnostic feature of BV and is produced by a subset of G. vaginalis strains. Although its genetic basis has not been formally identified, sialidase activity is presumed to derive from the sialidase A gene, named here nanH1 In this study, BLAST searches predicted two additional G. vaginalis sialidases, NanH2 and NanH3. When expressed in Escherichia coli, NanH2 and NanH3 both displayed broad abilities to cleave sialic acids from α2-3- and α2-6-linked N- and O-linked sialoglycans, including relevant mucosal substrates. In contrast, recombinant NanH1 had limited activity against synthetic and mucosal substrates under the conditions tested. Recombinant NanH2 was much more effective than NanH3 in cleaving sialic acids bearing a 9-O-acetyl ester. Similarly, G. vaginalis strains encoding NanH2 cleaved and foraged significantly more Neu5,9Ac2 than strains encoding only NanH3. Among a collection of 34 G. vaginalis isolates, nanH2, nanH3, or both were present in all 15 sialidase-positive strains but absent from all 19 sialidase-negative isolates, including 16 strains that were nanH1-positive. We conclude that NanH2 and NanH3 are the primary sources of sialidase activity in G. vaginalis and that these two enzymes can account for the previously described substrate breadth cleaved by sialidases in human vaginal specimens of women with BV. Finally, PCRs of nanH2 or nanH3 from human vaginal specimens had 81% sensitivity and 78% specificity in distinguishing between Lactobacillus dominance and BV, as determined by Nugent scoring.
Asunto(s)
Proteínas Bacterianas , Gardnerella vaginalis , Neuraminidasa , Vaginosis Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Gardnerella vaginalis/enzimología , Gardnerella vaginalis/genética , Humanos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/química , Neuraminidasa/genética , Neuraminidasa/metabolismo , Especificidad por Sustrato , Vaginosis Bacteriana/enzimología , Vaginosis Bacteriana/genética , Vaginosis Bacteriana/microbiologíaRESUMEN
The gut harbors many symbiotic, commensal, and pathogenic microbes that break down and metabolize host carbohydrates. Sialic acids are prominent outermost carbohydrates on host glycoproteins called mucins and protect underlying glycan chains from enzymatic degradation. Sialidases produced by some members of the colonic microbiota can promote the expansion of several potential pathogens (e.g. Clostridium difficile, Salmonella, and Escherichia coli) that do not produce sialidases. O-Acetyl ester modifications of sialic acids help resist the action of many sialidases and are present at high levels in the mammalian colon. However, some gut bacteria, in turn, produce sialylate-O-acetylesterases to remove them. Here, we investigated O-acetyl ester removal and sialic acid degradation by Bacteroidetes sialate-O-acetylesterases and sialidases, respectively, and subsequent utilization of host sialic acids by both commensal and pathogenic E. coli strains. In vitro foraging studies demonstrated that sialidase-dependent E. coli growth on mucin is enabled by Bacteroides EstA, a sialate O-acetylesterase acting on glycosidically linked sialylate-O-acetylesterase substrates, particularly at neutral pH. Biochemical studies suggested that spontaneous migration of O-acetyl esters on the sialic acid side chain, which can occur at colonic pH, may serve as a switch controlling EstA-assisted sialic acid liberation. Specifically, EstA did not act on O-acetyl esters in their initial 7-position. However, following migration to the 9-position, glycans with O-acetyl esters became susceptible to the sequential actions of bacterial esterases and sialidases. We conclude that EstA specifically unlocks the nutritive potential of 9-O-acetylated mucus sialic acids for foraging by bacteria that otherwise are prevented from accessing this carbon source.
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Proteínas Bacterianas/metabolismo , Bacteroides fragilis/enzimología , Bacteroides thetaiotaomicron/enzimología , Bacteroidetes/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Interacciones Microbianas , Mucinas/metabolismo , Neuraminidasa/metabolismo , Acetilación , Animales , Bacteroides fragilis/crecimiento & desarrollo , Bacteroides fragilis/fisiología , Bacteroides thetaiotaomicron/crecimiento & desarrollo , Bacteroides thetaiotaomicron/fisiología , Bacteroidetes/crecimiento & desarrollo , Bacteroidetes/fisiología , Bovinos , Escherichia coli Enterohemorrágica/crecimiento & desarrollo , Escherichia coli Enterohemorrágica/fisiología , Microbioma Gastrointestinal , Concentración de Iones de Hidrógeno , Hidrólisis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/genética , Polisacáridos Bacterianos/metabolismo , Proteínas Recombinantes/metabolismo , Streptococcus agalactiae/crecimiento & desarrollo , Streptococcus agalactiae/fisiología , Especificidad por SustratoRESUMEN
Gardnerella vaginalis is a predominant species in bacterial vaginosis, a dysbiosis of the vagina that is associated with adverse health outcomes, including preterm birth. Here, we present the draft genome sequences of 15 Gardnerella vaginalis strains (now available through BEI Resources) isolated from women with and without bacterial vaginosis.
RESUMEN
The presence of bacteria in urine can pose significant risks during pregnancy. However, there are few reference genome strains for many common urinary bacteria. We isolated 12 urinary strains of Streptococcus, Staphylococcus, Citrobacter, Gardnerella, and Lactobacillus These strains and their genomes are now available to the research community.
RESUMEN
The composition of the vaginal microbiota is an important health determinant. Several members of the phylum Actinobacteria have been implicated in bacterial vaginosis, a condition associated with many negative health outcomes. Here, we present 11 strains of vaginal Actinobacteria (now available through BEI Resources) along with draft genome sequences.
RESUMEN
The vagina is home to a wide variety of bacteria that have great potential to impact human health. Here, we announce reference strains (now available through BEI Resources) and draft genome sequences for 9 Gram-negative vaginal isolates from the taxa Citrobacter, Klebsiella, Fusobacterium, Proteus, and Prevotella.
RESUMEN
Sialic acids are nine-carbon backbone carbohydrates found in prominent outermost positions of glycosylated molecules in mammals. Mimicry of sialic acid (N-acetylneuraminic acid, Neu5Ac) enables some pathogenic bacteria to evade host defenses. Fusobacterium nucleatum is a ubiquitous oral bacterium also linked with invasive infections throughout the body. We employed multidisciplinary approaches to test predictions that F. nucleatum engages in de novo synthesis of sialic acids. Here we show that F. nucleatum sbsp. polymorphum ATCC10953 NeuB (putative Neu5Ac synthase) restores Neu5Ac synthesis to an Escherichia coli neuB mutant. Moreover, purified F. nucleatum NeuB participated in synthesis of Neu5Ac from N-acetylmannosamine and phosphoenolpyruvate in vitro Further studies support the interpretation that F. nucleatum ATCC10953 NeuA encodes a functional CMP-sialic acid synthetase and suggest that it may also contain a C-terminal sialic acid O-acetylesterase. We also performed BLAST queries of F. nucleatum genomes, revealing that only 4/31 strains encode a complete pathway for de novo Neu5Ac synthesis. Biochemical studies including mass spectrometry were consistent with the bioinformatic predictions, showing that F. nucleatum ATCC10953 synthesizes high levels of Neu5Ac, whereas ATCC23726 and ATCC25586 do not express detectable levels above background. While there are a number of examples of sialic acid mimicry in other phyla, these experiments provide the first biochemical and genetic evidence that a member of the phylum Fusobacterium can engage in de novo Neu5Ac synthesis.
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Fusobacterium/metabolismo , Ácido N-Acetilneuramínico/biosíntesis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Ácido N-Acetilneuramínico/químicaRESUMEN
Bacterial vaginosis (BV) is a polymicrobial imbalance of the vaginal microbiota associated with reproductive infections, preterm birth, and other adverse health outcomes. Sialidase activity in vaginal fluids is diagnostic of BV and sialic acid-rich components of mucus have protective and immunological roles. However, whereas mucus degradation is believed to be important in the etiology and complications associated with BV, the role(s) of sialidases and the participation of individual bacterial species in the degradation of mucus barriers in BV have not been investigated. Here we demonstrate that the BV-associated bacterium Gardnerella vaginalis uses sialidase to break down and deplete sialic acid-containing mucus components in the vagina. Biochemical evidence using purified sialoglycan substrates supports a model in which 1) G. vaginalis extracellular sialidase hydrolyzes mucosal sialoglycans, 2) liberated sialic acid (N-acetylneuraminic acid) is transported into the bacterium, a process inhibited by excess N-glycolylneuraminic acid, and 3) sialic acid catabolism is initiated by an intracellular aldolase/lyase mechanism. G. vaginalis engaged in sialoglycan foraging in vitro, in the presence of human vaginal mucus, and in vivo, in a murine vaginal model, in each case leading to depletion of sialic acids. Comparison of sialic acid levels in human vaginal specimens also demonstrated significant depletion of mucus sialic acids in women with BV compared with women with a "normal" lactobacilli-dominated microbiota. Taken together, these studies show that G. vaginalis utilizes sialidase to support the degradation, foraging, and depletion of protective host mucus barriers, and that this process of mucus barrier degradation and depletion also occurs in the clinical setting of BV.
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Proteínas Bacterianas/metabolismo , Gardnerella vaginalis/enzimología , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Proteoglicanos/metabolismo , Vaginosis Bacteriana/enzimología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiologíaRESUMEN
Bacterial vaginosis (BV) is a common polymicrobial imbalance of the vaginal flora associated with a wide variety of obstetric and gynecologic complications including serious infections and preterm birth. As evidenced by high recurrence rates following treatment, interventions for BV are still lacking. Several hydrolytic activities, including glycosidases and proteases, have been previously correlated with BV and have been hypothesized to degrade host sialoglycoproteins that participate in mucosal immune functions. Sialidase activity is most predictive of BV status and correlates strongly with adverse health outcomes. Here we combine clinical specimens with biochemical approaches to investigate secretory immunoglobulin A (SIgA) as a substrate of BV-associated glycosidases and proteases. We show that BV clinical specimens hydrolyze sialic acid from SIgA, but not in the presence of the sialidase inhibitor dehydro-deoxy-sialic acid. The collective action of BV-associated glycosidases exposes underlying mannose residues of SIgA, most apparent on the heavily N-glycosylated secretory component of the antibody. Terminal sialic acid residues on SIgA protect underlying carbohydrate residues from exposure and hydrolysis by exoglycosidases (galactosidase and hexosaminidase). It is known that both IgG and SIgA are present in the human reproductive tract. We show that the IgG heavy chain is more susceptible to proteolysis than its IgA counterpart. Gentle partial deglycosylation of the SIgA secretory component enhanced susceptibility to proteolysis. Together, these data support a model of BV in which SIgA is subject to stepwise exodeglycosylation and enhanced proteolysis, likely compromising the ability of the reproductive mucosa to neutralize and eliminate pathogens.
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Modelos Biológicos , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Proteolisis , Componente Secretorio/metabolismo , Vaginosis Bacteriana/metabolismo , Ensayos Clínicos como Asunto , Estudios de Cohortes , Femenino , Humanos , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Vaginosis Bacteriana/microbiologíaRESUMEN
Curli are extracellular amyloid fibres produced by Escherichia coli that are critical for biofilm formation and adhesion to biotic and abiotic surfaces. CsgA and CsgB are the major and minor curli subunits, respectively, while CsgE, CsgF and CsgG direct the extracellular localization and assembly of curli subunits into fibres. The secretion and stability of CsgA and CsgB are dependent on the outer membrane lipoprotein CsgG. Here, we identified functional interactions between CsgG and CsgE during curli secretion. We discovered that CsgG overexpression restored curli production to a csgE strain under curli-inducing conditions. In antibiotic sensitivity and protein secretion assays, CsgG expression alone allowed translocation of erythromycin and small periplasmic proteins across the outer membrane. Coexpression of CsgE with CsgG blocked non-specific protein and antibiotic passage across the outer membrane. However, CsgE did not block secretion of proteins containing a 22-amino-acid putative outer membrane secretion signal of CsgA (A22). Finally, using purified proteins, we found that CsgE prohibited the self-assembly of CsgA into amyloid fibres. Collectively, these data indicate that CsgE provides substrate specificity to the curli secretion pore CsgG, and acts directly on the secretion substrate CsgA to prevent premature subunit assembly.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Desnaturalización Proteica , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Proteínas de Transporte de Membrana/genética , Unión ProteicaRESUMEN
Elucidation of the early events in amyloidogenesis is key to understanding the pathology of, and developing therapies for, amyloid diseases. Critical informants about these early events are amyloid assembly proteins that facilitate the transition from monomer to amyloid fiber. Curli are a functional amyloid whose in vivo polymerization requires a dedicated nucleator protein, CsgB, and an assembly protein, CsgF. Here we demonstrate that without CsgF, curli subunits are released from the cell into the media and are inefficiently polymerized, resulting in fewer and mislocalized curli fibers. CsgF is secreted to the cell surface, where it mediates the cell-association and protease-resistance of the CsgB nucleator, suggesting that CsgF is required for specific localization and/or chaperoning of CsgB for full nucleator activity. CsgF is thus critical to achieve localized and efficient nucleation of fiber subunits into functional, cell-associated amyloid.