RESUMEN
BACKGROUND: Group B Streptococcus (GBS) is the leading cause of neonatal meningitis responsible for a substantial cause of death and disability worldwide. The vast majority of GBS neonatal meningitis cases are due to the CC17 hypervirulent clone. However, the cellular and molecular pathways involved in brain invasion by GBS CC17 isolates remain largely elusive. Here, we studied the specific interaction of the CC17 clone with the choroid plexus, the main component of the blood-cerebrospinal fluid (CSF) barrier. METHODS: The interaction of GBS CC17 or non-CC17 strains with choroid plexus cells was studied using an in vivo mouse model of meningitis and in vitro models of primary and transformed rodent choroid plexus epithelial cells (CPEC and Z310). In vivo interaction of GBS with the choroid plexus was assessed by microscopy. Bacterial invasion and cell barrier penetration were examined in vitro, as well as chemokines and cytokines in response to infection. RESULTS: GBS CC17 was found associated with the choroid plexus of the lateral, 3rd and 4th ventricles. Infection of choroid plexus epithelial cells revealed an efficient internalization of the bacteria into the cells with GBS CC17 displaying a greater ability to invade these cells than a non-CC17 strain. Internalization of the GBS CC17 strain involved the CC17-specific HvgA adhesin and occurred via a clathrin-dependent mechanism leading to transcellular transcytosis across the choroid plexus epithelial monolayer. CPEC infection resulted in the secretion of several chemokines, including CCL2, CCL3, CCL20, CX3CL1, and the matrix metalloproteinase MMP3, as well as immune cell infiltration. CONCLUSION: Our findings reveal a GBS strain-specific ability to infect the blood-CSF barrier, which appears to be an important site of bacterial entry and an active site of immune cell trafficking in response to infection.
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Plexo Coroideo , Streptococcus agalactiae , Plexo Coroideo/metabolismo , Plexo Coroideo/microbiología , Plexo Coroideo/inmunología , Animales , Streptococcus agalactiae/patogenicidad , Ratones , Adhesinas Bacterianas/metabolismo , Virulencia , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Barrera Hematoencefálica/microbiología , Barrera Hematoencefálica/metabolismo , Modelos Animales de Enfermedad , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/inmunología , Ratones Endogámicos C57BL , Transcitosis/fisiología , FemeninoRESUMEN
Extracellular cellular adhesins facilitate microbial aggregation; however, most of the information about extracellular adhesins is based on pure culture studies. In this study, we characterized the hydrophobic characteristics and distribution of the extracellular adhesins in environmental biofilms and flocs. The hydrophobic characteristics of the extracellular adhesins were studied by sonicating the microbial aggregates to disperse the cells and by fractionating them using the microbial adhesion to the hydrocarbon method. Furthermore, we probed environmental biofilms and flocs using immunohistochemistry coupled with confocal laser scanning microscopy for reimaging the microbial aggregates based on extracellular adhesins. Small flocs have a relatively dispersed distribution of extracellular adhesins (flagella, fimbriae, pili, and amyloid adhesins). The stratified distribution of extracellular adhesins was observed in environmental biofilms. It was observed that the pili and amyloid adhesins were predominantly present in the core of biofilms, whereas flagella and fimbriae were present in the outer layer of the microbial aggregates. The dispersion of microbial aggregates is one of the limiting factors that challenge the sustainable application of wastewater treatment processes. Greater attention to the components of extracellular protein (such as the adhesins) is required to understand the aggregation of dispersible environmental microbial aggregates.
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Biopelículas , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Fimbrias Bacterianas/metabolismoRESUMEN
Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.
The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.
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Antígenos Bacterianos , Infecciones Meningocócicas , Vacunas Meningococicas , Neisseria meningitidis Serogrupo B , Humanos , Argentina/epidemiología , Vacunas Meningococicas/inmunología , Vacunas Meningococicas/administración & dosificación , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Infecciones Meningocócicas/epidemiología , Lactante , Adolescente , Niño , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Preescolar , Adulto Joven , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/aislamiento & purificación , Neisseria meningitidis Serogrupo B/inmunología , Adulto , Femenino , Masculino , Secuenciación Completa del Genoma , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Genotipo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Persona de Mediana Edad , Porinas/genética , Porinas/inmunología , Determinación de Anticuerpos Séricos Bactericidas , Anciano , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Neisseria meningitidis/aislamiento & purificación , Neisseria meningitidis/clasificaciónRESUMEN
Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.
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Adhesinas Bacterianas , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Adhesión Bacteriana , Interacciones Huésped-Patógeno , Lectinas , Humanos , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Lectinas/metabolismo , Lectinas/genética , Lectinas/inmunología , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Unión Proteica , Ratones , Células CHO , Cricetulus , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Neisseria meningitidis/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/inmunología , Neisseria meningitidis Serogrupo B/metabolismoRESUMEN
Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.
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Adhesinas Bacterianas , Cromatografía de Afinidad , Haemophilus influenzae , Cromatografía de Afinidad/métodos , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/aislamiento & purificación , Humanos , Haemophilus influenzae/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/sangre , GlicosilaciónRESUMEN
Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.
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Adhesinas Bacterianas , Anticuerpos Antibacterianos , Bordetella pertussis , Vacuna contra la Tos Ferina , Vacunas de Partículas Similares a Virus , Tos Ferina , Animales , Bordetella pertussis/inmunología , Ratones , Tos Ferina/prevención & control , Tos Ferina/inmunología , Vacuna contra la Tos Ferina/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Ratones Endogámicos BALB C , Factores de Virulencia de Bordetella/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 µg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 µM), samarangenin B (71%, 20 µM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 µM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC50 12.78 µg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC50 3 µg/mL). On Lysine-specific gingipain, AQF has an IC50 15.89, EAF 14.15, and FLB7 6 µg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC50 11.34 (AQF), 11.66 (EAF), and 12.09 µg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg.
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Adhesinas Bacterianas , Adhesión Bacteriana , Biopelículas , Cisteína Endopeptidasas , Cisteína-Endopeptidasas Gingipaínas , Extractos Vegetales , Plumbaginaceae , Porphyromonas gingivalis , Factores de Virulencia , Biopelículas/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Humanos , Adhesinas Bacterianas/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/química , Plumbaginaceae/química , Raíces de Plantas/química , Proantocianidinas/farmacología , Proantocianidinas/aislamiento & purificación , Células KB , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/química , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificaciónRESUMEN
Periodontitis is a critical risk factor for the occurrence and development of diabetes. Porphyromonas gingivalis may participate in insulin resistance (IR) caused by periodontal inflammation, but the functional role and specific mechanisms of P. gingivalis in IR remain unclear. In the present study, clinical samples were analysed to determine the statistical correlation between P. gingivalis and IR occurrence. Through culturing of hepatocytes, myocytes, and adipocytes, and feeding mice P. gingivalis orally, the functional correlation between P. gingivalis and IR occurrence was further studied both in vitro and in vivo. Clinical data suggested that the amount of P. gingivalis isolated was correlated with the Homeostatic Model Assessment for IR score. In vitro studies suggested that coculture with P. gingivalis decreased glucose uptake and insulin receptor (INSR) protein expression in hepatocytes, myocytes, and adipocytes. Mice fed P. gingivalis tended to undergo IR. P. gingivalis was detectable in the liver, skeletal muscle, and adipose tissue of experimental mice. The distribution sites of gingipain coincided with the downregulation of INSR. Gingipain proteolysed the functional insulin-binding region of INSR. Coculture with P. gingivalis significantly decreased the INSR-insulin binding ability. Knocking out gingipain from P. gingivalis alleviated the negative effects of P. gingivalis on IR in vivo. Taken together, these findings indicate that distantly migrated P. gingivalis may directly proteolytically degrade INSR through gingipain, thereby leading to IR. The results provide a new strategy for preventing diabetes by targeting periodontal pathogens and provide new ideas for exploring novel mechanisms by which periodontal inflammation affects the systemic metabolic state.
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Cisteína-Endopeptidasas Gingipaínas , Resistencia a la Insulina , Porphyromonas gingivalis , Receptor de Insulina , Porphyromonas gingivalis/metabolismo , Receptor de Insulina/metabolismo , Animales , Ratones , Cisteína-Endopeptidasas Gingipaínas/metabolismo , Humanos , Masculino , Adhesinas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteolisis , Femenino , Adipocitos/metabolismo , Periodontitis/microbiología , Técnicas de CocultivoRESUMEN
BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.
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Adhesinas Bacterianas , Adhesión Bacteriana , Fibronectinas , Unión Proteica , Streptococcus agalactiae , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidad , Fibronectinas/metabolismo , Humanos , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Fibrinógeno/metabolismo , Fibrinógeno/genética , Células Epiteliales/microbiología , Femenino , Infecciones Estreptocócicas/microbiología , Factores de Virulencia/metabolismo , Factores de Virulencia/genéticaRESUMEN
Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.
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Coagulasa , Fibrinógeno , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/química , Coagulasa/metabolismo , Coagulasa/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Unión Proteica , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/química , Humanos , Estabilidad ProteicaRESUMEN
BACKGROUND: Uropathogenic Escherichia coli (UPEC) isolates, have a wide variety of virulence factors to promote colonization and survival in the urinary tract. This study aimed to evaluate adhesin genes, biofilm formation ability, antibiotic resistance profiles of UPEC strains, and the related risk factors in patients with UTIs caused by drug-resistant UPEC. METHODOLOGY: A total of 105 UPEC isolates were evaluated for biofilm formation using 96-well microtiter plates, the presence of adhesin genes by PCR assay and the antimicrobial susceptibility pattern using the disk diffusion method. Demographic and clinical characteristics of patients were investigated to identify predisposing factors for drug-resistant isolates. RESULTS: Out of 105 UPEC isolates, 84.8% were positive for biofilm formation. Biofilm-producing isolates exhibited a significantly higher prevalence of fimH, kpsMTII, csgA, afa/draBC, and pap adhesin genes compared to non-biofilm-producing strains (p < 0.05). The results also revealed that 52.4% of the isolates were ESBL-producing, and 84.8% were multidrug-resistant (MDR). Further analysis of antibiotic susceptibility among ESBL-producing strains showed the highest resistance rates to ampicillin, ciprofloxacin, and trimethoprim-sulfamethoxazole. Conversely, the highest susceptibility, in addition to carbapenems, was observed for fosfomycin, amikacin, cefoxitin, and nitrofurantoin. We identified hypertension as a potential risk factor for infection with ESBL-producing UPEC strains. CONCLUSIONS: Our results revealed a significant rate of drug resistance among UPEC isolates obtained from UTIs in our region. This underscores the importance of monitoring the empirical use of antibiotics and identifying specific risk factors in our geographical area to guide the selection of appropriate empirical treatment for UTIs.
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Biopelículas , Infecciones por Escherichia coli , Infecciones Urinarias , Escherichia coli Uropatógena , Humanos , Irán/epidemiología , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/efectos de los fármacos , Infecciones Urinarias/microbiología , Infecciones Urinarias/epidemiología , Femenino , Factores de Riesgo , Masculino , Biopelículas/crecimiento & desarrollo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/epidemiología , Adulto , Persona de Mediana Edad , Anciano , Adulto Joven , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Factores de Virulencia/genética , Adhesinas de Escherichia coli/genética , Adolescente , Niño , Adhesinas Bacterianas/genética , Anciano de 80 o más Años , Farmacorresistencia Bacteriana Múltiple/genética , Reacción en Cadena de la Polimerasa , PreescolarRESUMEN
BACKGROUND: Staphylococcus hominis is an opportunistic pathogen that expresses surface proteins, which are adhesive proteins that play a major role in biofilm formation. Biofilm is a protective layer that provides S. hominis bacteria with greater antibiotic resistance and promotes its adherence to biomedical surfaces, facilitating its entry into the bloodstream. OBJECTIVE: This research aimed to investigate the activity of Salicylic Acid (SA) and its effect on the gene expression of biofilm genes (FnbA and FnbB genes). METHODS: A total of 150 blood specimens were collected from patients. The specimens were cultured in broth media of the BacT/ALERT® system and subcultured on blood and chocolate agar. Bacteria were detected using the VITEK2 system. FnbA and FnbB genes were detected using PCR. The broth microdilution method performed the minimum inhibitory concentration (MIC) of Salicylic acid (SA) on S. hominis isolates with both genes. Detection of the gene expression levels of FnbA and FnbB genes was assessed using Real-Time PCR(RT-PCR). RESULTS: The results showed that out of the 150 specimens collected, 35 were S. hominis. The detection of S. hominis bacteria was performed by PCR amplification of two genes FnbA and FnbB and showed 100% and 17.14% of isolates were positive for genes FnbA and FnbB, respectively. The expression of FnbA and FnbB genes was decreased in samples treated with SA compared with untreated ones. CONCLUSION: In conclusion, there is a significant impact of SA on the prevention of biofilm formation of S. hominis through the suppression of gene expression, specifically FnbA and FnbB. This could enhance susceptibility to antimicrobial treatments. However, more research is required to determine whether SA leads to the selection of resistant bacteria.
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Biopelículas , Pruebas de Sensibilidad Microbiana , Ácido Salicílico , Staphylococcus hominis , Ácido Salicílico/farmacología , Humanos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Staphylococcus hominis/genética , Staphylococcus hominis/efectos de los fármacos , Adhesinas Bacterianas/genética , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Antibacterianos/farmacologíaRESUMEN
We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae. We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae. We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Proteínas Fimbrias , Fimbrias Bacterianas , Infecciones por Klebsiella , Klebsiella pneumoniae , Klebsiella pneumoniae/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/microbiología , Animales , Fimbrias Bacterianas/inmunología , Proteínas Fimbrias/inmunología , Anticuerpos Antibacterianos/inmunología , Ratones , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Fagocitosis , Técnicas de Visualización de Superficie Celular , Biblioteca de Péptidos , Adhesinas BacterianasRESUMEN
Arcobacter butzleri is a foodborne pathogen that mainly causes enteritis in humans, but the number of cases of bacteraemia has increased in recent years. However, there is still limited knowledge on the pathogenic mechanisms of this bacterium. To investigate how A. butzleri causes disease, single knockout mutants were constructed in the cadF, ABU_RS00335, ciaB, and flaAB genes, which might be involved in adhesion and invasion properties. These mutants and the isogenic wild-type (WT) were then tested for their ability to adhere and invade human Caco-2 and HT29-MTX cells. The adhesion and invasion of A. butzleri RM4018 strain was also visualized by a Leica CTR 6500 confocal microscope. The adhesion and invasion abilities of mutants lacking the invasion antigen CiaB or a functional flagellum were lower than those of the WTs. However, the extent of the decrease varied depending on the strain and/or cell line. Mutants lacking the fibronectin (FN)-binding protein CadF consistently exhibited reduced abilities, while the inactivation of the other studied FN-binding protein, ABU_RS00335, led to a reduction in only one of the two strains tested. Therefore, the ciaB and flaAB genes appear to be important for A. butzleri adhesion and invasion properties, while cadF appears to be indispensable.
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Adhesinas Bacterianas , Arcobacter , Adhesión Bacteriana , Flagelos , Adhesión Bacteriana/genética , Humanos , Arcobacter/genética , Células CACO-2 , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Flagelos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Inactivación de Genes , Células HT29 , Fibronectinas/metabolismo , Fibronectinas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Genes Bacterianos/genética , Células Epiteliales/microbiología , Virulencia/genéticaRESUMEN
B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis.
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Adhesinas Bacterianas , Adhesión Bacteriana , Biopelículas , Bordetella parapertussis , Células Epiteliales , Biopelículas/crecimiento & desarrollo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Bordetella parapertussis/genética , Bordetella parapertussis/metabolismo , Humanos , Células Epiteliales/microbiología , Viabilidad Microbiana , Tos Ferina/microbiología , Regulación Bacteriana de la Expresión Génica , Línea CelularRESUMEN
Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.
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Adhesinas Bacterianas , Anaplasma marginale , Dermacentor , Animales , Anaplasma marginale/genética , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Dermacentor/microbiología , Bovinos , Adhesión Bacteriana/fisiología , Anaplasmosis/microbiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Técnicas de Visualización de Superficie Celular , Interacciones Huésped-Patógeno , Enfermedades de los Bovinos/microbiologíaRESUMEN
Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.
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Heparina , Mycoplasma pneumoniae , Polisacáridos , Mycoplasma pneumoniae/efectos de los fármacos , Heparina/farmacología , Heparina/química , Polisacáridos/farmacología , Polisacáridos/química , Organismos Acuáticos , Humanos , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Neumonía por Mycoplasma/tratamiento farmacológico , Neumonía por Mycoplasma/microbiología , Antibacterianos/farmacología , Antibacterianos/química , Animales , Interacciones Huésped-Patógeno , Sulfatos/química , Sulfatos/farmacologíaRESUMEN
INTRODUCTION: Outer membrane protein (OMP) of Helicobacter pylori (H. pylori) i.e., blood group antigen binding adhesin (babA) is responsible for the attachment of H. pylori in the gastric epithelium. Its adherence is causative for gastric pathology such as gastritis, peptic ulcer disease (PUD), or digestive tract disorders like erosive reflux disease (ERD) and (NERD) non-erosive reflux disease and together called Gastroesophageal reflux disease (GERD). BabA manifests rapid and varied selection via substitution of amino acid in its Leb-carbohydrate binding domain (CBD) which enables better binding preferences for distinct human populations and ABO blood group phenotypes. The positive evolutionary selection of the pathogenic factor of this genetically diverse bacterium has enabled it to adapt to the host gastric environment. Analyzing the association of virulent genes (cagA, vacA) and babA will help us better understand bacteria's pathogenicity. METHOD: 109 H. pylori strains from patients with distinct gastrointestinal diseases were genotyped using Polymerase Chain Reaction(PCR) for cagA, vacA, and babA followed by Sanger sequencing and phylogenetic analysis. RESULT: In the babA + ve genotype, a statistically significant association with p = 0.04 and < 0.0001 is seen in gastritis and ERD respectively. A significant association of genotype vacAs1m2 (p = 0.0002) was seen in gastritis, vacAs1m1 (p = 0.02) in NERD, vacAs1m1 (p < 0.0001) and vacAs1m2 (p = 0.002) in ERD. This relationship helps to detect gastritis or ERD where BabA gene can be used as an independent marker for detecting their presence. CONCLUSION: The appearance of variants within distinct disease categories is due to local genetic variation.
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Adhesinas Bacterianas , Infecciones por Helicobacter , Helicobacter pylori , Filogenia , Humanos , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Helicobacter pylori/aislamiento & purificación , Adhesinas Bacterianas/genética , Infecciones por Helicobacter/microbiología , India , Masculino , Gastritis/microbiología , Femenino , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/genética , Antígenos Bacterianos/genética , Genotipo , Adulto , Persona de Mediana Edad , Proteínas Bacterianas/genéticaRESUMEN
Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions.
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Adhesinas Bacterianas , Helicobacter pylori , Leucina , Serina , Helicobacter pylori/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Humanos , Serina/genética , Serina/metabolismo , Leucina/genética , Leucina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/genética , AnimalesRESUMEN
The Rib domain, which is often found as tandem-repeat structural modules in surface proteins of Gram-positive bacteria, plays important roles in mediating interactions of bacteria with their environments and hosts. A comprehensive structural analysis of various Rib domains is essential to fully understand their impact on the structure and functionality of these bacterial adhesins. To date, structural information has been limited for this expansive group of domains. In this study, the high-resolution crystal structure of the second member of the long Rib domain, a unique subclass within the Rib-domain family, derived from Limosilactobacillus reuteri is presented. The data not only demonstrate a highly conserved structure within the long Rib domain, but also highlight an evolutionary convergence in structural architecture with other modular domains found in cell-adhesion molecules.