RESUMEN
Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.
Asunto(s)
Proteínas Bacterianas , Fimbrias Bacterianas , Flagelos , Xanthomonas , Xanthomonas/patogenicidad , Xanthomonas/genética , Virulencia/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Regulación Bacteriana de la Expresión Génica , Morfogénesis , Enfermedades de las Plantas/microbiología , Saccharum/microbiologíaRESUMEN
Due to the increasing occurrence of drug resistant urinary tract infections (UTI) among children, there is a need to investigate alternative effective treatment protocols such as nanoparticles. Flagella and fimbriae are primary factors contributing the virulence of urinary tract infecting bacteria. The aim of this study was to assess the antibacterial effects of zinc oxide nanoparticles which have been synthesized using both chemical and green methods on multi-drug resistant (MDR) uropathogenic bacteria encoding fli and fim genes and investigating their binding ability to bacterial appendage proteins. A total of 30 urine culture samples were collected from children under 2 years old diagnosed with urinary tract infection. The isolates underwent antibiotic suseptibility assessment and the isolates demonstrating MDR were subjected to molecular amplification of fimG (fimbrial) and fliD and fliT (flagellal) genes. The confirmation of cellular appendages was achieved through silver nitrate staining. The antibacterial efficacy of the synthetized nanoparticles was assessed using the micro and macrodilution methods. The successful binding of nanoparticles to bacterial appendage proteins was confirmed through mobility shift and membrane filter assays. The dimensions of chemically synthesized ZnO nanoparticles and green nanoparticles were measured at 30 nm and 85 nm, respectively, with the exhibition of hexagonal geometries. The nanoparticles synthesized through chemical and green methods exhibited minimum inhibitory concentrations (MIC) of 0.0062-0.025 g/L and 0.3 g/L, respectively. The ability of ZnO nanoparticles to bind bacterial appendage proteins and to combat MDR uropathogenic bacteria are promising for new treatment protocols against UTI in children in future.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Flagelos , Infecciones Urinarias , Óxido de Zinc , Óxido de Zinc/farmacología , Óxido de Zinc/química , Óxido de Zinc/metabolismo , Antibacterianos/farmacología , Humanos , Infecciones Urinarias/microbiología , Infecciones Urinarias/tratamiento farmacológico , Flagelos/efectos de los fármacos , Flagelos/genética , Flagelos/metabolismo , Pruebas de Sensibilidad Microbiana , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/efectos de los fármacos , Nanopartículas/química , Lactante , Nanopartículas del Metal/químicaRESUMEN
BACKGROUND: In the present study, we aimed to determine the frequency of the csgA, fimH, mrkD, foc, papaGI, papGII and papGIII genes, to provide and to design fimbrial adhesin gene (FAG) patterns and profiles for the isolated uropathogenic Escherichia coli (UPEC) strains. METHODS: The enrollment of 108 positive urine samples was performed during seven months, between January 2022 and July 2022. The UPEC strains were confirmed through the standard microbiological and biochemical tests. The antimicrobial susceptibility test was performed through the Kirby-Bauer disc diffusion method. Molecular screening of FAGs was done through the polymerase chain reaction technology. The statistical analyses including chi square and Fisher's exact tests were performed to interpret the obtained results in the present study. RESULTS: As the main results, the antimicrobial resistance (AMR) patterns, multi- (MDR) and extensively drug-resistance (XDR) patterns and FAG patterns were designed and provided. fimH (93.3%), csgA (90.4%) and papG (37.5%) (papGII (30.8%)) genes were recognized as the top three FAGs, respectively. Moreover, the frequency of csgA-fimH gene profile was identified as the top FAG pattern (46.2%) among the others. The isolates bearing csgA-fimH gene profile were armed with a versatile of phenotypic AMR patterns. In the current study, 27.8%, 69.4% and 1.9% of the UPEC isolates were detected as extended-spectrum ß-lactamases (ESBLs) producers, MDR and XDR strains, respectively. CONCLUSIONS: In conclusion, detection, providing and designing of patterns and profiles in association with FAGs, AMR feature in UPEC strains give us an effective option to have a successful and influential prevention for both of UTIs initiation and AMR feature.
Asunto(s)
Infecciones por Escherichia coli , Proteínas de Escherichia coli , Proteínas Fimbrias , Fimbrias Bacterianas , Infecciones Urinarias , Escherichia coli Uropatógena , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/efectos de los fármacos , Humanos , Proteínas de Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones Urinarias/microbiología , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/metabolismo , Femenino , Adulto , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Masculino , Farmacorresistencia Bacteriana Múltiple/genética , Persona de Mediana Edad , Adulto Joven , Adolescente , Proteínas BacterianasRESUMEN
INTRODUCTION: Pigs without intestinal receptors for F4 fimbriae are congenitally resistant to F4 fimbriae-bearing enterotoxigenic Escherichia coli (ETEC F4). In general, 50 % and 100 % of piglets born to resistant (RR) sows crossed with hetero- or homozygous susceptible (SR, SS) boars, respectively, are susceptible but do not receive colostral antibodies against F4 fimbriae unless the sows have been vaccinated. The question arises as to whether resistant sows produce protective amounts of F4 antifimbrial antibodies after vaccination. The serum and colostrum antibody titres of 12 resistant and 12 susceptible vaccinated gilts were compared. The effect of the receptor status of the dam and sire on the preweaning performance of 5027 piglets was evaluated using Agroscope's recordings. The sows of the experimental herd, where ETEC F4 was circulating, were vaccinated against ETEC twice during the first pregnancy and once during each following pregnancy. The log2 transformed F4 antibody titres in the serum obtained after the second vaccine injection as well as in the colostrum of the 12 resistant animals were lower than the titres of the susceptible animals (serum: F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019; colostrum: F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). The heat labile enterotoxin (LT) antibody titres after vaccination did not differ between susceptible and resistant animals (p > 0,10). Preweaning mortality in the offspring of RR sows × SS boars was slightly lower than in the offspring of SS sows × RR boars (P = 0,04), suggesting that the disease risk of susceptible piglets born to vaccinated resistant sows was not increased, even though they received colostrum with a slightly reduced content of antibody against F4 fimbriae.
INTRODUCTION: Les porcs dépourvus de récepteurs intestinaux pour les fimbriae F4 sont congénitalement résistants aux Escherichia coli entérotoxinogènes porteurs de fimbriae F4 (ETEC F4). En général, 50 % et 100 % des porcelets nés de truies résistantes (RR) croisées avec des verrats hétéro- ou homozygotes sensibles (SR, SS), respectivement, sont sensibles mais ne reçoivent pas d'anticorps colostraux contre les fimbriae F4, à moins que les truies n'aient été vaccinées. La question se pose de savoir si les truies résistantes produisent des quantités protectrices d'anticorps antifimbriae F4 après la vaccination. Les titres d'anticorps dans le sérum et le colostrum de 12 truies reproductrices vaccinées résistantes et de 12 truies reproductrices vaccinées sensibles ont été comparés et l'effet du statut récepteur de la mère et du père sur les performances avant sevrage de 5027 porcelets a été évalué. Les truies du troupeau expérimental, où circulait ETEC F4, ont été vaccinées deux fois au cours de la première gestation et une fois au cours de chaque gestation suivante contre ETEC. Les titres d'anticorps F4 transformés en log2 dans le sérum obtenu après la deuxième injection de vaccin ainsi que dans le colostrum des 12 animaux résistants étaient inférieurs aux titres des animaux sensibles (sérum : F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096 ; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019 ; colostrum : F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033 ; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). Les titres d'anticorps contre l'entérotoxine thermolabile (LT) après la vaccination ne différaient pas entre les animaux sensibles et résistants (p > 0,10). La mortalité avant sevrage dans la progéniture des truies RR × verrats SS était légèrement inférieure à celle de la progéniture des truies SS × verrats RR (P = 0,04), ce qui suggère que le risque de maladie des porcelets sensibles nés de truies résistantes vaccinées n'a pas été augmenté, même s'ils ont reçu du colostrum avec une teneur légèrement réduite en anticorps contre les fimbriae F4.
Asunto(s)
Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Vacunas contra Escherichia coli , Fimbrias Bacterianas , Enfermedades de los Porcinos , Animales , Porcinos , Infecciones por Escherichia coli/veterinaria , Infecciones por Escherichia coli/prevención & control , Infecciones por Escherichia coli/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/microbiología , Femenino , Vacunas contra Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Escherichia coli Enterotoxigénica/inmunología , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/genética , Embarazo , Calostro/inmunología , Anticuerpos Antibacterianos/sangre , DesteteRESUMEN
Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.
Asunto(s)
Proteínas Fimbrias , Fimbrias Bacterianas , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Adhesión Bacteriana , OperónRESUMEN
During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris. This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris. Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism. IMPORTANCE: Gram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae, SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance.
Asunto(s)
Actinomyces , Proteínas Bacterianas , Cisteína Endopeptidasas , Proteínas Fimbrias , Fimbrias Bacterianas , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , Actinomyces/genética , Actinomyces/enzimología , Actinomyces/metabolismo , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/genética , Pared Celular/metabolismo , Señales de Clasificación de ProteínaRESUMEN
Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.
Asunto(s)
Aeromonas hydrophila , Biopelículas , Fimbrias Bacterianas , Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Operón , Aeromonas hydrophila/genética , Aeromonas hydrophila/patogenicidad , Aeromonas hydrophila/fisiología , Biopelículas/crecimiento & desarrollo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Virulencia/genética , Animales , Infecciones por Bacterias Gramnegativas/microbiología , Enfermedades de los Peces/microbiología , Adhesión Bacteriana/genética , Bagres/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Inactivación de GenesRESUMEN
Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the OG1RF ΔsrtAΔhtrA background partially restores the observed defects of the ΔsrtAΔhtrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal resistance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.
Asunto(s)
Aminoaciltransferasas , Proteínas Bacterianas , Biopelículas , Cisteína Endopeptidasas , Enterococcus faecalis , Fimbrias Bacterianas , Chaperonas Moleculares , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Enterococcus faecalis/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biopelículas/crecimiento & desarrollo , Membrana Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Virulencia/genética , Antibacterianos/farmacología , Ceftriaxona/farmacologíaRESUMEN
Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.
Asunto(s)
Microscopía por Crioelectrón , Proteínas Fimbrias , Fimbrias Bacterianas , Modelos Moleculares , Plásmidos , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Plásmidos/metabolismo , Plásmidos/química , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Fosfolípidos/metabolismo , Fosfolípidos/química , Conjugación Genética , Escherichia coli/metabolismo , Escherichia coli/genética , Unión ProteicaRESUMEN
Type 1 fimbria, the short hair-like appendage assembled on the bacterial surface, plays a pivotal role in adhesion and invasion in Edwardsiella piscicida. The type III secretion system (T3SS), another bacterial surface appendage, facilitates E. piscicida's replication in vivo by delivering effectors into host cells. Our previous research demonstrated that E. piscicida T3SS protein EseJ inhibits adhesion and invasion of E. piscicida by suppressing type 1 fimbria. However, how EseJ suppresses type 1 fimbria remains elusive. In this study, a lacI-like operator (nt -245 to -1 of fimA) upstream of type 1 fimbrial operon in E. piscicida was identified, and EseJ inhibits type 1 fimbria through the lacI-like operator. Moreover, through DNA pull-down and electrophoretic mobility shift assay, an AraC-type T3SS regulator, EsrC, was screened and verified to bind to nt -145 to -126 and nt -50 to -1 of fimA, suppressing type 1 fimbria. EseJ is almost abolished upon the depletion of EsrC. EsrC and EseJ impede type 1 fimbria expression. Intriguingly, nutrition and microbiota-derived indole activate type 1 fimbria through downregulating T3SS, alleviating EsrC or EseJ's inhibitory effect on lacI-like operator of type 1 fimbrial operon. By this study, it is revealed that upon entering the gastrointestinal tract, rich nutrients and indole downregulate T3SS and thereof upregulate type 1 fimbria, stimulating efficient adhesion and invasion; upon being internalized into epithelium, the limit in indole and nutrition switches on T3SS and thereof switches off type 1 fimbria, facilitating effector delivery to guarantee E. piscicida's survival/replication in vivo.IMPORTANCEIn this work, we identified the lacI-like operator of type 1 fimbrial operon in E. piscicida, which was suppressed by the repressors-T3SS protein EseJ and EsrC. We unveiled that E. piscicida upregulates type 1 fimbria upon sensing rich nutrition and the microbiota-derived indole, thereof promoting the adhesion of E. piscicida. The increase of indole and nutrition promotes type 1 fimbria by downregulating T3SS. The decrease in EseJ and EsrC alleviates their suppression on type 1 fimbria, and vice versa.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas , Edwardsiella , Fimbrias Bacterianas , Operón , Sistemas de Secreción Tipo III , Edwardsiella/genética , Edwardsiella/fisiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Animales , Regulación Bacteriana de la Expresión Génica , Infecciones por Enterobacteriaceae/microbiologíaRESUMEN
In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.
Asunto(s)
Fimbrias Bacterianas , Osmorregulación , Trehalosa , Vejiga Urinaria , Infecciones Urinarias , Animales , Trehalosa/metabolismo , Ratones , Vejiga Urinaria/microbiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Infecciones Urinarias/microbiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Animales de Enfermedad , Femenino , Presión Osmótica , Escherichia coli Patógena Extraintestinal/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Urea/metabolismo , Trehalasa/metabolismo , Trehalasa/genética , Eliminación de Gen , Glucosa/metabolismoRESUMEN
Pili are bacterial surface structures important for surface adhesion. In the alphaproteobacterium Caulobacter crescentus, the global regulator CtrA activates transcription of roughly 100 genes, including pilA which codes for the pilin monomer that makes up the pilus filament. While most CtrA-activated promoters have a single CtrA-binding site at the - 35 position and are induced at the early to mid-predivisional cell stage, the pilA promoter has 3 additional upstream CtrA-binding sites and it is induced at the late predivisional cell stage. Reporter constructs where these additional sites were disrupted by deletion or mutation led to increased activity compared to the WT promoter. In synchronized cultures, these mutations caused pilA transcription to occur approximately 20 min earlier than WT. The results suggested that the site overlapping the - 35 position drives pilA gene expression while the other upstream CtrA-binding sites serve to reduce and delay expression. EMSA experiments showed that the - 35 Site has lower affinity for CtrAâ¼P compared to the other sites, suggesting binding site affinity may be involved in the delay mechanism. Mutating the upstream inhibitory CtrA-binding sites in the pilA promoter caused significantly higher numbers of pre-divisional cells to express pili, and phage survival assays showed this strain to be significantly more sensitive to pilitropic phage. These results suggest that pilA regulation evolved in C. crescentus to provide an ecological advantage within the context of phage infection.
Asunto(s)
Proteínas Bacterianas , Caulobacter crescentus , Proteínas Fimbrias , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Sitios de Unión , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Unión ProteicaRESUMEN
The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins.
Asunto(s)
Proteínas Bacterianas , Thermus thermophilus , Thermus thermophilus/metabolismo , Thermus thermophilus/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Unión Proteica , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/química , ADN Bacteriano/metabolismo , ADN Bacteriano/genéticaRESUMEN
Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction.
Asunto(s)
Fimbrias Bacterianas , Sulfolobus acidocaldarius , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/metabolismo , Sulfolobus acidocaldarius/fisiología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genéticaRESUMEN
Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.
Asunto(s)
Microscopía por Crioelectrón , Fimbrias Bacterianas , Sistemas de Secreción Tipo IV , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Fimbrias Bacterianas/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/química , Modelos Moleculares , Conjugación Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Escherichia coli/genética , Conformación ProteicaRESUMEN
Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic Escherichia coli. Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease.
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Infecciones por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli , Proteínas Fimbrias , Fimbrias Bacterianas , Regulación Bacteriana de la Expresión Génica , Factores de Virulencia , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Infecciones por Escherichia coli/microbiología , Virulencia/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Factores de Virulencia/genética , Animales , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Ratones , Humanos , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Femenino , Tracto Gastrointestinal/microbiologíaRESUMEN
The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.
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Proteínas Fimbrias , Streptococcus sanguis , Biopelículas/crecimiento & desarrollo , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Streptococcus sanguis/metabolismo , Streptococcus sanguis/genéticaRESUMEN
Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.
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Adhesión Bacteriana , Modelos Animales de Enfermedad , Células Epiteliales , Infecciones por Escherichia coli , Escherichia coli , Fimbrias Bacterianas , Regulación Bacteriana de la Expresión Génica , Animales , Ratones , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Escherichia coli/genética , Escherichia coli/patogenicidad , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Adhesión Bacteriana/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Virulencia/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Intestinos/microbiología , FemeninoRESUMEN
Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses. IMPORTANCE: The notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa. Since the cupA pilus is an important adhesin of P. aeruginosa, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.
Asunto(s)
Macrófagos , Fagocitosis , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Macrófagos/microbiología , Macrófagos/inmunología , Ratones , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/inmunología , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Humanos , Células RAW 264.7RESUMEN
Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.