Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.

Selective and easy detection of the Porphyromonas gingivalis fimA type II and IV genes by loop-mediated isothermal amplification.

Porphyromonas gingivalis fimbrillin (fimA) type II and IV, the definitive factors for periodontitis, are also found to be associated with systemic diseases. To detect the fimA type II and IV genes easily and rapidly, we used the loop-mediated isothermal amplification (LAMP) method. The LAMP method showed high specificity as DNA from the P. gingivalis HW24D1 strain could only be amplified by the type II-specific primers and that from the W83 strain could only be amplified by the type IV-specific primers. Pathogens, namely, Streptococcus sobrinus, S. mutans, and Candida species, lack the type II and IV genes, and hence, were not detected by the LAMP reaction. Both bacterial cells and purified DNA could be used in the LAMP reaction. The LAMP reaction was highly sensitive and both type II and type IV genes could be detected in 1000 DNA molecules. In the bacterial suspensions of HW24D1 and W83 strains, type II and type IV genes, respectively, could be detected in 100 bacterial cells. We examined the type II and type IV genes in the dental plaques from 22 P. gingivalis-positive patients using the LAMP method. Only one person was found to be positive for the type II gene (4.5%). For the type IV gene, 3 positive cases (13.6%) were identified. Moreover, type II and type IV genes could be detected simultaneously using a multiplex amplification primer of fimA type II and type IV, under visible light. Thus, we established a selective and easy method to detect P. gingivalis fimA type II and IV genes using LAMP.

Purification of Type IV Pili and Pilin Subunits.

The Type IV pili are displayed peritrichously on the surfaces of Neisseria gonorrhoeae cells. Here we present protocols for isolating and purifying Type IV pili and dissociating them into PilE pilin subunits. Pilus filaments are isolated from the bacterial cell surface by mechanical shearing and purified by differential precipitation and centrifugation. PilE subunits are extracted by treating the purified pili with detergent to disrupt the hydrophobic interactions holding them together in the filaments. Purified pili and pilin subunits can be used for structural, biophysical, or biochemical characterization and as antigens for antibody production.

Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy.

Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. OBJECTIVES: The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. MATERIAL AND METHODS: Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. RESULTS: Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). CONCLUSION: The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Frequency of Porphyromonas gingivalis fimA in smokers and nonsmokers after periodontal therapy

Abstract Porphyromonas gingivalis is one of the most important Gram-negative anaerobe bacteria involved in the pathogenesis of periodontitis. P. gingivalis has an arsenal of specialized virulence factors that contribute to its pathogenicity. Among them, fimbriae play a role in the initial attachment and organization of biofilms. Different genotypes of fimA have been related to length of fimbriae and pathogenicity of the bacterium. Objectives The aim of this study was to identify 5 types of fimA genotype strains in smokers and nonsmokers with periodontitis, before and after periodontal therapy. Material and Methods Thirty-one patients with periodontitis harboring P. gingivalis were selected: 16 nonsmokers (NS) and 15 smokers (SM). Clinical and microbiological parameters were evaluated at baseline and 3 months after periodontal treatment, namely: plaque index, bleeding on probe, probing depth, gingival recession and clinical attachment level. The frequency of P. gingivalis and fimA genotype strains were determined by polymerase chain reaction. Results Type I fimA was detected in the majority of SM and NS at baseline, and the frequency did not diminish after 3 months of treatment. The frequency of type II genotype was higher in SM than NS at baseline. After 3 months, statistical reduction was observed only for types II and V fimA genotypes in SM. The highest association was found between types I and II at baseline for NS (37.5%) and SM (53.3%). Conclusion The most prevalent P. gingivalis fimA genotypes detected in periodontal and smoker patients were genotypes I and II. However, the presence of fimA genotype II was higher in SM. Periodontal treatment was effective in controlling periodontal disease and reducing type II and V P. gingivalis fimA.

Typical & atypical enteropathogenic Escherichia coli in diarrhoea & their role as carrier in children under five.

BACKGROUND & OBJECTIVES: Multidrug-resistant enteropathogenic Escherichia coli (EPEC) is responsible for a large number of cases of infantile diarrhoea in developing countries, causing failure in treatment with consequent health burden and resulting in a large number of deaths every year. This study was undertaken to determine the proportion of typical and atypical EPEC in under five children with diarrhoea and controls, their function as a carriage and to identify virulent genes associated with them. METHODS: During the study period, 120 stool samples including 80 from controls children were collected and analyzed for the presence of EPEC using standard bacteriological methods. Isolates were subjected to antimicrobial testing by disc diffusion method. Isolates confirmed as E. coli by phenotypic method were further tested for the presence of attaching and effacing (eae) and bundle-forming pilus (bfpA) genes by real-time SYBR Green-based polymerase chain reaction. RESULTS: All isolates were tested for the presence of EPEC. The frequency of typical EPEC was 20 and 16.25 per cent whereas the frequency of atypical EPEC strains was 5 and 23.75 per cent in patients and controls, respectively (PbfpA was seen in 45 and 18.75 per cent isolates of diarrhoeal patients and controls, respectively. INTERPRETATION & CONCLUSIONS: Our results showed that typical EPEC was a common cause of diarrhoea, but at the same time, atypical EPEC was emerging as colonizers in the intestine of children with and without diarrhoea in and around Delhi. Children can be considered asymptomatic carriers of these pathogens and can transmit them to other susceptible children. Adequate steps need to be taken to stop these strains from developing and spreading further.

Role of Mfa5 in Expression of Mfa1 Fimbriae in Porphyromonas gingivalis.

Fimbriae are protein-based filamentous appendages that protrude from the bacterial cell surface and facilitate host adhesion. Two types of fimbriae, FimA and Mfa1, of the periodontal pathogen Porphyromonas gingivalis are responsible for adherence to other bacteria and to host cells in the oral cavity. Both fimbrial forms are composed of 5 proteins, but there is limited information about their polymerization mechanisms. Here, the authors evaluated the function of Mfa5, one of the Mfa1 fimbrial accessory proteins. Using mfa5 gene disruption and complementation studies, the authors revealed that Mfa5 affects the incorporation of other accessory proteins, Mfa3 and Mfa4, into fibers and the expression of fimbriae on the cell surface. Mfa5 is predicted to have a C-terminal domain (CTD) that uses the type IX secretion system (T9SS), which is limited to this organism and related Bacteroidetes species, for translocation across the outer membrane. To determine the relationship between the putative Mfa5 CTD and the T9SS, mutants were constructed with in-frame deletion of the CTD and deletion of porU, a C-terminal signal peptidase linked to T9SS-mediated secretion. The ∆CTD-expressing strain presented a similar phenotype to the mfa5 disruption mutant with reduced expression of fimbriae lacking all accessory proteins. The ∆porU mutants and the ∆CTD-expressing strain showed intracellular accumulation of Mfa5. These results indicate that Mfa5 function requires T9SS-mediated translocation across the outer membrane, which is dependent on the CTD, and subsequent incorporation into fibers. These findings suggest the presence of a novel polymerization mechanism of the P. gingivalis fimbriae.

Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established.

Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera.

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

[The detection of occurrence rate of genes coding capability to form pili binding in auto-strains of Escherichia coli].

E. coli is a commensal of intestine of the vertebrata. The exchange of genetic material of different types of bacteria between themselves and with other representatives of family of Enterobacteriaceae in intestinal ecosystem results in development of types of normal colibacillus with genetic characteristics of pathogenicity that can serve as a theoretical substantiation to attribute such strains to pathobionts. The entero-pathogenic colibacillus continues be an important cause of diarrhea in children in developing countries. The gene responsible for formation of pili binding is a necessary condition for virulence of entero-pathogenic colibacillus. The polymerase chain reaction was applied to examine 316 strains of different types of E. coli (normal, with weak enzyme activity and hemolytic activity) isolated from healthy children and children with functional disorders of gastro-intestinal tract for presence of genes coding capability to form pill binding. The presence of this gene in different biochemical types of E. coli permits to establish the fact of formation of reservoir of pathogenicity in indigent microbiota of intestinal biocenosis.

Purification, crystallization and preliminary X-ray diffraction analysis of SpaD, a backbone-pilin subunit encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG.

SpaD is the predicted backbone-pilin subunit of the SpaFED pilus, whose loci are encoded by the fimbrial spaFED operon in Lactobacillus rhamnosus GG, a Gram-positive gut-adapted commensal strain with perceived probiotic benefits. In this study, soluble recombinant SpaD protein was overproduced in Escherichia coli and then purified by Ni2+-chelating affinity and gel-filtration chromatography. After limited proteolysis with α-chymotrypsin, good-quality crystals of SpaD were obtained which diffracted beyond 2.0 Šresolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=50.11, b=83.27, c=149.65 Å. For phasing, sodium iodide-derivatized crystals were prepared using the halide quick-soaking method and diffraction data were collected in-house to a resolution of 2.2 Å. An interpretable electron-density map was successfully obtained using single-wavelength anomalous diffraction (SAD).

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili.

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the ß-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

Purification, crystallization and preliminary crystallographic analysis of the SpaA backbone-pilin subunit from probiotic Lactobacillus rhamnosus GG.

Lactobacillus rhamnosus GG, a widely used Gram-positive probiotic strain, is clinically well known for its perceived health-promoting effects. It has recently been shown to display proteinaceous pilus fibres (called SpaCBA) on its cell surface. Structurally, SpaCBA pili possess a characteristic three-pilin polymerized architecture, with repeating SpaA major pilins that form the backbone and two types of minor subunits (SpaB and SpaC). In this study, recombinant SpaA protein was purified, characterized and crystallized. The crystals diffracted to a resolution of 2.0 Šand belonged to space group C2, with unit-cell parameters a=227.9, b=63.2, c=104.3 Å, ß=95.1°.

Specific DNA recognition mediated by a type IV pilin.

Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable gram-negative bacteria, including the human pathogen Neisseria meningitidis, have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought.

[Reproducibility of Fim2 and Fim3 antigens determination in Bordetella pertussis by serotyping method]. / Ocena porównywalnosci oznaczen obecnosci antygenów Fim2 i Fim3 Bordetella pertussis metoda serotypowania.

INTRODUCTION: Serotyping is a commonly used method to characterize the presence of Fimbriae 2 and 3 in Bordetella pertussis strains for epidemiological purposes and optimal choice of strain composition of the pertussis whole-cell vaccine. Monoclonal antisera against Fim2 and Fim3 are recommended to be used for microplate serotyping instead ofpolyclonal. Reliable evaluation offimbriae expressed by B. pertussis strains influence interpretation of vaccine-driven strain evolution. METHODS: To evaluate the impact of tests conditions on the reproducibility of serotyping, results of serotyping based on a standardized protocol for microplate agglutination with monoclonal antisera performed in three different accredited laboratories were compared. For the study isolates of three vaccine strains of B. pertussis deposited within seed lot system originating from different liofilization lots were compared. RESULTS: Lack of the complete agreement on serotyping results among three labs might relates to the differences of media used, subjective reading, test conditions, and specificity of the reagents. CONCLUSIONS: Serotyping results should be interpreted with caution and the type of media and culture conditions used should be precisely recommended after validation studies. Inconsistent results should be confirmed using an alternative technique, eg. ELISA or by reference laboratory.

Antibody responses to individual Bordetella pertussis fimbrial antigen Fim2 or Fim3 following immunization with the five-component acellular pertussis vaccine or to pertussis disease.

Bordetella pertussis expresses two serologically distinct fimbriae (Fim2 and Fim3) which are included in the Sanofi Pasteur 5-component acellular pertussis vaccine, and antibody responses to these antigens have been shown to be associated with protection. Studies to date have assessed the IgG response to this vaccine using a copurified mixture of Fim2 and Fim3, and the response to the individual antigens has not been characterized. We have purified separate Fim2 and Fim3 from strains that express either Fim2 or Fim3 and have used these antigens in an enzyme-linked immunosorbent assay (ELISA) to quantify IgG responses following immunization with 5-component acellular pertussis vaccine in 15-month-old, 4- to 6-year-old, and 11- to 18-year-old subjects. All individuals showed increases in Fim2 and Fim3 IgG concentrations following immunization, with 3-fold-greater Fim2 than Fim3 IgG concentrations seen in the younger two age groups. Fim2 IgG concentrations were 1.5-fold greater than Fim3 IgG concentrations in the 11- to 18-year-olds. We have also compared Fim2 and Fim3 IgG concentrations in individuals with prolonged cough who were diagnosed as having recent pertussis using a pertussis toxin (Ptx) IgG ELISA with individuals with prolonged cough but without elevated Ptx IgG concentrations. Individuals with evidence of recent pertussis had greater Fim3 IgG concentrations, consistent with the predominant serotype of isolates obtained in the United Kingdom. However, a surprising number of individuals had moderate Fim2 IgG concentrations despite very few isolates of that serotype obtained in the sampling period.

Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhoea and controls in Teresina/PI, Brazil.

This 3.5-year prospective study was conducted to ascertain the level of attaching and effacing Escherichia coli (AEEC) associated diarrhoea in children from Teresina, a northeastern state of Brazil. Passed faecal specimens from 400 patients (250 with and 150 without diarrhoea) up to 60 months of age attending from 2004 to 2007 at two public hospitals were investigated. Conventional microbiology methods and PCR were employed. Escherichia coli was isolated from 390 children, 240 of them with diarrhoea. A total of 117 AEEC strains were cultivated from specimens from 63 children, 37 with and 26 without diarrhoea. No association between AEEC and diarrhoea was observed. Atypical enteropathogenic E. coli (a-EPEC) (79.4%) was more commonly found than typical EPEC (t-EPEC). Association between EPEC and EPEC subtypes and diarrhoea was not detected. Mixed infection by t-EPEC and a-EPEC and infection by Shiga toxin-producing E. coli (STEC) were rare. Enteropathogenic E. coli was more common in males and in children aged less than 12 months. Correlation between serotyping and PCR results was 0.19. High resistance rates of AEEC to ampicillin, cephalotin, and trimethoprim-sulfamethoxazole were found. In conclusion, EPEC is very common in children with diarrhoea and controls in the population we studied, with a-EPEC predominating. This diarrhoeagenic E. coli (DEC) pathotype is more common in infant males and is resistant to drugs frequently used in clinical practice.

Fimbrial Adhesins Produced by Atypical Enteropathogenic Escherichia coli Strains

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrheaworldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. Inthis study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Braziland Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic andcommensal E. coli. Here, the most prevalent fimbrial genes found, in descending order, were hcpA (98.6%), ecpA(86%), fimA (76%), elfA (72%), and csgA (19.7%). Phenotypic expression of pili in aEPEC strains was assessedby several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli lamininbindingfimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in59% (by yeast agglutination) and 2.8% (by Congo red binding and immunofluorescence) of the strains,respectively. The E. coli common pilus (ECP) was evidenced in 36.6% of the strains on bacteria adhering toHeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence forsome aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strainsinvolving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins andindicates that these strains bear several pilus operons that could potentially be expressed in different nichesfavoring colonization and survival in and outside the host.