Curli fimbria: an Escherichia coli adhesin associated with human cystitis

Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (csgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of -D-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 csgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.(AU)

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology.

PsaA, a candidate antigen for a vaccine against pneumonia, is well-conserved in all Streptococcus pneumoniae serotypes. A sequence of two-level experimental designs was used to evaluate medium composition and seed conditions to optimize the expression of soluble mature PsaA in E. coli. A face-centered central composite design was first used to evaluate the effects of yeast extract (5 and 23.6 g/L), tryptone (0 and 10 g/L), and glucose (1 and 10 g/L), with replicate experiments at the central point (14.3 g/L yeast extract, 5 g/L tryptone, 5.5 g/L glucose). Next, a central composite design was used to analyze the influence of NaCl concentration (0, 5, and 10 g/L) compared with potassium salts (9.4 g/L K(2)HPO(4)/2.2 g/L KH(2)PO(4)), and seed growth (7 and 16 h). Tryptone had no significant effect and was removed from the medium. Yeast extract and glucose were optimized at their intermediate concentrations, resulting in an animal-derived material-free culture medium containing 15 g/L yeast extract, 8 g/L glucose, 50 µg/mL kanamycin, and 0.4% glycerol, yielding 1 g/L rPsaA after 16 h induction at 25°C in shake flasks at 200 rpm. All the seed age and salt conditions produced similar yields, indicating that no variation had a statistically significant effect on expression. Instead of growing the seed culture for 16 h (until saturation), the process can be conducted with 7 h seed growth until the exponential phase. These results enhanced the process productivity and reduced costs, with 5 g/L NaCl being used rather than potassium salts.

Fimbrial adhesins produced by atypical enteropathogenic Escherichia coli strains.

Atypical enteropathogenic Escherichia coli (aEPEC) has emerged as a significant cause of pediatric diarrhea worldwide; however, information regarding its adherence mechanisms to the human gut mucosa is lacking. In this study, we investigated the prevalence of several (fimA, ecpA, csgA, elfA, and hcpA) fimbrial genes in 71 aEPEC strains isolated from children with diarrhea (54 strains) and healthy individuals (17 strains) in Brazil and Australia by PCR. These genes are associated with adhesion and/or biofilm formation of pathogenic and commensal 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 assessed by several approaches. We were not able to detect the hemorrhagic coli pilus (HCP) or the E. coli laminin-binding fimbriae (ELF) in these strains by using immunofluorescence. Type 1 pili and curli were detected in 59% (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 to HeLa cells by immunofluorescence, suggesting that ECP could play an important role in cell adherence for some aEPEC strains. This study highlights the complex nature of the adherence mechanisms of aEPEC strains involving the coordinated function of fimbrial (e.g., ECP) and nonfimbrial (e.g., intimin) adhesins and indicates that these strains bear several pilus operons that could potentially be expressed in different niches favoring colonization and survival in and outside the host.

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage.

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.

Iron stress increases Bordetella pertussis mucin-binding capacity and attachment to respiratory epithelial cells.

Whooping cough is a reemerging infectious disease of the respiratory tract caused by Bordetella pertussis. The incomplete understanding of the molecular mechanisms of host colonization hampers the efforts to control this disease. Among the environmental factors that commonly determine the bacterial phenotype, the concentration of essential nutrients is of particular importance. Iron, a crucial and scarce nutrient in the natural environment of B. pertussis, has been found to induce substantial phenotypic changes in this pathogen. However, the relevance of this phenotype for the interaction with host cells was never investigated. Using an in vitro model for bacterial attachment, it was shown that the attachment capacity of B. pertussis to epithelial respiratory cells is enhanced under iron stress conditions. Attachment is mediated by iron-induced surface-exposed proteins with sialic acid-binding capacity. The results further suggest that some of these iron-induced surface-associated proteins are immunogenic and may represent attractive vaccine candidates.

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells.

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A.

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 10(9) CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

[Isolation and characterization of Shiga-toxin-producing Escherichia coli from frozen hamburgers and soft cheeses]. / Aislamiento y caracterización de Escherichia coli productor de toxina Shiga en hamburguesas supercongeladas y quesos de pasta blanda.

Shiga toxin producing-Escherichia coli (STEC), an important emerging foodborne pathogen, has been associated with bloody and non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and milk are the most common vehicles of infection. In the present study, the prevalence of STEC in 95 samples of frozen hamburgers and in 114 samples of soft cheese was established in 8.4% and 0.9%, respectively. The genotypic and phenotypic characteristics of the strains were determined. The virulence genes stx1, stx2, eaeA and EHEC-hlyA were identified by PCR and by colony blot hybridization assays. Serotyping, antimicrobial susceptibility and production of Stx using specific cytotoxicity assays on Vero cells were also determined. All STEC strains were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was prevalent (77.8%), and four different O:H serotypes were found, comprising: O8:H19 (5 strains), O113:H21 (1), O8:H16 (1), and O39:H49 (1). One STEC strain was nontypable. Although soft cheese complimented the microbiological quality controls for the coliform counts, the detection of STEC in one sample raises doubts concerning the effectiveness of the current quality controls. These data contribute to the implementation of strategies for the prevention and control of HUS.

Aislamiento y caracterización de Escherichia coli productor de toxina Shiga en hamburguesas supercongeladas y quesos de pasta blanda / Isolation and characterization of Shiga-toxin-producing Escherichia coli from frozen hamburgers and soft cheeses

Shiga toxin producing-Escherichia coli (STEC), an important emerging foodborne pathogen, has been associated with bloody and non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. The cattle have been shown to be a major reservoir of STEC and raw foods such as ground beef and milk are the most common vehicles of infection. In the present study, the prevalence of STEC in 95 samples of frozen hamburgers and in 114 samples of soft cheese was established in 8.4 and 0.9, respectively. The genotypic and phenotypic characteristics of the strains were determined. The virulence genes stx1, stx2, eaeA and EHEC-hlyA were identified by PCR and by colony blot hybridization assays. Serotyping, antimicrobial susceptibility and production of Stx using specific cytotoxicity assays on Vero cells were also determined. All STEC strains were characterized as eaeA-/EHEC-hlyA+. The stx2 genotype was prevalent (77.8), and four different O:H serotypes were found, comprising: O8:H19 (5 strains), O113:H21 (1), O8:H16 (1), and O39:H49 (1). One STEC strain was nontypable. Although soft cheese complimented the microbiological quality controls for the coliform counts, the detection of STEC in one sample raises doubts concerning the effectiveness of the current quality controls. These data contribute to the implementation of strategies for the prevention and control of HUS.(AU)

CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15.

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H- strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.

Influence of culture media and air supply on the production of adhesins by Moraxella bovis

Estudou-se a influência do meio de cultivo e do fornecimento de ar na produçäo de adesinas hemaglutinantes de Moraxella bovis GF9 cultivada em fementador. Os mais altos rendimentos de adesinas e massa bacteriana obtiveram-se em meios preparados com peptona de carne, infuso de cérebro e coraçäo (BHI) e médio com peptona e extrato de carne (DCF), respectivamente. A influência do fornecimento de ar foi diferente nos meios estudados. Em BHI, a produçäo de adesinas foi 2.5 vezes maior nos cultivos aerados a 1 volume de ar por volume de meio (vvm) que com 0,5 vvm. Em DCF os títulos näo alcançaram a metade dos obtidos em BHI, enquanto que em meio de Mueller Hinton (MH) e caldo soja tripticaseina (TSB) näo se produziram adesinas. O título mais alto se obteve em BHI aerado a 1 vvm às 12 horas de cultivo. Os níveis de oxigênio dissolvido cairam a 7 e 30 por cento ao início da fase exponencial, nos cultivos aerados a 0.5 e 1 vvm., respectivamente