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Biomedical subjects

J P Nataro

Publications and source records attributed to J P Nataro.

At least 19 recordsLinked to original sources
PubMed2006

Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM.

Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

Agar↗
PubMed2001

Enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are an increasingly important cause of diarrhoea. E. coli belonging to this category cause watery diarrhoea, which is often persistent and can be inflammatory. EAEC have been implicated in sporadic diarrhoea in children and adults, in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. EAEC are defined by their ability to adhere to epithelial cells in a characteristic "stacked-brick" pattern but are otherwise highly heterogeneous. Genes that could contribute to the pathogenicity of EAEC encode adhesins, toxins, and other factors, all of which are only partially conserved. Practicable tools are needed to improve diagnosis and identify risk factors. EAEC-infected individuals can be treated with fluoroquinolones but there is a need to examine alternative treatment protocols.

Adhesins, Escherichia coli↗
PubMed2001

Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) forms thick biofilms on the intestinal mucosa. Here, we show that most EAEC strains form a biofilm on glass or plastic surfaces when grown in cell culture medium with high sugar and osmolarity. Biofilm-forming ability in two prototype EAEC strains required aggregative adherence fimbriae (AAF), although many other EAEC strains that do not express AAF also developed biofilms under these conditions. Ten thousand transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the seven remaining insertions, five mapped to one of two unsuspected loci. Two insertions involved the E. coli chromosomal fis gene, a DNA-binding protein that is involved in growth phase-dependent regulation. Using reverse transcription-polymerase chain reaction (RT-PCR), we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. Biofilm formation also required the product of the yafK gene, which is predicted to encode a secreted 28 kDa protein. The yafK product is required for transcription of AAF/II-encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears to form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.

Adhesins, Escherichia coli↗
PubMed2001

A longitudinal study of Giardia lamblia infection in north-east Brazilian children.

OBJECTIVE: To evaluate the epidemiology of Giardia lamblia infection, investigate factors which might be associated with clinical manifestations and recurrence, and examine the role of copathogens in disease course. METHODS: Prospective 4-year cohort study of children born in an urban slum in north-eastern Brazil. RESULTS: Of 157 children followed for > or = 3 months, 43 (27.4%) were infected with Giardia. The organism was identified in 8.8% of all stool specimens, and although found with similar frequency in non-diarrhoeal (7.4%) and diarrhoeal stools (9.7%), was more common in children with persistent (20.6%) than acute diarrhoea (7.6%, P=0.002). Recurrent or relapsing infections were common (46%). Children with symptomatic infections had significantly lower weight-for-age and height-for-age than asymptomatic children. Copathogens were not associated with disease course. CONCLUSION With its protean clinical manifestations, Giardia may be associated with substantial morbidity amongst children in Brazil.

Animals↗
PubMed2001

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin.

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

Animals↗
PubMed2001

Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells.

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet.

Amino Acid Motifs↗
PubMed2001

Short report: characterization of enteroaggregative Escherichia coli isolates from Iranian children.

We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Iranian infants and children. To better understand the characteristics of EAEC in Iran, we analyzed EAEC isolates for the presence of pAA plasmid-borne factors. Ninety-eight E. coli strains that displayed the aggregative adherence (AA) pattern on HeLa cells were hybridized with the CVD432 (AA) probe and with genes encoding enteroaggregative heat-stable enterotoxin-1 and aggregative adherence fimbriae (AAF) I and II. Our data suggest that AAF/II is common in this population and that AAF/I and AAF/II can sometimes be detected in the same E. coli isolate. Surprisingly, we have found that AA probe-negative strains in Iran share virulence factors with AA probe-positive isolates and therefore may be more similar to probe-positive strains than previously believed.

Bacterial Adhesion↗
PubMed2000

Enteroaggregative escherichia coli virulence factors in traveler's diarrhea strains.

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhea in Spanish travelers to developing countries. In this study, the polymerase chain reaction was used to test EAEC isolates for genes encoding putative virulence factors, including EAEC adhesins, the plasmid-encoded toxin (Pet), a heat-stable enterotoxin (EAST), and Shigella enterotoxins 1 and 2 (ShET1 and ShET2). Findings included a low prevalence of genes for Pet (4.3%), ShET2 (4.3%), and the adherence factor AAF/II (8.7%). The overlapping genes encoding the ShET1 and the Pic mucinase were present in most EAEC strains tested (56.5%); however, some strains that carried this locus did not produce both proteins, as determined by Western immunoblot. Surprisingly, ShET1 and ShET2 genes were also found in other E. coli pathotypes, as was the EAST toxin locus. These findings underscore the heterogeneity of EAEC strains and suggest that the ShET1 may be an important virulence factor in traveler's diarrhea.

Adhesins, Escherichia coli↗
PubMed2000

Characterization of the AfaD-like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra-intestinal infections.

The afimbrial adhesive sheath, encoded by the afa-3 gene cluster, is composed of two proteins with different roles in bacterium-HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa-expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD-related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli.

Adhesins, Bacterial↗
PubMed2000

Persistent diarrhea signals a critical period of increased diarrhea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil.

Persistent diarrhea (PD; duration >/=14 days) is a growing part of the global burden of diarrheal diseases. A 45-month prospective cohort study (with illness, nutritional, and microbiologic surveillance) was conducted in a shantytown in northeastern Brazil, to elucidate the epidemiology, nutritional impact, and causes of PD in early childhood (0-3 years of age). A nested case-control design was used to examine children's diarrhea burden and nutritional status before and after a first PD illness. PD illnesses accounted for 8% of episodes and 34% of days of diarrhea. First PD illnesses were preceded by a doubling of acute diarrhea burdens, were followed by further 2.6-3.5-fold increased diarrhea burdens for 18 months, and were associated with acute weight shortfalls. Exclusively breast-fed children had 8-fold lower diarrhea rates than did weaned children. PD-associated etiologic agents included Cryptosporidium, Giardia, enteric adenoviruses, and enterotoxigenic Escherichia coli. PD signals growth shortfalls and increased diarrhea burdens; children with PD merit extended support, and the illness warrants further study to elucidate its prevention, treatment, and impact.

Bacterial Infections↗
PubMed2000

Identification of sat, an autotransporter toxin produced by uropathogenic Escherichia coli.

Urinary tract infection (UTI) is a very common extraintestinal infection, and Escherichia coli is by far the most common causative organism. Uropathogenic E. coli possess traits that distinguish them from commensal strains of E. coli, such as secretion systems that allow virulence factors to be targeted to extracytoplasmic compartments. One of at least five characterized secretion mechanisms is the autotransporter system, which involves translocation of a protein across the inner membrane, presumably via the sec system, and across the outer membrane through a beta-barrel porin structure formed by the carboxy-terminus autotransporter domain. We identified a 107 kDa protein that was expressed significantly more often by E. coli strains associated with the clinical syndrome of acute pyelonephritis than by faecal strains (P = 0.029). We isolated the protein from E. coli CFT073, a strain cultured from the blood and urine of a patient with acute pyelonephritis. The N-terminal amino acid sequence showed highest similarity to two known SPATE (serine protease autotransporters of Enterobacteriaceae) proteins, Pet and EspC. Using a 509 bp probe from the 5' region of pet, 10 cosmid clones of an E. coli CFT073 gene library were positive for hybridization. From one cosmid clone, a 7.5 kb EcoRI restriction fragment, which reacted strongly with the probe, was shown to include the entire 3885 bp gene. The predicted 142 kDa protein product possesses the three domains that are typical of SPATE autotransporters: an unusually long signal sequence of 49 amino acids; a 107 kDa passenger domain containing a consensus serine protease active site (GDSGSG); and a C-terminal autotransporter domain of 30 kDa. The protein exhibited serine protease activity and displayed cytopathic activity on VERO primary kidney, HK-2 bladder and HEp-2 cell lines; the name Sat (secreted autotransporter toxin) was derived from these properties. In addition, Sat antibodies were present in the serum of mice infected with E. coli CFT073. Based upon its association with pathogenic isolates, its cytopathic phenotype and its ability to elicit a strong antibody response after infection, we postulate that Sat represents a novel virulence determinant of uropathogenic E. coli.

Amino Acid Sequence↗
PubMed2000

Heterogeneous virulence of enteroaggregative Escherichia coli strains isolated from children in Southwest Nigeria.

Enteroaggregative Escherichia coli (EAEC) has been implicated in acute and persistent diarrhea, and most strains harbor a member of a partially-conserved plasmid family (called pAA). We studied EAEC isolated from Nigerian children aged <5 years to elucidate the roles of plasmid and chromosomal EAEC loci. We tested a total of 131 EAEC strains isolated from acute diarrhea case patients and control subjects for hybridization with 8 pAA plasmid-derived and 2 chromosomal gene probes, for several in vitro phenotypes and for resistance to antimicrobial agents. Using by multiple logistic regression, we found genes encoding the AAF/II fimbriae to be strongly associated with diarrhea in this population. EAEC strains appear to be of heterogeneous virulence, and data suggest that AAF/II may be a marker for pathogenic strains.

Case-Control Studies↗
PubMed2000

Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption.

Pet toxin is a serine protease from enteroaggregative Escherichia coli which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of alpha- and beta-spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the alpha- and beta-spectrin chains. The same degradation effect was observed in alpha-fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.

Amino Acid Sequence↗
PubMed2000

Shigella flexneri 2a strain CVD 1207, with specific deletions in virG, sen, set, and guaBA, is highly attenuated in humans.

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated Shigella flexneri 2a vaccine candidate with specific deletion mutations in virG, sen, set, and guaBA. CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (DeltavirG), does not produce enterotoxin (Deltasen and Deltaset), and has limited proliferation in vivo (DeltaguaBA). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10(6), 10(7), 10(8), 10(9), or 10(10) CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10(8) CFU. In comparison, one of 12 subjects who received 10(9) CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10(10) CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10(8) to 10(10) CFU excreted the vaccine; in 23 of 25, the duration of excretion was </=3 days. A dose-related, immunoglobulin A antibody-secreting cell (ASC) response to S. flexneri 2a O-specific lipopolysaccharide was seen, with geometric mean peak values of 6.1 to 35.2 ASCs/10(6) peripheral blood mononuclear cells (PBMC) among recipients of 10(7) to 10(10) CFU. The cytokine response to Shigella-specific antigens observed in volunteers' PBMC following vaccination suggested a Th1 pattern with stimulation of gamma interferon and absence of interleukin 4 (IL-4) or IL-5. CVD 1207 represents a Shigella live oral vaccine strain prepared from wild-type S. flexneri 2a by rational use of recombinant DNA technology that achieves a remarkable degree of attenuation compared with earlier recombinant strains, even when administered at high dosage.

Adolescent↗