Nabendu Sekhar Chatterjee

Intestinal Adherence of Vibrio cholerae Involves a Coordinated Interaction between Colonization Factor GbpA and Mucin

ABSTRACT The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N-acetyl-d-glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 μM. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-κB nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae.

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present study examined the intestinal uptake of thiamine (vitamin B1) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na+ independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 μM and maximal velocity of 13.37 ± 0.94 pmol ⋅ mg protein-1 ⋅ 3 min-1; 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca2+/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na+ independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.The present study examined the intestinal uptake of thiamine (vitamin B(1)) using the human-derived intestinal epithelial cells Caco-2 as an in vitro model system. Thiamine uptake was found to be 1) temperature and energy dependent and occurred with minimal metabolic alteration; 2) pH sensitive; 3) Na(+) independent; 4) saturable as a function of concentration with an apparent Michaelis-Menten constant of 3.18 +/- 0.56 microM and maximal velocity of 13.37 +/- 0.94 pmol. mg protein(-1). 3 min(-1); 5) inhibited by the thiamine structural analogs amprolium and oxythiamine, but not by unrelated organic cations tetraethylammonium, N-methylnicotinamide, and choline; and 6) inhibited in a competitive manner by amiloride with an inhibition constant of 0.2 mM. The role of specific protein kinase-mediated pathways in the regulation of thiamine uptake by Caco-2 cells was also examined using specific modulators of these pathways. The results showed possible involvement of a Ca(2+)/calmodulin (CaM)-mediated pathway in the regulation of thiamine uptake. No role for protein kinase C- and protein tyrosine kinase-mediated pathways in the regulation of thiamine uptake was evident. These results demonstrate the involvement of a carrier-mediated system for thiamine uptake by Caco-2 intestinal epithelial cells. This system is Na(+) independent and is different from the transport systems of organic cations. Furthermore, a CaM-mediated pathway appears to play a role in regulating thiamine uptake in these cells.

Folate uptake in the human intestine: promoter activity and effect of folate deficiency.

The intestinal folate absorption process occurs via a specialized mechanism that involves the reduced folate carrier (RFC). In humans, multiple variants of the hRFC (driven by multiple promoters) have been identified with variant I being the prominent form expressed in the intestine. While it is known that promoter B (pB) of hRFC drives the expression of this variant, little is known about the minimal region required for basal activity of this promoter in human intestinal epithelial cells. Also not known is whether folate absorption in the human intestine is up‐regulated during folate deficiency (as occur in animal studies), and if so, whether transcriptional mechanisms via activation of hRFC pB are involved in such regulation. To address these issues, we have used deletion constructs of the hRFC pB and determined their activity in two human intestinal epithelial cell lines: the colon‐derived Caco‐2 cells, and the duodenum‐derived HuTu‐80 cells. Our results showed that activity of hRFC pB to be significantly higher in Caco‐2 cells compared to HuTu‐80 cells, a finding that corresponds with a higher level of folate uptake and endogenous hRFC mRNA levels in the former compared to the latter cell type. The minimal region required for basal activity of hRFC pB in Caco‐2 cells was found to be encoded in a sequence between −1088 and −1043, while in HuTu‐80 cells it was encoded in a sequence between −1431 and −1088. Growing Caco‐2 cells in a folate deficient medium led to a significant and specific up‐regulation in folate uptake. This up‐regulation was associated with a parallel increase in hRFC protein and mRNA levels, and in the activity of hRFC pB. The most responsive sequence of pB to the effect of folate deficiency was found to be encoded in a sequence between −2016 and −1431, i.e., outside the minimal region of the pB. These results show that different minimal regions for hRFC pB are utilized by different intestinal epithelial cells. In addition, folate‐deficiency was found to up‐regulate folate uptake by human intestinal epithelial cells and that this regulation involves activation of hRFC pB. J. Cell. Physiol. 196: 403–408, 2003.

PCR-Based Identification of Common Colonization Factor Antigens of Enterotoxigenic Escherichia coli

ABSTRACT Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes.

Cigarette smoke induces p-benzoquinone–albumin adduct in blood serum: Implications on structure and ligand binding properties

Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as ɛ-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60, the number of p-BQ incorporated is 40. The formation of HSA-p-BQ adduct has been demonstrated by absorption spectroscopy, MALDI-MS and MALDI-TOF-TOF-MS analyses. Upon complexation with p-BQ, the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of the reasons why transport and distribution of lipids and drugs are impaired in smokers.

Characterization and Studies of the Cellular Interaction of Native Colonization Factor CS6 Purified from a Clinical Isolate of Enterotoxigenic Escherichia coli

ABSTRACT CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.

Allelic variation in colonization factor CS6 of enterotoxigenic Escherichia coli isolated from patients with acute diarrhoea and controls

Colonization factor antigens (CFAs) are important virulence factors in enterotoxigenic Escherichia coli (ETEC). Using a multiplex PCR and RT-PCR, this study tested the presence of common colonization factor-encoding genes and their expression in 50 ETEC strains isolated from stool specimens. The samples were from patients (children) with acute diarrhoea (cases) admitted to the Infectious Disease Hospital (Kolkata, India) and from normal children (controls) under 5 years of age from the community. The results indicated that coli surface antigen 6 (CS6) was the most prevalent CFA (78 %) expressed by these ETEC strains. Sequence analysis of both of the CS6 structural genes, i.e. cssA and cssB, in different ETEC isolates revealed the presence of point mutations in a systematic fashion. Based on the analysis of these variations, it was found that CssA had three alleles and CssB had two. Based on the allelic variations, subtyping of CS6 into AIBI, AIIBII, AIIIBI, AIBII and AIIIBII is proposed. The point mutations in the different alleles were reflected in a partial alteration in the secondary structure of both subunits, as determined by computational analysis. The functional significance of these changes was confirmed with cellular binding studies in Caco-2 cells with representative ETEC isolates. CS6 with AI or AIII allelic subtypes showed a higher binding capacity than AII, whereas BI showed stronger binding than BII. The AII and BII alleles were mostly detected in controls rather than in cases. The antibody specificity of BI and BII also varied due to alteration of the amino acids. Thus, CS6 variants are formed as a result of different allelic combinations of CssA and CssB, and these changes at the functional level might be important in the development of an effective ETEC vaccine.

The Vibrio cholerae extracellular chitinase ChiA2 is important for survival and pathogenesis in the host intestine.

In aquatic environments, Vibrio cholerae colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of chiA2 is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of V. cholerae chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for V. cholerae survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that V. cholerae could utilize mucin as a nutrient source. In comparison to the wild type strain, ΔchiA2 mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the ΔchiA2 mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the ΔchiA2 mutant and wild type V. cholerae was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by V. cholerae in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by V. cholerae for growth and survival in the host intestine.

Entire sequence of the colonization factor coli surface antigen 6-encoding plasmid pCss165 from an enterotoxigenic Escherichia coli clinical isolate

Coli surface antigen 6 (CS6) is one of the most prevalent colonization factors among enterotoxigenic Escherichia coli (ETEC) isolated in developing countries. Although it is known that CS6 is encoded by a plasmid, there are no reports on the sequence analysis of the CS6-encoding plasmid or genes exhibiting similar behavior to CS6. Here, we report the isolation of the CS6-encoding plasmid, pCss165Kan, from 4266 ΔcssB::kanamycin (Km) and its complete nucleotide sequence. This plasmid consisted of 165,311bp and 222 predicted coding sequences. Remarkably, there were many insertion sequence (IS) elements, which comprised 24.4% of the entire sequence. Virulence-associated genes such as heat-stable enterotoxin, homologues of ATP-binding cassette transporter in enteroaggregative E. coli (EAEC), and ETEC autotransporter A were also present, although the ETEC autotransporter A gene was disrupted by the integration of IS629. We found that 2 transcriptional regulators belonging to the AraC family were not involved in CS6 expression. Interestingly, pCss165 had conjugative transfer genes, as well as 3 toxin-antitoxin systems that potentially exclude other plasmid-free host bacteria. These genes might be involved in the prevalence of CS6 among ETEC isolates.

Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers

Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.