The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Structural and functional characterization of colonization factors AIBI-CS6 and AIIBII-CS6 of enterotoxigenic Escherichia coli.

Over time, the structure and function of the broadly dispersed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) have become more significant. CS6 is composed of tightly-associated subunits, CssA and CssB which due to presence of natural point mutation gave rise to CS6 subtypes. In contrast to the other ETEC CFs, CS6 is an afimbrial, spherical-shaped oligomers of (CssA-CssB)n complex where 'n' is concentration dependent. In this study, we have compared AIBI-CS6 and AIIBII-CS6 structurally and functionally. The Mw of CssAI was 18.5 kDa but Mw of CssAII was 15.1 kDa. Both CssBI and CssBII had Mw of 15.9 kDa. The substitution of Gly39 with Ala39 in CssAI leads to reduction in Mw from 18.5 to 15.1 kDa. Due to higher Mw of CssAI, the size of AIBI concentration-dependent oligomers should be higher. However, the Mw of AIIBII oligomers were higher and AIIBII also showed higher oligomeric forms compared to AIBI both in native PAGE and electron microscopy. The oligomers of both subtypes could withstand greater temperatures and denaturant concentrations. In terms of cellular response, the levels of inflammatory cytokines were significantly higher in case of AIBI-CS6 expressing ETEC as compared to AIIBII-CS6 expressing ETEC both in vitro and in vivo. When inflammatory cytokines were evaluated after infecting suckling mice with these ETEC strains, the results were consistent. In conclusion, even though there was subtle structural difference between AIBI-CS6 and AIIBII-CS6 due to natural point mutations but ETEC strains expressing these subtypes displayed great variability in pathogenicity.

A review of venous thromboembolism in India.

Venous thromboembolism (VTE), which entails the formation of a thrombus (blood clot) in a vein, has a significant disease burden worldwide. While VTE has traditionally been considered to predominantly affect Caucasian populations, recent studies have indicated a gradual shift in the disease burden towards Asian populations, with added significance of it being a key driver of post-operative mortality. It is imperative to develop a sound understanding of the various factors that affect VTE in stratified local populations. However, there is a glaring paucity of quality data on VTE and its ramifications among Indians - both in terms of quality of life and cost of healthcare. This review aims to throw light on the disease burden, epidemiology, risk factors, environmental factors, food and nutrition that plays a key role in VTE. We also explored the association of VTE with coronavirus disease 2019 to grasp the interplay between the two most significant public health crises of our time. It is vital to place a special emphasis on future research on VTE in India to plug the gaps, which exist in our current knowledge of the disease, particularly with respect to Indian population.

Distribution of virulence factors and its relatedness towards the antimicrobial response of enterotoxigenic Escherichia coli strains isolated from patients in Kolkata, India.

AIM: Enterotoxigenic Escherichia coli (ETEC) is one of the most widely recognized diarrhoeal pathogens in developing countries. The advancement of ETEC vaccine development depends on the antigenic determinants of the ETEC isolates from a particular geographical region. So, the aim here was to comprehend the distribution of virulence determinants of the clinical ETEC strains of this region. Additionally, an attempt was made to find any correlation with the antimicrobial response pattern. METHODS AND RESULTS: Multiplex PCR was employed to identify virulence determinants followed by confirmatory singleplex PCR. For observation of antibiotic response, the Kirby-Bauer method was used. Out of 379 strains, 46% of strains harboured both the enterotoxins ST and LT, whereas 15% were LT only. Among the major colonization factors (CFs), CS6 (41%) was the most prevalent followed by CFA/I (35%) and CFA/III was the lowest (3%). Among the minor CFs, CS21 (25%) was most prevalent, while CS15 showed the lowest (3%) presence. Among the non-classical virulence factors, EatA (69%) was predominant. ETEC strains harbouring CS6 showed resistance towards the commonly used drug Ciprofloxacin (70%). CONCLUSION: CS6 and elt+est toxin genes co-occurred covering 51% of the isolates. CS21 was found in most strains with est genes (43%). EatA was found to occur frequently when ST was present alone or with LT. CS6-harbouring strains showed an independent correlation to antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study would aid in identifying the commonly circulating ETEC isolates of Kolkata, India, and their prevalent virulence determinants. Knowledge of antibiotic resistance patterns would also help in the appropriate use of antibiotics. Furthermore, the study would aid in identifying the multivalent antigens suitable for region-specific ETEC vaccines with maximum coverage.

Iron influences the expression of colonization factor CS6 of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen of acute watery diarrhoea. The pathogenicity of ETEC is linked to adherence to the small intestine by colonization factors (CFs) and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). CS6 is one of the most common CFs in our region and worldwide. Iron availability functions as an environmental cue for enteropathogenic bacteria, signalling arrival within the human host. Therefore, iron could modify the expression of CS6 in the intestine. The objective of this study was to determine the effect of iron availability on CS6 expression in ETEC. This would help in understanding the importance of iron during ETEC pathogenesis. ETEC strain harbouring CS6 was cultured under increasing concentrations of iron salt to assess the effect on CS6 RNA expression by quantitative RT-PCR, protein expression by ELISA, promoter activity by ß-galactosidase activity, and epithelial adhesion on HT-29 cells. RNA expression of CS6 was maximum in presence of 0.2 mM iron (II) salt. The expression increased by 50-fold, which also reduced under iron-chelation conditions and an increased iron concentration of 0.4 mM or more. The surface expression of CS6 also increased by 60-fold in presence of 0.2 mM iron. The upregulation of CS6 promoter activity by 25-fold under this experimental condition was in accordance with the induction of CS6 RNA and protein. This increased CS6 expression was independent of ETEC strains. Bacterial adhesion to HT-29 epithelial cells was also enhanced by five-fold in the presence of 0.2 mM iron salt. These findings suggest that CS6 expression is dependent on iron concentration. However, with further increases in iron concentration beyond 0.2 mM CS6 expression is decreased, suggesting that there might be a strong regulatory mechanism for CS6 expression under different iron concentrations.

Multifunctional transcription factor CytR of Vibrio cholerae is important for pathogenesis.

Vibrio cholerae, the Gram-negative facultative pathogen, resides in the aquatic environment and infects humans and causes diarrhoeagenic cholera. Although the environment differs drastically, V. cholerae thrives in both of these conditions aptly and chitinases play a vital role in their persistence and nutrient acquisition. Chitinases also play a role in V. cholerae pathogenesis. Chitinases and its downstream chitin utilization genes are regulated by sensor histidine kinase ChiS, which also plays a significant role in pathogenesis. Recent exploration suggests that CytR, a transcription factor of the LacI family in V. cholerae, also regulates chitinase secretion in environmental conditions. Since chitinases and chitinase regulator ChiS is involved in pathogenesis, CytR might also play a significant role in pathogenicity. However, the role of CytR in pathogenesis is yet to be known. This study explores the regulation of CytR on the activation of ChiS in the presence of mucin and its role in pathogenesis. Therefore, we created a CytR isogenic mutant strain of V. cholerae (CytR¯) and found considerably less ß-hexosaminidase enzyme production, which is an indicator of ChiS activity. The CytR¯ strain greatly reduced the expression of chitinases chiA1 and chiA2 in mucin-supplemented media. Electron microscopy showed that the CytR¯ strain was aflagellate. The expression of flagellar-synthesis regulatory genes flrB, flrC and class III flagellar-synthesis genes were reduced in the CytR¯ strain. The isogenic CytR mutant showed less growth compared to the wild-type in mucin-supplemented media as well as demonstrated highly retarded motility and reduced mucin-layer penetration. The CytR mutant revealed decreased adherence to the HT-29 cell line. In animal models, reduced fluid accumulation and colonization were observed during infection with the CytR¯ strain due to reduced expression of ctxB, toxT and tcpA. Collectively these data suggest that CytR plays an important role in V. cholerae pathogenesis.

Chitin-induced T6SS in Vibrio cholerae is dependent on ChiS activation.

Vibrio cholerae regularly colonizes the chitinous exoskeleton of crustacean shells in the aquatic region. The type 6 secretion system (T6SS) in V. cholerae is an interbacterial killing device. This system is thought to provide a competitive advantage to V. cholerae in a polymicrobial community of the aquatic region under nutrient-poor conditions. V. cholerae chitin sensing is known to be initiated by the activation of a two-component sensor histidine kinase ChiS in the presence of GlcNAc2 (N,N'-diacetylchitobiose) residues generated by the action of chitinases on chitin. It is known that T6SS in V. cholerae is generally induced by chitin. However, the effect of ChiS activation on T6SS is unknown. Here, we found that ChiS inactivation resulted in impaired bacterial killing and reduced expression of T6SS genes. Active ChiS positively affected T6SS-mediated natural transformation in V. cholerae. ChiS depletion or inactivation also resulted in reduced colonization on insoluble chitin surfaces. Therefore, we have shown that V. cholerae colonization on chitinous surfaces activates ChiS, which promotes T6SS-dependent bacterial killing and horizontal gene transfer. We also highlight the importance of chitinases in T6SS upregulation.

Functional Role of N- and C-Terminal Amino Acids in the Structural Subunits of Colonization Factor CS6 Expressed by Enterotoxigenic Escherichia coli.

UNLABELLED: CS6 is a common colonization factor expressed by enterotoxigenic Escherichia coli It is a two-subunit protein consisting of CssA and CssB in an equal stoichiometry, assembled via the chaperone-usher pathway into an afimbrial, oligomeric assembly on the bacterial cell surface. A recent structural study has predicted the involvement of the N- and C-terminal regions of the CS6 subunits in its assembly. Here, we identified the functionally important residues in the N- and C-terminal regions of the CssA and CssB subunits during CS6 assembly by alanine scanning mutagenesis. Bacteria expressing mutant proteins were tested for binding with Caco-2 cells, and the results were analyzed with respect to the surface expression of mutant CS6. In this assay, many mutant proteins were not expressed on the surface while some showed reduced expression. It appeared that some, but not all, of the residues in both the N and C termini of CssA and CssB played an important role in the intermolecular interactions between these two structural subunits, as well as chaperone protein CssC. Our results demonstrated that T20, K25, F27, S36, Y143, and V147 were important for the stability of CssA, probably through interaction of CssC. We also found that I22, V29, and I33 of CssA and G154, Y156, L160, V162, F164, and Y165 of CssB were responsible for CssA-CssB intermolecular interactions. In addition, some of the hydrophobic residues in the C terminus of CssA and the N terminus of CssB were involved in the stabilization of higher-order complex formation. Overall, the results presented here might help in understanding the pathway used to assemble CS6 and predict its structure. IMPORTANCE: Unlike most other colonization factors, CS6 is nonfimbrial, and in a sense, its subunit composition and assembly are also unique. Here we report that both the N- and C-terminal amino acid residues of CssA and CssB play a critical role in the intermolecular interactions between them and assembly proteins. We found mainly that alternate hydrophobic residues present in these motifs are essential for the interaction between the structural subunits, as well as the chaperone and usher assembly proteins. Our results indicate the involvement of the side chains of identified amino acids in CS6 assembly. This study adds a step toward understanding the interactions between structural subunits of CS6 and assembly proteins during CS6 biogenesis.

Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli.

The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis.

Vibrio cholera survival in an aquatic environment depends on chitin utilization pathway that requires two factors, chitin binding protein and chitinases. The chitinases and the chitin utilization pathway are regulated by a two-component sensor histidine kinase ChiS in V. cholerae. In recent studies these two factors are also shown to be involved in V. cholerae pathogenesis. However, the role played by their upstream regulator ChiS in pathogenesis is yet to be known. In this study, we investigated the activation of ChiS in presence of mucin and its functional role in pathogenesis. We found ChiS is activated in mucin supplemented media. The isogenic chiS mutant (ChiS-) showed less growth compared to the wild type strain (ChiS+) in the presence of mucin supplemented media. The ChiS- strain also showed highly retarded motility as well as mucin layer penetration in vitro. Our result also showed that ChiS was important for adherence and survival in HT-29 cell. These observations indicate that ChiS is activated in presence of intestinal mucin and subsequently switch on the chitin utilization pathway. In animal models, our results also supported the in vitro observation. We found reduced fluid accumulation and colonization during infection with ChiS- strain. We also found ChiS- mutant with reduced expression of ctxA, toxT and tcpA. The cumulative effect of these events made V. cholerae ChiS- strain hypovirulent. Hence, we propose that ChiS plays a vital role in V. cholerae pathogenesis.

Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli.

Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1ß and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.

Role of Vibrio cholerae exochitinase ChiA2 in horizontal gene transfer.

Vibrio cholerae exochitinase ChiA2 plays a key role in acquisition of nutrients by chitin hydrolysis in the natural environment as well as in pathogenesis in the intestinal milieu. In this study we demonstrate the importance of ChiA2 in horizontal gene transfer in the natural environment. We found that the expression of ChiA2 and TfoX, the central regulator of V. cholerae horizontal gene transfer, varied with changes in environmental conditions. The activity of ChiA2 was also dependent on these conditions. In 3 different environmental conditions tested here, we observed that the supporting environmental condition for maximum expression and activity of ChiA2 was 20 °C, pH 5.5, and 100 mmol/L salinity in the presence of chitin. The same condition also induced TfoX expression and was favorable for horizontal gene transfer in V. cholerae. High-performance liquid chromatography analysis showed that ChiA2 released a significant amount of (GlcNAc)2 from chitin hydrolysis under the favorable condition. We hypothesized that under the favorable environmental condition, ChiA2 was upregulated and maximally active to produce a significant amount of (GlcNAc)2 from chitin. The same environmental condition also induced tfoX expression, followed by its translational activation by the (GlcNAc)2 produced, leading to efficient horizontal gene transfer.

Two specific amino acid variations in colonization factor CS6 subtypes of enterotoxigenic Escherichia coli results in differential binding and pathogenicity.

CS6 is the predominant colonization factor of enterotoxigenic Escherichia coli (ETEC). We report the existence of multiple CS6 subtypes caused by natural point mutations in cssA and cssB, the structural genes for CS6. The subtype AIBI was mostly associated with ETEC isolated from diarrhoeal cases, whereas AIIBII was mostly found in asymptomatic controls. Here we explore the rationale behind this association. ETEC isolates expressing AIIBII showed weaker adherence to intestinal epithelial cells compared with ETEC expressing AIBI. AIIBII expression on the ETEC cell surface was threefold less than AIBI. We found that alanine at position 37 in CssAII, in conjunction with asparagine at position 97 in CssBII, was responsible for the decreased levels of AIIBII on the bacterial surface. In addition, purified AIIBII showed fourfold less mucin binding compared with AIBI. The asparagine at position 97 in CssBII was also accountable for the decreased mucin binding by AIIBII. Reduced fluid accumulation and colonization occurred during infection with ETEC expressing AIIBII in animal models. Together these results indicate that the differential adherence between AIBI and AIIBII was a cumulative effect of decreased surface-level expression and mucin binding of AIIBII due to two specific amino acid variations. As a consequence, ETEC expressing these two subtypes displayed differential pathogenicity. We speculate that this might explain the subjective association of AIBI with ETEC from diarrhoeal cases and AIIBII with asymptomatic controls.

A review of clinical trials registered in India from 2008 to 2022 to describe the first-in-human trials.

Aim: This analysis was conducted to review the number, and describe the characteristics of first-in-human (FIH) Phase 1 clinical trials registered in India from 2008 to 2022. Materials and Methods: The data were extracted from the Clinical Trials Registry - India database for all FIH Phase 1 clinical trials registered between 2008 and 2022. Early-phase trials that were not FIH trials (e.g., pharmacokinetic studies and drug-drug interaction studies) were excluded from the study. Results: A total of 1891 trials were retrieved and 220 were included in the analysis. Most of the investigational products were drugs (55%) followed by vaccines (38.2%). The most common therapeutic class of drugs was cancer chemotherapy (19.8%), followed by antimicrobial chemotherapy and endocrinology (18.2% each). The most common vaccine was the influenza vaccine (21.4%), followed by the measles-mumps-rubella vaccine (15.5%). The pharmaceutical industry was the predominant sponsor for most (91%) of the Phase 1 trials. Of the top five sites where most of the Phase 1 trials were conducted, three were private nonacademic centers (cumulatively 31%) and two were tertiary care medical colleges (cumulatively 9%). Conclusion: Phase 1 clinical trials seem to be conducted in India predominantly with industry sponsorship. There is a need to have an alternate ecosystem to take forward molecules that do not receive adequate attention from the industry and molecules that are of national health priority other than areas such as chemotherapy, antimicrobials, and endocrinology. The Indian Council of Medical Research is setting up Phase 1 clinical trial capacity for molecules that predominantly may arise from nonindustry channels.

Prescription-based cost analysis of medicines for cardiovascular risk factors at Indian Council of Medical Research-Rational Use of Medicine Centre Hospitals of India.

OBJECTIVES: India has taken several initiatives to provide health care to its population while keeping the related expenditure minimum. Since cardiovascular diseases are the most prevalent chronic conditions, in the present study, we aimed to analyze the difference in prices of medicines prescribed for three cardiovascular risk factors, based on (a) listed and not listed in the National List of Essential Medicines (NLEM) and (b) generic and branded drugs. MATERIALS AND METHODS: Outpatient prescriptions for diabetes mellitus, hypertension, and dyslipidemia were retrospectively analyzed from 12 tertiary centers. The prices of medicines prescribed were compared based on presence or absence in NLEM India-2015 and prescribing by generic versus brand name. The price was standardized and presented as average price per medicine per year for a given medicine. The results are presented in Indian rupee (INR) and as median (range). RESULTS: Of the 4,736 prescriptions collected, 843 contained oral antidiabetic, antihypertensive, and/or hypolipidemic medicines. The price per medicine per year for NLEM oral antidiabetics was INR 2849 (2593-3104) and for non-NLEM was INR 5343 (2964-14364). It was INR 806 (243-2132) for generic and INR 3809 (1968-14364) for branded antidiabetics. Antihypertensives and hypolipidemics followed the trend. The price of branded non-NLEM medicines was 5-22 times higher compared to generic NLEM which, for a population of 1.37 billion, would translate to a potential saving of 346.8 billion INR for statins. The variability was significant for sulfonylureas, angiotensin receptor blockers, beta-blockers, diuretics, and statins (P < 0.0001). CONCLUSION: The study highlights an urgent need for intervention to actualize the maximum benefit of government policies and minimize the out-of-pocket expenditure on medicines.

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.

Real-time PCR-based mismatch amplification mutation assay for specific detection of CS6-expressing allelic variants of enterotoxigenic Escherichia coli and its application in assessing diarrheal cases and asymptomatic controls.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.

Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence.

Enterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA-CssB-CssC complex is necessary for recognition by CssD and transport of CssA-CssB to the outer membrane as a colonization factor.

Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1.

Cholera continues to be an important public health concern in developing countries where proper hygiene and sanitation are compromised. This severe diarrheal disease is caused by the Gram-negative pathogen Vibrio cholerae belonging to serogroups O1 and O139. Cholera toxin (CT) is the prime virulence factor and is directly responsible for the disease manifestation. The ctxB gene encodes cholera toxin B subunit (CTB) whereas the A subunit (CTA) is the product of ctxA gene. Enzymatic action of CT depends on binding of B pentamers to the lipid-based receptor ganglioside GM1. In recent years, emergence of V. cholerae Haitian variant strains with ctxB7 allele and their rapid spread throughout the globe has been linked to various cholera outbreaks in Africa and Asia. These strains produce classical type (WT) CTB except for an additional mutation in the signal sequence region where an asparagine (N) residue replaces a histidine (H) at the 20th amino acid position (H20N) of CTB precursor (pre-CTB). Here we report that Haitian variant V. cholerae O1 strains isolated in Kolkata produced higher amount of CT compared to contemporary O1 El Tor variant strains under in vitro virulence inducing conditions. We observed that the ctxB7 allele, itself plays a pivotal role in higher CT production. Based on our in silico analysis, we hypothesized that higher accumulation of toxin subunits from ctxB7 allele might be attributed to the structural alteration at the CTB signal peptide region of pre-H20N CTB. Overall, this study provides plausible explanation regarding the hypertoxigenic phenotype of the Haitian variant strains which have spread globally, possibly through positive selection for increased pathogenic traits.