The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.     

Distinct repeat motifs at the C-terminal region of CagA of Helicobacter pylori strains isolated from diseased patients and asymptomatic individuals in West Bengal, India

Background

Infection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India.

Results

Seventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants.

Conclusion

Our analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.     

Storing drinking-water in copper pots kills contaminating diarrhoeagenic bacteria

Microbially-unsafe water is still a major concern in most developing countries. Although many water-purification methods exist, these are expensive and beyond the reach of many people, especially in rural areas. Ayurveda recommends the use of copper for storing drinking-water. Therefore, the objective of this study was to evaluate the effect of copper pot on microbially-contaminated drinking-water. The antibacterial effect of copper pot against important diarrhoeagenic bacteria, including Vibrio cholerae O1, Shigella flexneri 2a, enterotoxigenic Escherichia coli, enteropathogenic E. coli, Salmonella enterica Typhi, and Salmonella Paratyphi is reported. When drinking-water (pH 7.83 +/- 0.4; source: ground) was contaminated with 500 CFU/mL of the above bacteria and stored in copper pots for 16 hours at room temperature, no bacteria could be recovered on the culture medium. Recovery failed even after resuscitation in enrichment broth, followed by plating on selective media, indicating loss of culturability. This is the first report on the effect of copper on S. flexneri 2a, enteropathogenic E. coli, and Salmonella Paratyphi. After 16 hours, there was a slight increase in the pH of water from 7.83 to 7.93 in the copper pots while the other physicochemical parameters remained unchanged. Copper content (177 +/- 16 ppb) in water stored in copper pots was well within the permissible limits of the World Health Organization. Copper holds promise as a point-of-use solution for microbial purification of drinking-water, especially in developing countries.     

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.     

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca²⁺ current, suggesting interactions of transducin with the synaptic machinery.     

Evidence of Reduced β-Cell Function in Asian Indians With Mild Dysglycemia

OBJECTIVE

To examine β-cell function across a spectrum of glycemia among Asian Indians, a population experiencing type 2 diabetes development at young ages despite low BMI.

RESEARCH DESIGN AND METHODS

One-thousand two-hundred sixty-four individuals without known diabetes in the Diabetes Community Lifestyle Improvement Program in Chennai, India, had a 75-g oral glucose tolerance test, with glucose and insulin measured at 0, 30, and 120 min. Type 2 diabetes, isolated impaired fasting glucose (iIFG), isolated impaired glucose tolerance (iIGT), combined impaired fasting glucose and impaired glucose tolerance, and normal glucose tolerance (NGT) were defined by American Diabetes Association guidelines. Measures included insulin resistance and sensitivity (homeostasis model assessment of insulin resistance [HOMA-IR], modified Matsuda Index, 1/fasting insulin) and β-cell function (oral disposition index = [Δinsulin_0–30/Δglucose_0–30] × [1/fasting insulin]).

RESULTS

Mean age was 44.2 years (SD, 9.3) and BMI 27.4 kg/m² (SD, 3.8); 341 individuals had NGT, 672 had iIFG, IGT, or IFG plus IGT, and 251 had diabetes. Patterns of insulin resistance or sensitivity were similar across glycemic categories. With mild dysglycemia, the absolute differences in age- and sex-adjusted oral disposition index (NGT vs. iIFG, 38%; NGT vs. iIGT, 32%) were greater than the differences in HOMA-IR (NGT vs. iIFG, 25%; NGT vs. iIGT, 23%; each P < 0.0001). Compared with NGT and adjusted for age, sex, BMI, waist circumference, and family history, the odds of mild dysglycemia were more significant per SD of oral disposition index (iIFG: odds ratio [OR], 0.36; 95% CI, 0.23–0.55; iIGT: OR, 0.37; 95% CI, 0.24–0.56) than per SD of HOMA-IR (iIFG: OR, 1.69; 95% CI, 1.23–2.33; iIGT: OR, 1.53; 95% CI, 1.11–2.11).

CONCLUSIONS

Asian Indians with mild dysglycemia have reduced β-cell function, regardless of age, adiposity, insulin sensitivity, or family history. Strategies in diabetes prevention should minimize loss of β-cell function.Type 2 diabetes mellitus is a global problem, with 80% of all cases worldwide occurring in low- and middle-income countries (1). However, despite the increasing prevalence of type 2 diabetes, the etiology of the disease remains incompletely understood. Previously invoked as the driving feature of diabetes, increased insulin resistance can trigger increased insulin production to maintain normoglycemia and, over time, can strain β cells to the point at which insulin production is no longer adequate (2–4), i.e., β-cell “exhaustion.” Characteristics associated with insulin resistance, particularly older age, obesity, and physical inactivity, are strong risk factors for diabetes (5).Yet, poor β-cell function also may have more of a primary role in diabetes development. The inadequate β-cell response to physiologic needs for insulin not only may be an acquired feature (e.g., as a result of insulin resistance) but also, at least in some individuals, may be an inherent feature. β-Cell dysfunction has been detected early in the pathogenesis of the disease (6), with recent cross-sectional and longitudinal studies detecting dysfunction in people with prediabetes or even normoglycemia (7–10). Supported by recent genetic discoveries (11), these studies suggest that some individuals have an underlying susceptibility to poor β-cell function (12) and that β-cell dysfunction may be an early driving metabolic feature of diabetes development.Most studies of diabetes pathogenesis have been conducted in populations of European descent; however, more people have diabetes in other populations worldwide. Asian Indians, in particular, experience high rates of type 2 diabetes (13) at younger ages and lower BMI values (14) compared with other populations. They have high basal insulin levels (15) that are not entirely explained by obesity or adverse fat distribution (16), which are commonly cited factors related to insulin resistance. Considering these characteristics, Asian Indians may be an ideal population to utilize for developing a better understanding of the relative roles of β-cell function and insulin resistance in the pathogenesis of type 2 diabetes. Previous studies that have examined the etiology of diabetes in Asian Indians have produced conflicting findings. Altered β-cell function has been associated with impaired glucose tolerance (IGT) (17), has not been associated with IGT (18,19), and has been associated with impaired fasting glucose (IFG) but not IGT (20). Furthermore, β-cell function has not always been evaluated rigorously in Asian Indians (i.e., expressed relative to the insulin resistance of each individual) (21). We investigated the associations between the pathophysiologic mechanisms of insulin resistance and β-cell function with glycemic status in a large cohort (n = 1,264) of Asian Indians in Chennai, India.     

Enrichment of collagen and gelatin degrading activities in the plasma membranes of human cancer cells

Interactions between connective tissue substrates and proteinases localized to the surface of cancer cells are implicated in cancer invasion. In this report we have compared the enrichment of collagen and gelatin degrading activities and cysteine proteinase(s) in well-characterized (enzyme markers and electron microscopy) subcellular membrane fractions isolated from human small cell lung cancer lines (NCI-H69 and NCI-H82) and the RWP-1 pancreatic cancer line. With each cell line collagenolytic, gelatinolytic, and cysteine proteinase activities were enriched 5- to 128-fold in the plasma membrane fractions with differences noted between microvilli versus smooth membrane profiles. Incubation of tumor plasma membranes with methyl-3H-labeled collagen resulted in extensive degradation of the gamma, beta, alpha 1, and alpha 2 chains, suggesting the combined action of metalloproteinases. Treatment of tumor plasma membranes with the chaotropic agent, 2 M KCl, did not diminish membrane collagen- or gelatin-degrading activity, but extensively leached out the cysteine proteinase, suggesting that the latter enzyme is not an integral membrane protein. Enzyme inhibitors specific for metalloproteinases and cysteine proteinase were used to corroborate enzymatic classification. In conclusion, we have demonstrated variations in the localization of proteinases in the plasma membrane domains of different human cancer cells.     

Gelatin-degrading type IV collagenase isolated from human small cell lung cancer

The goal of this study has been to identify and characterize metalloproteinases from a highly metastatic human small cell lung cancer cell line. The cytosol isolated from NCI-H82 lung cancer cells propagated as solid tumors in nude mice contained a gelatinolytic enzyme that was subjected to ammonium sulfate precipitation, zinc chelate Sepharose column chromatography, anion exchange chromatography and gel permeation chromatography. This purification scheme resulted in a 280-fold enrichment of an active gelatin and type IV collagen-degrading enzyme. On gelatin zymography two bands of gelatinolytic activity were detected, corresponding to Mr of 75,000 and 63,000. Gelatinolytic activity was inhibited by metal chelators, tetracyclines, and serum. On immunoblotting using an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase, the tumor enzyme was identified as type IV collagenase. A second tumor metalloproteinase of Mr = 29,000, which degraded proteoglycan substrates, was also isolated.     

Purification of a gelatin-degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts

Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated c-Harvey-ras oncogene from T24 bladder cancer cells. This tumor proteinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass = 67,000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67,000 and 62,000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase.