Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Intestinal Microbial Diversity during Early-Life Colonization Shapes Long-Term IgE Levels

Microbial exposure following birth profoundly impacts mammalian immune system development. Microbiota alterations are associated with increased incidence of allergic and autoimmune disorders with elevated serum IgE as a hallmark. The previously reported abnormally high serum IgE levels in germ-free mice suggests that immunoregulatory signals from microbiota are required to control basal IgE levels. We report that germ-free mice and those with low-diversity microbiota develop elevated serum IgE levels in early life. B cells in neonatal germ-free mice undergo isotype switching to IgE at mucosal sites in a CD4 T-cell- and IL-4-dependent manner. A critical level of microbial diversity following birth is required in order to inhibit IgE induction. Elevated IgE levels in germ-free mice lead to increased mast-cell-surface-bound IgE and exaggerated oral-induced systemic anaphylaxis. Thus, appropriate intestinal microbial stimuli during early life are critical for inducing an immunoregulatory network that protects from induction of IgE at mucosal sites.

Colonization Map, Northwest Algeria, 1883 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Département d'Oran : colonisation, par A.D. Langlois ; dressée sous le patronage de la Société de Géographie d'Oran. It was published by Challamel et Cie. in 1883. Scale [ca. 1:800,000]. Covers northwest Algeria. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the Europe Lambert Conformal Conic coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, roads, railroads and stations, canals, ruins, mines, territorial claims, shoreline features, forests, ground cover, and more. Includes also statistical tables on population and agriculture.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Surface Elevation Change Processes in the Snohomish River Estuary, Washington

Estuaries provide crucial ecosystem functions and contain significant socio-economic value. Within Washington State, estuaries supply rearing habitat for juvenile salmon during their transition period from freshwater to open sea. In order to properly manage wetland resources and restore salmon habitat, the mechanisms through which estuaries evolve and adapt to pressures from climate change, most notably eustatic sea level rise, must be understood. Estuaries maintain elevation relative to sea level rise through vertical accretion of sediment. This report investigates the processes that contribute to local surface elevation change in the Snohomish Estuary, conveys preliminary surface elevation change results from RTK GPS monitoring, and describes how surface elevation change will be monitored with a network of RSET-MH’s. Part of the tidal wetlands within the Snohomish River Estuary were converted for agricultural and industrial purposes in the 1800’s, which resulted in subsidence of organic soils and loss of habitat. The Tulalip Tribes, the National Oceanic and Atmospheric Administration (NOAA), Northwest Indian Fisheries Commission (NWIFC), and the Environmental Protection Agency (EPA) are conducting a large-scale restoration project to improve ecosystem health and restore juvenile salmon habitat. A study by Crooks et al. (2014) used 210Pb and carbon densities within sediment cores to estimate wetland re-building capacities, sediment accretion rates, and carbon sequestration potential within the Snohomish Estuary. This report uses the aforementioned study in combination with research on crustal movement, tidal patterns, sediment supply, and sea level rise predictions in the Puget Sound to project how surface elevation will change in the Snohomish Estuary with respect to sea level rise. Anthropogenic modification of the floodplain has reduced the quantity of vegetation and functional connectivity within the Snohomish Estuary. There have been losses up to 99% in vegetation coverage from historic extents within the estuary in both freshwater and mesohaline environments. Hydrographic monitoring conducted by NOAA and the Tulalip Tribe shows that 85% of the historic wetland area is not connected to the main stem of the Snohomish (Jason Hall 2014, unpublished data, NOAA). As vegetation colonization and functional connectivity of the floodplains of the Snohomish estuary is re-established through passive and active restoration, sediment transport and accretion is expected to increase. Under the Intergovernmental Panel on Climate Change (IPCC) “medium- probability” scenario sea level is projected to rise at a rate of 4.28 mm/year in the Puget Sound. Sea level rise in the Snohomish Estuary will be exacerbated from crustal deformation from subsidence and post-glacial rebound, which are measured to be -1.4 mm/year and -0.02 mm/year, respectively. Sediment accretion rates calculated by Crooks et al. (2014) and RTK GPS monitoring of surface elevation change of the Marysville Mitigation site from 2011-2014 measured vertical accretion rates that range from -48-19 mm/year and have high spatial variability. Sediment supply is estimated at 490 thousand tons/year, which may be an under-estimate because of the exclusion of tidal transport in this value. The higher rates of sediment accretion measured in the Snohomish Estuary suggest that the Snohomish will likely match or exceed the pace of sea level rise under “medium-probability” projections. The network of RSET-MH instruments will track surface elevation change within the estuary, and provide a more robust dataset on rates of surface elevation change to quantify how vertical accretion and subsidence are contributing to surface elevation change on a landscape scale.

In vitro studies on the colonization of bovine colonic mucosa by Shiga-toxigenic Escherichia coli (STEC)

This study investigated host-related factors that influence intestinal colonization by Shiga-toxigenic E. coli (STEC). A quantitative colonization assay was developed to comparatively measure attachment of STEC to bovine colonic tissues maintained in vitro. No differences were determined in colonization susceptibility between tissues derived from weaning calves and adult cattle, or for tissues from cattle fed grain and forage-based rations. Substrate conditions designed to represent various intra-enteric environments were tested for their effect on STEC/mucosal interaction. Under conditions corresponding to a well-fed ruminant (high volatile fatty acid and lactate concentrations, low pH), significantly less STEC colonized the mucosal surface of colonic biopsies. These results may help explain why fasted. poorly or intermittently fed cattle and pre-ruminant calves excrete STEC to a greater degree. Studies on the ecology of STEC within the ruminant gut help identify mechanisms to reduce their threat to public health.