Adherence and invasion of Bacteroidales isolated from the human intestinal tract

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.

Coupled human and natural system dynamics as key to the sustainability of Lake Victoria's ecosystem services

East Africa’s Lake Victoria provides resources and services to millions of people on the lake’s shores and abroad. In particular, the lake’s fisheries are an important source of protein, employment, and international economic connections for the whole region. Nonetheless, stock dynamics are poorly understood and currently unpredictable. Furthermore, fishery dynamics are intricately connected to other supporting services of the lake as well as to lakeshore societies and economies. Much research has been carried out piecemeal on different aspects of Lake Victoria’s system; e.g., societies, biodiversity, fisheries, and eutrophication. However, to disentangle drivers and dynamics of change in this complex system, we need to put these pieces together and analyze the system as a whole. We did so by first building a qualitative model of the lake’s social-ecological system. We then investigated the model system through a qualitative loop analysis, and finally examined effects of changes on the system state and structure. The model and its contextual analysis allowed us to investigate system-wide chain reactions resulting from disturbances. Importantly, we built a tool that can be used to analyze the cascading effects of management options and establish the requirements for their success. We found that high connectedness of the system at the exploitation level, through fisheries having multiple target stocks, can increase the stocks’ vulnerability to exploitation but reduce society’s vulnerability to variability in individual stocks. We describe how there are multiple pathways to any change in the system, which makes it difficult to identify the root cause of changes but also broadens the management toolkit. Also, we illustrate how nutrient enrichment is not a self-regulating process, and that explicit management is necessary to halt or reverse eutrophication. This model is simple and usable to assess system-wide effects of management policies, and can serve as a paving stone for future quantitative analyses of system dynamics at local scales.

A legacy of human-induced ecosystem changes: spatial processes drive the taxonomic and functional diversities of testate amoebae in Sphagnum peatlands of the Galápagos

Aim Our aims were to compare the composition of testate amoeba (TA) communities from Santa Cruz Island, Galápagos Archipelago, which are likely in existence only as a result of anthropogenic habitat transformation, with similar naturally occurring communities from northern and southern continental peatlands. Additionally, we aimed at assessing the importance of niche-based and dispersal-based processes in determining community composition and taxonomic and functional diversity. Location The humid highlands of the central island of Santa Cruz, Galápagos Archipelago. Methods We survey the alpha, beta and gamma taxonomic and functional diversities of TA, and the changes in functional traits along a gradient of wet to dry habitats. We compare the TA community composition, abundance and frequency recorded in the insular peatlands with that recorded in continental peatlands of Northern and Southern Hemispheres. We use generalized linear models to determine how environmental conditions influence taxonomic and functional diversity as well as the mean values of functional traits within communities. We finally apply variance partitioning to assess the relative importance of niche- and dispersal-based processes in determining community composition. Results TA communities in Santa Cruz Island were different from their Northern Hemisphere and South American counterparts with most genera considered as characteristic for Northern Hemisphere and South American Sphagnum peatlands missing or very rare in the Galápagos. Functional traits were most correlated with elevation and site topography and alpha functional diversity to the type of material sampled and site topography. Community composition was more strongly correlated with spatial variables than with environmental ones. Main conclusions TA communities of the Sphagnum peatlands of Santa Cruz Island and the mechanisms shaping these communities contrast with Northern Hemisphere and South American peatlands. Soil moisture was not a strong predictor of community composition most likely because rainfall and clouds provide sufficient moisture. Dispersal limitation was more important than environmental filtering because of the isolation of the insular peatlands from continental ones and the young ecological history of these ecosystems.

Cloning of human adenosine kinase cDNA: sequence similarity to microbial ribokinases and fructokinases.

Adenosine kinase catalyzes the phosphorylation of adenosine to AMP and hence is a potentially important regulator of extracellular adenosine concentrations. Despite extensive characterization of the kinetic properties of the enzyme, its primary structure has never been elucidated. Full-length cDNA clones encoding catalytically active adenosine kinase were obtained from lymphocyte, placental, and liver cDNA libraries. Corresponding mRNA species of 1.3 and 1.8 kb were noted on Northern blots of all tissues examined and were attributable to alternative polyadenylylation sites at the 3' end of the gene. The encoding protein consists of 345 amino acids with a calculated molecular size of 38.7 kDa and does not contain any sequence similarities to other well-characterized mammalian nucleoside kinases, setting it apart from this family of structurally and functionally related proteins. In contrast, two regions were identified with significant sequence identity to microbial ribokinase and fructokinases and a bacterial inosine/guanosine kinase. Thus, adenosine kinase is a structurally distinct mammalian nucleoside kinase that appears to be akin to sugar kinases of microbial origin.

Transcytosis of cholera toxin subunits across model human intestinal epithelia.

Cholera toxin (CT) elicits a massive secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and ultimately activating basolateral effectors (adenylate cyclase). The mechanism of signal transduction from apical to basolateral membrane, however, remains undefined. We have previously shown that CT action on the polarized human intestinal epithelial cell line T84 requires endocytosis and processing in multiple intracellular compartments. Our aim in the present study was to test the hypothesis that CT may actually move to its site of action on the basolateral membrane by vesicular traffic. After binding apical receptors, CT entered basolaterally directed transcytotic vesicles. Both CT B subunits and to a lesser extent CT A subunits were delivered intact to the serosal surface of the basolateral membrane. The toxin did not traverse the monolayer by diffusion through intercellular junctions. Transcytosis of CT B subunits displayed nearly identical time course and temperature dependency with that of CT-induced Cl- secretion--suggesting the two may be related. These data identify a mechanism that may explain the link between the toxin's apical receptor and basolateral effector.

Human intestinal epithelial cells express functional cytokine receptors sharing the common gamma c chain of the interleukin 2 receptor.

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.

Investigation of the absorption pathways of some poorly absorbed drugs using human intestinal (CAC0-2) cell monolayers

Absorption across the gastro-intestinal epithelium is via two pathways; the transcellular and paracellular pathway. Caco-2 cells, when cultured on polycarbonate filters, formed a confluent monolayer with many properties of differentiated intestinal epithelial cells, As a model of human gastro-intestinaJ tract epithelia they were used to elucidate and characterise the transepithelial transport of two protein kinase C inhibitors, N-(3-chlorophenyl)-4-[2-(3-hydroxypropylamino)-4-pyridyl]-2-pyrimidinamin (CHPP) and N-benzoyl-staurosporine (NBS), and the polypeptide, human calcitonin. Lanthanum ions are proposed as a paracellular pathway inhibitor and tested with D-mannitol permeability and transepithelial electrical resistance measurements. The effect La3+ has on the carrier-mediated transport of D-glucose and Sodium taurocholate as well as the vesicularly transcytosed horseradish peroxidase was also investigated. As expected, 2 mM apical La3+ increases transepithelial electrical resistance 1.S-fold and decreases mannitol permeability by 63.0 % ± 1.37 %. This inhibition was not repeated by other cations. Apical 2 mM La3+ was found to decrease carrier-mediated D-glucose and taurocholate permeability by only 8.7 % ± 1.6 %, 26.3 % ± 5.0 %. There was no inhibitory effect on testosterone or PEG 4000 permeability observed with La3+. However, for horseradish peroxidase and human calcitonin permeability was decreased by 98.7 % ± 11.7%, and 96.2 % ± 0.8 % respectively by 2 mM La3+. Indicating that human calcitonin could also be transported by vesicular transcytosis. The addition of 2 mM La3+ to the apical surface of Caco-2 monolayers produces a paracellular pathway inhibition. Therefore, La3+ could be a useful additional tool in delineating the transepithelial pathway of passive drug absorption.

Interaction of quinolone antibiotics with the human intestinal CACO-2 cell model

The transport of a group of quinolone antibiotics across the human intestinal model, Caco-2 cells, was investigated. It was found that the transport of the quinolones generally correlated with the lipophilicity of the compounds, indicating the passive diffusional transcellular processes were involved. However, it was observed that the transport in both directions apical-to-basolateral and basolateral-to-apical was not equivalent, and polarised transport occurred. For all the quinolones studied except, BMS-284756-01, it was found that the basolateral-to-apical transport was significantly greater than the apical-to-basolateral transport. This finding suggested that the quinolones underwent a process of active secretion. The pKas and logPs for the quinolones were determined using potentiometric titrations. The measured logP values were compared with those determined using theoretical methods. The theoretical methods for calculating logP including the Moriguchi method correlated poorly with the measured logP values. Further investigations revealed that there may be an active transporter involved in the apical-to-basolateral transport of quinolones as well. This mechanism was sensitive to competing quinolones, but, it was unaffected by the metabolic inhibitor combination of sodium azide (15mM) with 2-deoxy-D-glucose (50mM). The basolateral-to-apical transport of quinolones was found to be sensitive to inhibition by a number of different inhibitors. The metabolic inhibitors, sodium azide (15mM) with 2-deoxy-D-glucose (50mM) and 2,4-dinitrophenol (1mM), were able to reduce the basolateral-to-apical transport of quinolones. A reduction in temperature from 37°C to 2°C caused an 80-fold decrease in the transport of gatifloxacin in both directions, however, this effect was not sufficient to abolish the greater basolateral-to-apical secretion. As with apical-to-basolateral transport, it was found that quinolones competed with gatifloxacin for basolateral-to-apical transport, both ofloxacin (100μM) and norfloxacin (100μM) significantly (P<0.003) decreased the basolateral-to-apical transport of gatifloxacin; however, ciprofloxacin (100μM and 300μM) had no effect. A number of inhibitors of various transport systems were also investigated. It was found that the anion transport inhibitor, probenecid (100 μM) had a significant inhibitory effect on the basolateral-to-apical transport of ciprofloxacin (P=0.039), while the cation transport inhibitor cimetidine (100μM and 500μM) had no effect. The organic anion exchange inhibitor 4,4'diisothiocyanostilbene-2-2' -disulphonic acid DIDS (400μM) also had a significant inhibitory effect (P=O.O 13). The PgP inhibitor and anion exchange inhibitor verapamil (400Mμ) was able to completely abolish the basolateral-to-apical secretion of gatifloxacin and bring it into line with the apical-to-basolateral flux. In conclusion, the apical-to-basolateral and basolateral-toapical transport of quinolones involved an active component. The basolateral-to-apical secretion was abolished by a verapamil (400μM), a bisubstrate for PgP and the anion transporter.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Exploring diarrhoea, enteral nutrition and intestinal microbial flora relationships in critically ill patients

Diarrhoea is a common complication observed in critically ill patients. Relationships between diarrhoea, enteral nutrition and aerobic intestinal microflora have been disconnectedly examined in this patient cohort. This research used a two-study, observational design to examine these associations. Higher diarrhoea incidence rates were observed when patients received enteral tube feeding, had abnormal serum blood results, received multiple medications and had aerobic microflora dysbiosis. Further, significant aerobic intestinal microflora changes were observed over time in patients who experienced diarrhoea. These results establish a platform for further work to improve the intestinal health of critically ill patients.