Capacity to adhere to and invade human epithelial cells, as related to the presence of virulence genes in, motility of, and biofilm formation ofCampylobacter jejunistrains isolated from chicken and cattle

Campylobacter jejuni is a zoonotic pathogen transmitted through the “farm to fork” route. Outbreaks are generally associated with the consumption of chicken meat; however, dairy cows, birds, wild and domestic food animals, and pets are other important sources. Currently, there are not enough data comparing the virulence of strains isolated from these reservoirs. In this study, we compared C. jejuni strains isolated from broiler chickens and dairy cattle by determining their ability to adhere to and invade in vitro human colonic epithelial cells in the T84 cell line with their motility, formation of biofilms, and presence of eight virulence genes. A Wilcoxon Rank Sum test was performed to establish the relationship between presence of the studied genes and cellular invasion and adhesion, as well as differences between the animal species of origin of the isolate. A Spearman correlation was performed to assess the relationship between invasion and motility, along with invasion and biofilm generation. The virB11 gene was positively associated with the adherence capacity of the strains (mean difference = 0.21, p = 0.006), and strains isolated from chickens showed a significant difference for adherence compared with strains isolated from cattle (p = 0.0001). Our results indicate that strains of C. jejuni have a difference in their adherence capacity depending on the animal reservoir from which they came, with chicken isolates displaying higher virulence than dairy cattle isolates.

Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma

Considerable progress has been made in understanding the roles of Helicobacter pylori in inflammation and gastric cancer; however, far less is known about the roles of enterohepatic Helicobacter species (EHS) in carcinogenesis and their zoonotic or pathogenic potential. We determined the prevalence of EHS infection in a cohort of geriatric rhesus monkeys in which intestinal adenocarcinoma (IAC) is common and investigated the association between EHS infection and IAC. The cohort consisted of 36 animals, 14 of which (age 26–35 years) had IAC. Of the 36 rhesus, 35 (97 %) were positive for EHS using PCR or bacterial isolation from faeces, colonic or tumour tissues. Only a single rhesus, which had IAC, was negative for EHS by all detection methods. The EHS identified by 16S rRNA sequencing in this study were from three Helicobacter taxa: Helicobacter macacae (previously rhesus monkey taxon 1), Helicobacter sp. rhesus monkey taxon 2, previously described from strain MIT 99-5507, and Helicobacter sp. rhesus monkey taxon 4, related to Helicobacter fennelliae. Thirteen of 14 monkeys with IAC were positive for either H. macacae (7/13, 54 %), EHS rhesus monkey taxon 4 (4/13, 31 %) or a mixture of the two EHS (2/13, 15 %). These results indicate that EHS are prevalent among aged rhesus macaques with IAC. Using Helicobacter genus-specific florescent in situ hybridization, EHS were detected on the surface of colonic epithelia of infected monkeys. All Helicobacter isolates, including H. macacae, effectively adhered to, invaded, and significantly induced proinflammatory genes, including IL-8, IL-6, TNF-α and iNOS, while downregulating genes involved in the function of inflammasomes, particularly IL-1β, CASPASE-1, NRLP3, NLRP6 and NLRC4 in the human colonic T84 cell line (P<0.0001). These results suggest that EHS may represent an aetiological agent mediating diarrhoea, chronic inflammation, and possibly intestinal cancer in non-human primates, and may play a role in similar disease syndromes in humans. Downregulation of inflammasome function may represent an EHS strategy for long-term persistence in the host and play a role in inducing pathological changes in the host’s lower bowel.

Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1

Although colonic lumen NH4+levels are high, 15–44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH4+. More extensive studies have elucidated the transport of NH4+in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH4+secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH4+and maintain an apical-to-basolateral NH4+gradient. NH4+transport in the secretory direction was supported by basolateral NH4+loading on NKCC1, Na+-K+-ATPase, and the NH4+transporter, RhBG. NH4+was transported on NKCC1 in T84 cells nearly as well as K+as determined by bumetanide-sensitive86Rb-uptake.86Rb-uptake and ouabain-sensitive current measurement indicated that NH4+is transported by Na+-K+-ATPase in these cells to an equal extent as K+. T84 cells expressed mRNA for the basolateral NH4+transporter RhBG and the apical NH4+transporter RhCG. Net NH4+transport in the secretory direction determined by14C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH4+transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH4+because of surface cell NH4+absorption. These findings suggest that we rethink the present limited understanding of NH4+handling by the distal colon as being due solely to passive absorption.

Soluble Extracts from Helicobacter pylori Induce Dome Formation in Polarized Intestinal Epithelial Monolayers in a Laminin-Dependent Manner

ABSTRACT Helicobacter pylori colonizes the stomach at the interface between the mucus layer and the apical pole of gastric epithelial cells. A number of secreted and shed products from the bacteria, such as proteins and lipopolysaccharide, are likely to have a role in the pathogenesis at the epithelial level. To determine the physiological response of transporting polarized epithelia to released soluble factors from the bacterium, we used the T84 cell line. Monolayers of T84 cells were exposed to soluble extracts from H. pylori. The extracts induced rapid “dome” formation as well as an immediate decrease in transepithelial electrical resistance. Domes are fluid-filled blister-like structures unique to polarized epithelia. Their formation has been linked to sodium-transporting events as well as to diminished adherence of the cells to the substrate. H. pylori-induced dome formation in T84 monolayers was exacerbated by amiloride and inhibited by ouabain. Furthermore, it was associated with changes in the expression of the laminin binding α6β4 integrin and the 67-kDa laminin receptor. Domes formed primarily on laminin-coated filters, rather than on fibronectin or collagen matrices, and their formation was inhibited by preincubating the bacterial extract with soluble laminin. This effect was specific to H. pylori and independent of the urease, vacA, cagA, and Lewis phenotype of the strains. These data indicate that released elements from H. pylori can alter the physiological balance and integrity of the epithelium in the absence of an underlying immune response.

Guanylyl Cyclase C Receptor: Regulation of Catalytic Activity by ATP

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.

Positive and negative regulation of chloride secretion in T84 cells

This review discusses recent findings regarding the mechanisms and regulation of chloride secretion in the T84 cell line, a widely used model for the study of transepithelial chloride transport, the properties of the cystic fibrosis gene product (cystic fibrosis transmembrane conductance regulator), and the interactions of inflammatory cell types with the intestinal epithelium. First, I review the features of the chloride secretory mechanism in this cell line, both as originally described by Dharmsathaphorn and co-workers and as modified by more recent findings. Second, I cover what is known of the intracellular regulation of the secretory process. Third, I review the ways in which the cell line has been utilized to dissect pathways of immune-epithelial interactions that may contribute to the pathogenesis of inflammatory diarrhea. Finally, I suggest areas of investigation with this cell line that may prove ripe for further study. The physiological and pathophysiological implications of findings obtained with the cell line are also discussed.