The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N-acetyl-D-glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 M. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-B nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae.Vibrio cholerae is the causative agent of the potentially lethal disease cholera. V. cholerae strains belonging to serogroups O1 and O139 are mainly responsible for cholera epidemics, while strains of other serogroups may cause sporadic outbreaks of the disease. Although the O139 strain has evolved recently, V. cholerae O1 biotype El Tor strains have still been responsible for most of the epidemics in recent years (20,26). In order to cause the disease, V. cholerae must adhere to the intestinal mucus barrier (52). The ability of V. cholerae to adhere to animal cells has been studied before (26,42), and various adherence factors have been proposed, including the virulence-associated toxin-coregulated pilus (5), outer membrane proteins (26, 42), and lipopolysaccharide (LPS) (11). Attachment of V. cholerae to abiotic surfaces has also been recently described (50). However, there is still no information about the factor(s) responsible for initial adherence of the bacteria to the intestine and whether the host plays any role in aiding the colonization of the intestine by the bacteria.Vibrios are marine organisms that adhere to chitin in the environment (12, 33) and utilize chitin as the sole source of nitrogen and carbon by using a family of glycosyl hydrolases, called chitinases (21). Genome analysis of V. cholerae O1 El Tor has revealed the presence of seven such chitinase genes (7), some of which have been characterized (27,37). One of these genes is the putative chitinase gene with locus number VCA0811, the product of which has been recently identified as a common adhesion molecule for both chitinou...

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology.

gene expresses a plasma membrane protein involved in the transport of the watersoluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region Enterotoxigenic Escherichia coli (ETEC) infection is the leading cause of infantile diarrhea in developing countries and an important etiologic agent for traveler's diarrhea. ETEC accounts for approximately 210 million diarrhea episodes and 380,000 deaths annually (35). Community-based studies conducted in developing countries with children younger than 5 years have shown that ETEC was the most frequently isolated enteropathogen (34, 35). As a cause of traveler's diarrhea, ETEC was found to be associated with 40 to 70% of the cases, with drastic outcome in terms of morbidity and economic consequences (34).In order to initiate pathogenesis, ETEC strains must adhere to the small intestine (14). This event is mediated by several proteinaceous surface antigens, collectively known as colonization factor antigens (CFAs) (6). To date, more than 25 distinct colonization factors have been identified, of which CS6 is the most prevalent in many countries (7,20,22). Many of the colonization factors have morphology of fimbriae or pili (14). However, the morphology of CS6 has not so far been defined. CS6 was assumed to be either a nonfimbrial or a short oligomeric assembly that does not protrude enough to be visualized under an electron microscope (17). Functional CS6 is expressed and transported to the bacterial surface in a chaperone-usher pathway. CssC and CssD are the chaperone and usher proteins, respectively, that help surface expression of the CS6 structural subunits, CssA and CssB (33).The role of CS6 in intestinal adherence has been demonstrated using CS6-expressing whole bacteria, but the receptor specificity is still unknown (11). A recent report has shown that when CssB is mutated, binding of bacteria to a colonic cell line (CaCo-2) is reduced slightly compared to that of the bacteria expressing whole CS6 (30).Here, we have purified CS6 to homogeneity from a clinical isolate of ETEC and separated its subunits (CssA and CssB) for the first time. We have characterized CS6 in its native form and demonstrated that fibronectin (Fn) is the interacting matrix for adherence....

Background: Pantothenate transport is essential for Plasmodium development. The transporter that mediates entry of pantothenate is unknown. Results: PfPAT encodes the primary pantothenate transporter of P. falciparum. Conclusion:PfPAT plays an essential function in parasite development and thus is a valid target for antimalarial therapy. Significance: PfPAT is the first pantothenate transporter identified and characterized in protozoan parasites and a valid target for therapy.