Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ϳ8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot ؊ ) phenotype and is specific for the Na ؉ -driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na ؉ bioenergetics, such as the function of the primary Na ؉ pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na ؉ -driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.Cholera is an acute waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. This Gramnegative pathogen continues to be a major public health concern in areas of endemicity in South Asia and Africa, with an estimated 5 million cases and more than 100,000 deaths per year. Cases of severe cholera are commonly treated with antibiotics to diminish the duration of its life-threatening clinical symptoms. In this regard, the emergence of multipleantibiotic-resistant V. cholerae O1 and O139 strains has been recognized as a major concern (12,43,45,49). The availability of novel prophylactic measures and/or adjunctive therapies could contribute to diminishing the burden of cholera and antibiotic resistance.V. cholerae strains that cause epidemic cholera exhibit three major virulence traits: (i) production of cholera toxin (CT), (ii) expression of the toxin-coregulated pilus (TCP), and (iii) expression of a single, fast-rotating sheathed polar flagellum driven by sodium motive force (SMF) (34). CT is an AD...

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ⌬hapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.Cholera is an acute waterborne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. Infecting V. cholerae cells adhere to the intestinal mucosa, where they express major virulence factors such as the toxin coregulated pilus (TCP) (21) and cholera toxin (CT), which is largely responsible for the profuse rice-watery diarrhea typical of this illness (12,26). Cholera patients shed V. cholerae in their stools, which usually contain about 10 8 hyperinfective V. cholerae cells per ml (38). The V. cholerae cells can then survive and persist in fresh water and estuarine aquatic ecosystems to eventually gain entrance to a new host.The Zn-dependent metalloprotease hemagglutinin (HA)/ protease (18) has been proposed to facilitate V. cholerae detachment from the intestinal mucosa when infecting V. cholerae cells reach a high density (4,14,43,44). Inactivation of hapA, which encodes HA/protease, has been shown to enhance adherence to mucin-coated polystyrene plates (43), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (4), and colonization of the suckling mouse intestine (41, 43). The mucinase activity of HA/protease (13), together with its capacity to cleave the mucin-binding adhesin GbpA at a high cell density, provides a mechanism that supports the "detachase" function attributed to this protein (25). In addition, HA/protease has been reported to perturb the paracellular barrier of cultured intestinal epithelial cells (37, 55) by acting on tight junction-associated proteins (56). In agreement with this finding, we have shown that HA/protease enhances enterotoxicity in the rabbit ileal loop model (43).The expression of flagellar motility ...

Apoptosis plays an important role in eliminating UV-damaged keratinocytes, but its role in UV-induced immune suppression is not clear. Langerhans cells (LCs) may function as inducers of immune suppression. We have shown that LCs derived from mice deficient in the proapoptotic Bid (BH3-interacting death domain protein) gene (Bid KO) resist apoptosis and induce amplified immune responses. In this report, we examined responses in Bid KO mice to UVB exposure. Acute UV exposure led Bid KO mice to develop fewer apoptotic cells and retain a greater fraction of LCs in the epidermal layer of skin in comparison to wild-type mice. Bid KO mice were also markedly resistant to local and systemic UV tolerance induction to hapten sensitization and contact hypersensitivity responses. Elicitation responses and inflammation at skin sensitization sites in UV-treated Bid KO mice were equal to or greater than nonsuppressed control responses. In Bid KO mice, LCs accumulated in lymph nodes to greater numbers, demonstrated longer lifespans, and contained fewer DNA-damaged cells. These studies provide evidence that Bid activation is a critical upstream mediator in UV-induced keratinocyte and LC apoptosis and that its absence abrogates UV-induced immune tolerance.

Avian species have a unique integument covered with feathers. Skin morphogenesis is a successive and complex process. To date, most studies have focused on a single developmental point or stage. Fewer studies have focused on whole transcriptomes based on the time-course of embryo integument development. To analyze the global changes in gene expression profiles, we sequenced the transcriptome of chicken embryo skin samples from day 6 to day 21 of incubation and identified 5830 differentially expressed genes (DEGs). Hierarchical clustering showed that E6 to E14 is the critical period of feather follicle morphogenesis. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs, two kinds of Wnt signaling pathways (a canonical pathway and a non-canonical pathway) changed during feather follicle and feather morphogenesis. The gene expression level of inhibitors and ligands related to the Wnt signaling pathway varied significantly during embryonic development. The results revealed a staggered phase relationship between the canonical pathway and the non-canonical pathway from E9 to E14. These analyses shed new light on the gene regulatory mechanism and provided fundamental data related to integument morphogenesis of chickens.

ABSTRACT. Previous molecular genetic studies of the goat hair life cycle have focused primarily on a limited number of genes and proteins. To identify additional genes that may play important roles in hair follicle cycle regulation, Illumina sequencing technology was used to catalog differential gene expression profiles in the hair growth cycle (anagen to catagen) of goat, comparing the primary hair follicle with the secondary hair follicle. There were 13,769 and 12,240 unigenes assembled from the reads obtained from primary hair follicle and secondary hair follicle, respectively. Genes encoding keratin proteins and keratin-associated proteins were the 17905 Hair follicle transcriptome in goat ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 17904-17915 (2015) most highly expressed. A total of 5899 genes were differentially expressed in anagen vs catagen primary hair follicles, with 532 genes up-regulated and 5367 genes down-regulated. A total of 5208 genes were differentially expressed in anagen vs catagen secondary hair follicle, including 545 genes that were up-regulated and 4663 genes that were down-regulated. Numerous hair growth genes are expressed in the goat hair follicle, of which 73 genes showed co-up-regulation in both hair follicles during the anagen stage. Many of these up-regulated genes, such as STC2, VEGFR, and ROR2, are known to be transfactors in the process of cell differentiation and in the cell cycle. The differential gene expression profiles between primary hair follicles and secondary hair follicles obtained provide a foundation for future studies examining the network of gene expression controlling hair growth cycle in Cashmere goat.