The domain of unknown function (DUF) YP_001302112.1, a protein secreted by the human intestinal microbita, has been determined by NMR and represents the first structure for the Pfam PF14466. Its NMR structure is classified as a new fold, which nonetheless shows limited similarities with representatives of the PLAT/LH2 domains from PF01477 and the C2 domains from PF00168, both of which bind Ca(2+) for their physiological functions. Further experiments revealed affinity of YP_001302112.1 for Ca(2+) , and the NMR structure in the presence of CaCl2 was better defined than that of the apo-protein. Overall, these NMR structures establish a new connection between structural representatives from two widely different Pfams that include the calcium-binding domain of a sialidase from Vibrio cholera and the α-toxin from Clostridium perfrigens, whereby these two proteins have only 7% sequence identity. Furthermore, it provides information toward the functional annotation of YP_001302112.1, based on its capacity to bind Ca(2+) , and thus adds to the structural and functional coverage of the protein sequence universe.

Introduction The number of reports of intestinal infections caused by Aeromonas spp. has increased significantly in recent years. In most clinical laboratories, identification of these bacteria is carried out by general phenotypic tests that sometimes do not accurately differentiate Aeromonas and Vibrio. Methods A duplex-polymerase chain reaction (PCR) was developed directed to 2 targets identifying Aeromonas spp. pathogenic to humans. Results The duplex-PCR results were reproducible and specific for Aeromonas spp. pathogenic to humans. Conclusions This method will allow differentiation between Vibrio and Aeromonas spp. in patients with in cholera-like symptoms and can also be used in water quality monitoring.

The D1 dopamine receptor (D1R) has been proposed to form a hetero-oligomer with the D2 dopamine receptor (D2R), which in turn results in a complex that couples to PLC-mediated intracellular calcium release. We have sought to elucidate the pharmacology and mechanism of action of this putative signaling pathway. Dopamine dose response curves assaying intracellular calcium mobilization in cells heterologously expressing the D1 and D2 subtypes either alone or in combination, and employing subtype selective ligands, revealed that concurrent stimulation is required for coupling. Surprisingly, characterization of a putative D1-D2 heteromer-selective ligand, SKF83959, found no stimulation of calcium release, but a broad range of cross-reactivity with other GPCRs. In contrast, SKF83959 appeared to be an antagonist of calcium mobilization. Over-expression of Gqα with the D1 and D2 DARs enhanced the dopamine-stimulated calcium response. However, this was also observed in cells expressing Gqα with only the D1R. Inactivation of Gi or Gs with pertussis or cholera toxin, respectively, was found to largely, but not entirely reduce the calcium response in D1R and D2R co-transfected cells. Moreover, we found that sequestration of Gβγ subunits through over-expression of GRK2 mutants either completely or largely eliminated dopamine-stimulated calcium mobilization. Our data suggests that the mechanism of D1R-D2R-mediated calcium signaling involves more than receptor-mediated Gq protein activation, may largely involve downstream signaling pathways, and may not be completely heteromer-specific. In addition SKF83959 may not exhibit selective activation of D1-D2 heteromers, and its significant cross-reactivity to other receptors warrants careful interpretation of its use in vivo.

We previously found that inhibition of the TCA cycle, either through mutations or chemical inhibition, increased toxT transcription in Vibrio cholerae. In this study, we found that the addition of malonate, an inhibitor of succinate dehydrogenase (SDH), decreased toxT transcription in V. cholerae, an observation inconsistent with the previous pattern observed. Unlike another SDH inhibitor, 2-thenoyltrifluoroacetone (TTFA), which increased toxT transcription and slightly inhibited V. cholerae growth, malonate inhibited toxT transcription in both the wild-type strain and TCA cycle mutants, suggesting malonate-mediated inhibition of virulence gene expression is independent to TCA cycle activity. Addition of malonate also inhibited ctxB and tcpA expressions but did not affect aphA, aphB, tcpP and toxR expressions. Malonate inhibited cholera toxin (CT) production in both V. cholerae classical biotype strains O395N1 and CA401, and El Tor biotype strain, N16961. Consistent with previous reports, we confirmed that these strains of V. cholerae did not utilize malonate as a primary carbon source. However, we found that the addition of malonate to the growth medium stimulated V. cholerae growth. All together, these results suggest that metabolizing malonate as a nutrient source negatively affects virulence gene expression in V. cholerae.

BACKGROUND:

Pasteurella multocida is the etiologic agent of fowl cholera, a highly contagious and severe disease of poultry causing significant mortality and morbidity throughout the world. All types of poultry are susceptible to fowl cholera. Turkeys are most susceptible to the peracute/acute forms of the disease while chickens are most susceptible to the acute and chronic forms of the disease. The whole genome of the Pm70 strain of P. multocida was sequenced and annotated in 2001. The Pm70 strain is not virulent to chickens and turkeys. In contrast, strains X73 and P1059 are highly virulent to turkeys, chickens, and other poultry species. In this study, we sequenced the genomes of P. multocida strains X73 and P1059 and undertook a detailed comparative genome analysis with the avirulent Pm70 strain. The goal of this study was to identify candidate genes in the virulent strains that may be involved in pathogenicity of fowl cholera disease.

RESULTS:

Comparison of virulent versus avirulent avian P. multocida genomes revealed 336 unique genes among the P1059 and/or X73 genomes compared to strain Pm70. Genes of interest within this subset included those encoding an L-fucose transport and utilization system, several novel sugar transport systems, and several novel hemagglutinins including one designated PfhB4. Additionally, substantial amino acid variation was observed in many core outer membrane proteins and single nucleotide polymorphism analysis confirmed a higher dN/dS ratio within proteins localized to the outer membrane.

CONCLUSIONS:

Comparative analyses of highly virulent versus avirulent avian P. multocida identified a number of genomic differences that may shed light on the ability of highly virulent strains to cause disease in the avian host, including those that could be associated with enhanced virulence or fitness.

Abstract Urban and rural areas have distinctive health problems, which require consideration. To examine sociocultural features of cholera and its community context, a semi-structured explanatory model interview based on vignettes depicting typical clinical features of cholera was used to interview 379 urban and rural respondents in Western Kenya. Findings included common and distinctive urban and rural ideas about cholera, and its prevention and treatment. The three most commonly perceived causes among urban and rural respondents collectively were drinking contaminated water, living in a dirty environment and lacking latrines. However, a dirty environment and flies were more prominently perceived causes among urban respondents. Rural respondents were less likely to identify additional symptoms and more likely to identify biomedically irrelevant perceived causes of cholera. Oral rehydration therapy was the most frequently reported home treatment. Health facilities were recommended unanimously at both sites. For prevention, rural respondents were more likely to suggest medicines, and urban respondents were more likely to suggest health education and clean food. Findings indicate community priority, demand for and potential effectiveness of enhanced efforts to control cholera in Western Kenya, and they suggest strategies that are particularly well suited for control of cholera in urban and rural areas.

The urinary bladder is innervated by parasympathetic preganglionic neurons (PPNs) that express μ-opioid receptors (MOR) in the sacral parasympathetic nucleus (SPN) at lumbosacral segments L6-S1. The SPN also contains endomorphin 2 (EM2)-immunoreactive (IR) fibers and terminals. EM2 is the endogenous ligand of MOR. In the present study, retrograde tract-tracing with cholera toxin subunit b (CTb) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) via the pelvic nerve combined with immunohistochemical staining for EM2 and MOR to identify PPNs within the SPN as well as synaptic connections between the EM2-IR terminals and MOR-expressing PPNs in the SPN of the rat. After CTb was injected into the pelvic nerve, CTb retrogradely labeled neurons were almost exclusively located in the lateral part of the intermediolateral gray matter at L6-S1 of the lumbosacral spinal cord. All of the them also expressed MOR. EM2-IR terminals formed symmetric synapses with MOR-IR, WGA-HRP-labeled and WGA-HRP/MOR double-labeled neuronal cell bodies and dendrites within the SPN. These results provided morphological evidence that EM2-containing axon terminals formed symmetric synapses with MOR-expressing PPNs in the SPN. The present results also show that EM2 and MOR might be involved in both the homeostatic control and information transmission of micturition.

We have detected biological toxins using localized surface plasmon resonance (LSPR) and synthetic glycosyl ceramides (β-lactoside, globosyl trisaccharide (Gb3), or GM1 pentasaccharide) attached to gold (Au) nanoparticles. The particle diameters ranged from 5-100 nm. The detection sensitivity for three toxins (ricin, Shiga toxin, and cholera toxin) was found to depend not only on the attached glycoside but also on the diameter of the Au nanoparticles. For the detection of ricin, the 20-nm β-lactoside-coated Au nanoparticle exhibited the highest LSPR response, whereas 40-nm Gb3- and GM1-coated Au nanoparticles gave the best results for Shiga toxin and cholera toxin, respectively. In addition, a blocking process on the nanoparticle surface greatly improved the detection sensitivity for cholera toxin. The LSPR system enabled us to detect ricin at 30 ng/mL, Shiga toxin at 10 ng/mL, and the cholera toxin at 20 ng/mL.

The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.

Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.