Calcium Enhances Bile Salt-Dependent Virulence Activation in Vibrio cholerae.

Vibrio cholerae is the causative bacteria of the diarrheal disease cholera, but it also persists in aquatic environments, where it displays an expression profile that is distinct from that during infection. Upon entry into the host, a tightly regulated circuit coordinates the induction of two major virulence factors: cholera toxin and a toxin-coregulated pilus (TCP). It has been shown that a set of bile salts, including taurocholate, serve as host signals to activate V. cholerae virulence through inducing the activity of the transmembrane virulence regulator TcpP. In this study, we investigated the role of calcium, an abundant mental ion in the gut, in the regulation of virulence. We show that whereas Ca2+ alone does not affect virulence, Ca2+ enhances bile salt-dependent virulence activation for V. cholerae The induction of TCP by murine intestinal contents is counteracted when Ca2+ is depleted by the high-affinity calcium chelator EGTA, suggesting that the calcium present in the gut is a relevant signal for V. cholerae virulence induction in vivo We further show that Ca2+ enhances virulence by promoting bile salt-induced TcpP-TcpP interaction. Moreover, fluorescence recovery after photobleaching (FRAP) analysis demonstrated that exposure to bile salts and Ca2+ together decreases the recovery rate for fluorescently labeled TcpP, but not for another inner membrane protein (TatA). Together, these data support a model in which physiological levels of Ca2+ may result in altered bile salt-induced TcpP protein movement and activity, ultimately leading to an increased expression of virulence.

Yield and cold storage of Trichoderma conidia is influenced by substrate pH and storage temperature.

In this study we examined the influence of the ambient pH during morphogenesis on conidial yield of Trichoderma sp. "atroviride B" LU132 and T. hamatum LU593 and storage at low temperatures. The ambient pH of the growth media had a dramatic influence on the level of Trichoderma conidiation and this was dependent on the strain and growth media. On malt-extract agar, LU593 yield decreased with increasing pH (3-6), whereas yield increased with increasing pH for LU132. During solid substrate production the reverse was true for LU132 whereby yield decreased with increasing pH. The germination potential of the conidia decreased significantly over time in cold storage and the rate of decline was a factor of the strain, pH during morphogenesis, growth media, and storage temperature.

Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization.

Vibrio cholerae causes human infection through ingestion of contaminated food and water, leading to the devastating diarrheal disease cholera. V. cholerae forms matrix-encased aggregates, known as biofilms, in the native aquatic environment. While the formation of V. cholerae biofilms has been well studied, little is known about the dispersal from biofilms, particularly upon entry into the host. In this study, we found that the exposure of mature biofilms to physiologic levels of the bile salt taurocholate, a host signal for the virulence gene induction of V. cholerae, induces an increase in the number of detached cells with a concomitant decrease in biofilm mass. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix. The inhibition of protein synthesis did not alter rates of detachment, suggesting that V. cholerae undergoes a passive dispersal. Cell-free media from taurocholate-exposed biofilms contains a larger amount of free polysaccharide, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, we found that V. cholerae is only able to induce virulence in response to taurocholate after exit from the biofilm. Thus, we propose a model in which V. cholerae ingested as a biofilm has coopted the host-derived bile salt signal to detach from the biofilm and go on to activate virulence.

Quorum sensing regulatory cascades control Vibrio fluvialis pathogenesis.

Quorum sensing (QS) is a process by which individual bacteria are able to communicate with one another, thereby enabling the population as a whole to coordinate gene regulation and subsequent phenotypic outcomes. Communication is accomplished through production and detection of small molecules in the extracellular milieu. In many bacteria, particularly Vibrio species, multiple QS systems result in multiple signals, as well as cross talk between systems. In this study, we identify two QS systems in the halophilic enteric pathogen Vibrio fluvialis: one acyl-homoserine lactone (AHL) based and one CAI-1/AI-2 based. We show that a LuxI homolog, VfqI, primarily produces 3-oxo-C10-HSL, which is sensed by a LuxR homolog, VfqR. VfqR-AHL is required to activate vfqI expression and autorepress vfqR expression. In addition, we have shown that similar to that in V. cholerae and V. harveyi, V. fluvialis produces CAI-1 and AI-2 signal molecules to activate the expression of a V. cholerae HapR homolog through LuxO. Although VfqR-AHL does not regulate hapR expression, HapR can repress vfqR transcription. Furthermore, we found that QS in V. fluvialis positively regulates production of two potential virulence factors, an extracellular protease and hemolysin. QS also affects cytotoxic activity against epithelial tissue cultures. These data suggest that V. fluvialis integrates QS regulatory pathways to play important physiological roles in pathogenesis.

Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae.

Toxin-antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.

Microbiota talks cholera out of the gut.

In a recent Nature paper, Hsiao et al., 2014 examine microbiota contribution during and after cholera and identify commensals correlated with healing and reconstitution of the microbial community. One particular species may use intercellular communication to stymie Vibrio cholerae pathogenesis, indicating how the microbiota can restrict pathogens.

The NorR regulon is critical for Vibrio cholerae resistance to nitric oxide and sustained colonization of the intestines.

UNLABELLED: Vibrio cholerae, the cause of an often fatal infectious diarrhea, remains a large global public health threat. Little is known about the challenges V. cholerae encounters during colonization of the intestines, which genes are important for overcoming these challenges, and how these genes are regulated. In this study, we examined the V. cholerae response to nitric oxide (NO), an antibacterial molecule derived during infection from various sources, including host inducible NO synthase (iNOS). We demonstrate that the regulatory protein NorR regulates the expression of NO detoxification genes hmpA and nnrS, and that all three are critical for resisting low levels of NO stress under microaerobic conditions in vitro. We also show that prxA, a gene previously thought to be important for NO detoxification, plays no role in NO resistance under microaerobic conditions and is upregulated by H(2)O(2), not NO. Furthermore, in an adult mouse model of prolonged colonization, hmpA and norR were important for the resistance of both iNOS- and non-iNOS-derived stresses. Our data demonstrate that NO detoxification systems play a critical role in the survival of V. cholerae under microaerobic conditions resembling those of an infectious setting and during colonization of the intestines over time periods similar to that of an actual V. cholerae infection. IMPORTANCE: Little is known about what environmental stresses Vibrio cholerae, the etiologic agent of cholera, encounters during infection, and even less is known about how V. cholerae senses and counters these stresses. Most prior studies of V. cholerae infection relied on the 24-h infant mouse model, which does not allow the analysis of survival over time periods comparable to that of an actual V. cholerae infection. In this study, we used a sustained mouse colonization model to identify nitric oxide resistance as a function critical for the survival of V. cholerae in the intestines and further identified the genes responsible for sensing and detoxifying this stress.

Characterisation of a Trichoderma hamatum monooxygenase gene involved in antagonistic activity against fungal plant pathogens.

A monooxygenase gene was isolated from a biocontrol strain of Trichoderma hamatum and its role in biocontrol was investigated. The gene had homologues in other fungal genomes, but was not closely related to any fully characterised gene. The T. hamatum monooxygenase gene was expressed specifically in response to the plant pathogens Sclerotinia sclerotiorum, Sclerotinia minor and Sclerotium cepivorum, but not in response to Botrytis cinerea or T. hamatum. Expression of the gene did not occur until contact had been made between the two fungal species. Homologues in T. atroviride and T. virens showed similar expression patterns. Expression of the gene in response to S. sclerotiorum was influenced by pH, with a peak of expression at pH 4, and was subject to nitrogen catabolite repression. Disruption of the monooxygenase gene did not affect the growth or morphology of T. hamatum, but caused a decrease in its ability to inhibit the growth and sclerotial production of S. sclerotiorum. The monooxygenase gene had a role in the antagonistic activity of Trichoderma species against specific fungal plant pathogens and is therefore a potentially important factor in biocontrol by Trichoderma species.