No abstract

Contact with food animals was associated with SARS-CoV infection in the People’s Republic of China.

Vibrio cholerae is the causative agent of cholera, which continues to be a major public health concern in Asia, Africa and Latin America. The bacterium can persist outside the human host and alternates between planktonic and biofilm community lifestyles. Transition between the different lifestyles is mediated by multiple signal transduction pathways including quorum sensing. Expression of the Zn-metalloprotease haemagglutinin (HA)/protease is subject to a dual regulation which involves the quorum-sensing regulator HapR and the cAMP receptor protein. In a previous study, we observed that a mutant defective in the cAMP-receptor protein (CRP) expressed lower levels of HapR. To further investigate the role of CRP in modulating HapR and other signal transduction pathways, we performed global gene expression profiling of a Dcrp mutant of El Tor biotype V. cholerae. Here we show that CRP is required for the biosynthesis of cholera autoinducer 1 (CAI-1) and affects the expression of multiple HapR-regulated genes. As expected, the Dcrp mutant produced more cholera toxin and enhanced biofilm. Expression of flagellar genes, reported to be affected in DhapR mutants, was diminished in the Dcrp mutant. However, an epistasis analysis indicated that cAMP-CRP affects motility by a mechanism independent of HapR. Inactivation of crp inhibited the expression of multiple genes reported to be strongly induced in vivo and to affect the ability of V. cholerae to colonize the small intestine and cause disease. These genes included ompU, ompT and ompW encoding outer-membrane proteins, the alternative sigma factor s E required for intestinal colonization, and genes involved in anaerobic energy metabolism. Our results indicate that CRP plays a crucial role in the V. cholerae life cycle by affecting quorum sensing and multiple genes required for survival of V. cholerae in the human host and the environment.

This document has been produced by the Severe Acute Respiratory Syndrome (SARS) Epidemiology Working Group and the participants at the Global Meeting on the Epidemiology of SARS, 16-17 May 2003.

Cyclic dinucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Although phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP-specific PDEs in V. cholerae (herein designated as V-cGAP1/2/3). V-cGAPs are HD-GYP domain-containing proteins and specifically break 3′3′-cGAMP, but not other forms of cGAMP. 3′3′-cGAMP is first linearized by all three V-cGAPs to produce 5′-pApG, which is further hydrolyzed into 5′-ApG by V-cGAP1. In this two-step reaction, V-cGAP1 functions as both a PDE and a 5′-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3′3′-cGAMP suggests the existence of specific PDEs for other cGAMPs, including 2′3′-cGAMP in mammalian cells. The absolute requirement of the GYP motif for 3′3′-cGAMP degradation suggests that HD domain-containing PDEs in eukaryotes are probably unable to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3′3′-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize a similar strategy. These results will provide valuable information for identification and characterization of mammalian 2′3′-cGAMP-specific PDEs in future studies.

Vibrio fluvialis is an emerging foodborne pathogen of increasing public health concern. The mechanism(s) that contribute to the bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SSs) are known to contribute to bacterial pathogenicity by exerting toxic effects on host cells or competing bacterial species. In this study, we characterized the genetic organization and prevalence of two T6SS gene clusters (VflT6SS1 and VflT6SS2) in V. fluvialis. VflT6SS2 harbors three “orphan” hcp-vgrG modules and was more prevalent than VflT6SS1 in our isolates. We showed that VflT6SS2 is functionally active under low (25°C) and warm (30°C) temperatures by detecting the secretion of a T6SS substrate, Hcp. This finding suggests that VflT6SS2 may play an important role in the survival of the bacterium in the aquatic environment. The secretion of Hcp is growth phase-dependent and occurs in a narrow range of the growth phase (OD600 from 1.0 to 2.0). Osmolarity also regulates the function of VflT6SS2, as evidenced by our finding that increasing salinity (from 170 to 855 mM of NaCl) and exposure to high osmolarity KCl, sucrose, trehalose, or mannitol (equivalent to 340 mM of NaCl) induced significant secretion of Hcp under growth at 30°C. Furthermore, we found that although VflT6SS2 was inactive at a higher temperature (37°C), it became activated at this temperature if higher salinity conditions were present (from 513 to 855 mM of NaCl), indicating that it may be able to function under certain conditions in the infected host. Finally, we showed that the functional expression of VflT6SS2 is associated with anti-bacterial activity. This activity is Hcp-dependent and requires vasH, a transcriptional regulator of T6SS. In sum, our study demonstrates that VflT6SS2 provides V. fluvialis with an enhanced competitive fitness in the marine environment, and its activity is regulated by environmental signals, such as temperature and osmolarity.

No abstract

Inactivation of the quorum-sensing regulator HapR causes Vibrio cholerae El Tor biotype strain C7258 to adopt a rugose colonial morphology that correlates with enhanced biofilm formation. V. cholerae mutants lacking the cyclic AMP (cAMP) receptor protein (CRP) produce very little HapR, which results in elevated expression of Vibrio exopolysaccharide (vps) genes and biofilm compared to the wild type. However, ⌬crp mutants still exhibited smooth colonial morphology and expressed reduced levels of vps genes compared to isogenic hapR mutants. In this study we demonstrate that deletion of crp and cya (adenylate cyclase) converts a rugose ⌬hapR mutant to a smooth one. The smooth ⌬hapR ⌬crp and ⌬hapR ⌬cya double mutants could be converted back to rugose by complementation with crp and cya, respectively. CRP was found to enhance the expression of VpsR, a strong activator of vps expression, but to diminish transcription of VpsT. Ectopic expression of VpsR in smooth ⌬hapR ⌬crp and ⌬hapR ⌬cya double mutants restored rugose colonial morphology. Lowering intracellular cAMP levels in a ⌬hapR mutant by the addition of glucose diminished VpsR expression and colonial rugosity. On the basis of our results, we propose a model for the regulatory input of CRP on exopolysaccharide biosynthesis.Cholera is a paradigm waterborne disease caused by Vibrio cholerae serogroups O1 and O139, which continue to cause seasonal outbreaks in heavily populated regions in Asia, Africa, and Latin America. V. cholerae is transmitted through contaminated food and drinking water as well as person to person through the fecal-oral route. The existence of an aquatic reservoir of serogroup O1 and O139 toxigenic strains has not been established. However, their capacity to survive and persist in estuarine and brackish waters is widely accepted (6,8,22). V. cholerae has been shown to alternate between a free-swimming planktonic lifestyle and biofilm communities attached to biotic and abiotic surfaces. In the biofilm lifestyle, V. cholerae has been found in association with phytoplankton and zooplankton (11,12). In addition, large clumps of aggregated partially dormant V. cholerae cells can be detected in surface water as biofilms that resist cultivation in conventional microbiological media (7). These aggregates can be recovered as virulent V. cholerae cells by inoculation into rabbit ileal loops (7).It has been shown that V. cholerae cells in biofilm communities are more resistant to environmental stresses and protozoan predation (11,12,13,18,31). The V. cholerae rugose colonial morphology variant initially described by White (27) has been shown to produces more exopolysaccharide and biofilm than the smooth colonial variant (28). Furthermore, the rugose variant has been demonstrated to be more resistant to chlorinated water (20,21,28) and osmotic and oxidative stresses than the smooth variant (26, 28).The rugose colonial morphology has been demonstrated to be a highly multifactorial phenotype subject to complex regulatory mechanisms. The genes responsible for...