SummaryThe facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA , the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfAdependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes . These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.

Geostationary infrared (IR) satellite data are used to provide estimates of the symmetric and total low-level wind fields in tropical cyclones, constructed from estimations of an azimuthally averaged radius of maximum wind (RMAX), a symmetric tangential wind speed at a radius of 182 km (V182), a storm motion vector, and the maximum intensity (VMAX). The algorithm is derived using geostationary IR data from 405 cases from 87 tropical systems in the Atlantic and east Pacific Ocean basins during the 1995-2003 hurricane seasons that had corresponding aircraft data available. The algorithm is tested on 50 cases from seven tropical storms and hurricanes during the 2004 season. Aircraft-reconnaissance-measured RMAX and V182 are used as dependent variables in a multiple linear regression technique, and VMAX and the storm motion vector are estimated using conventional methods. Estimates of RMAX and V182 exhibit mean absolute errors (MAEs) of 27.3 km and 6.5 kt, respectively, for the dependent samples. A modified combined Rankine vortex model is used to estimate the one-dimensional symmetric tangential wind field from VMAX, RMAX, and V182. Next, the storm motion vector is added to the symmetric wind to produce estimates of the total wind field. The MAE of the IR total wind retrievals is 10.4 kt, and the variance explained is 53%, when compared with the two-dimensional wind fields from the aircraft data for the independent cases.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious disease in immunocompromised individuals, pregnant women, and neonates. Bacterial virulence is mediated by the expression of specific gene products that facilitate entry into host cells and enable bacterial replication; the majority of these gene products are regulated by a transcriptional activator known as PrfA. L. monocytogenes strains containing prfA E77K or prfA G155S mutations exhibit increased expression of virulence genes in broth culture and are hypervirulent in mice. To define the scope of the influences of the prfA E77K and prfA G155S mutations on L. monocytogenes pathogenesis, multiple aspects of bacterial invasion and intracellular growth were examined. Enhanced bacterial invasion of host epithelial cells was dependent on the expression of a number of surface proteins previously associated with invasion, including InlA, InlB, and ActA. In addition to these surface proteins, increased production of the hly-encoded secreted hemolysin listeriolysin O (LLO) was also found to significantly enhance bacterial invasion into epithelial cell lines for both prfA mutant strains. Although prfA E77K and prfA G155S strains were similar in their invasive phenotypes, the infection of epithelial cells with prfA E77K strains resulted in host cell plasma membrane damage, whereas prfA G155S strains did not alter plasma membrane integrity. Bacterial infection of human epithelial cells, in which the production of LLO is not required for bacterial entry into the cytosol, indicated that prfA E77K cytotoxic effects were mediated via LLO. Both prfA E77K and prfA G155S strains were more efficient than wild-type bacteria in gaining access to the host cell cytosol and in initiating the polymerization of host cell actin, and both were capable of mediating LLO-independent lysis of host cell vacuoles in cell lines for which L. monocytogenes vacuole disruption normally requires LLO activity. These experiments illuminate the diverse facets of L. monocytogenes pathogenesis that are significantly enhanced by the constitutive activation of PrfA via prfA mutations and underscore the critical role of this protein in promoting L. monocytogenes virulence.

Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose. Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand. The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose. Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster. A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself. A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation. Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of beta-glucosides in other bacteria. Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon. These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L. monocytogenes.