Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised ironoverloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.Vibrio vulnificus is an opportunistic human pathogen that preferentially affects patients that have underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta-thalassemia, and heavy alcohol drinkers (6,19,32). This bacterium frequently causes fatal primary septicemia with very rapid progress, resulting in a mortality rate of more than 50% within a few days (6,12,20,33). The common theme in most of these patients is that iron is present at higher than physiological levels. Some of the confirmed or putative virulence factors required for in vivo survival and growth of V. vulnificus include capsule (30, 38), protease (8), flagella (9, 24), pili (22), and siderophore vulnibactin (14). More recently it was reported that antibodies against the V. vulnificus HlyU protein were present in serum of convalescent patients who survived V. vulnificus septicemia and that an hlyU mutation resulted in a 53-fold increase of the 50% lethal dose (LD 50 ) of V. vulnificus in the iron-normal mouse model (7). It was already known that the Vibrio cholerae HlyU predicted protein, containing a putative helix-turn-helix motif (34), activated the expression of the hemolysin gene hlyA as well as a hemolysincoregulated protein gene hcp (35,36). The HlyU homologue in V. vulnificus was reported to up-regulate the cytolysin/hemolysin gene vvhA and a gene encoding an elastolytic protease (7), while the hlyU mutant showed a significant decrease in cytotoxic activity to HeLa cells. It is worth mentioning that the purified V. vulnificus cytolysin (VvhA) exhibited cytolytic activity against Chinese hamster ovary cells (5) and could kill mice at low dosages by intravenous administration (10). However, when the vvhA gene was mutated, the VvhA cytolysinnegative strain still showed the same LD 50 in both iron-normal and iron-overloaded mouse models (37). Furthermore, the cytotoxicity for Hep-2 cells in the cytolysin-negative strain was comparable to that of the wild type (4). There...

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1?2 M the growth curves were similar to those of its wild-type control, at 1?5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.

Radiation therapy is a source of tumor antigen release that has the potential to serve as an endogenous tumor vaccination event. In preclinical models radiation therapy synergizes with checkpoint inhibitors to cure tumors via CD8 T cell responses. To evaluate the immune response initiated by radiation therapy, we used a range of approaches to block the pre-existing immune response artifact initiated by tumor implantation. We demonstrate that blocking immune responses at tumor implantation blocks development of a tumor-resident antigen specific T cell population and prevents tumor cure by radiation therapy combined with checkpoint immunotherapy. These data demonstrate that this treatment combination relies on a pre-existing immune response to cure tumors, and may not be a solution for patients without pre-existing immunity.

Vibrio vulnificus multiplies rapidly in host tissues under iron-overloaded conditions. To understand the effects of iron in the physiology of this pathogen, we performed a genome-wide transcriptional analysis of V. vulnificus growing at three different iron concentrations, i.e., iron-limiting [Trypticase soy broth with 1.5% NaCl (TSBS) plus ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)], low-iron (1 g Fe/ml; TSBS), and iron-rich (38 g Fe/ml; TSBS plus ferric ammonium citrate) concentrations. A few genes were upregulated under the last two conditions, while several genes were expressed differentially under only one of them. A gene upregulated under both conditions encodes the outer membrane porin, OmpH, while others are related to the biosynthesis of amino sugars. An ompH mutant showed sensitivity to sodium dodecyl sulfate (SDS) and polymyxin B and also had a reduced competitive index compared with the wild type in the iron-overloaded mice. Under iron-limiting conditions, two of the TonB systems involved in vulnibactin transport were induced. These genes were essential for virulence in the iron-overloaded mice inoculated subcutaneously, underscoring the importance of active iron transport in infection, even under the high-iron conditions of this animal model. Furthermore, we demonstrated that a RyhB homologue is also essential for virulence in the iron-overloaded mouse. This novel information on the role of genes induced under iron limitation in the iron-overloaded mouse model and the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions shed light on the many strategies used by this pathogen to multiply rapidly in the susceptible host.Vibrio vulnificus is an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels (17,43). This estuarine bacterium preferentially affects individuals with underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta thalassemia. In humans, this pathogen frequently causes fatal primary septicemia with a very rapid progress, resulting in a mortality rate of Ͼ50% within a few days (17). The common theme in susceptible individuals appears to be high serum iron levels; however, changes in the innate immune response in the host cannot be discarded (17,19).Iron is an important element in almost all known organisms. In many bacterial pathogens, the ability to acquire this metal from the host is an important virulence factor (7). Seminal work in Oliver's laboratory showed the importance of iron in V. vulnificus infections (74), as the laboratory identified two siderophores, vulnibactin and a hydroxamate-type siderophore, both involved in iron acquisition (59). 70) studied the role of the siderophore vulnibactin in the uptake of iron from transferrin and hemoglobin and identified genes involved in vulnibactin biosynthesis and transport as well as the heme receptor and Fur, a regulator that represses several genes under iron-limiting conditions (13). More recently, K...