Paul A. Gulig

Molecular analysis of spv virulence genes of the salmonella virulence plasmids

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium. Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon‐poor media, and optimal expression is dependent on the katF locus. The cirulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Phage Therapy of Local and Systemic Disease Caused by Vibrio vulnificus in Iron-Dextran-Treated Mice

ABSTRACT Vibrio vulnificus is a gram-negative bacterium that contaminates filter-feeding shellfish such as oysters. After ingestion of contaminated oysters, predisposed people may experience highly lethal septicemia. Contamination of wounds with the bacteria can result in devastating necrotizing fasciitis, which can progress to septicemia. The extremely rapid progression of these diseases can render antibiotic treatment ineffective, and death is a frequent outcome. In this study, we examined the potential use of bacteriophages as therapeutic agents against V. vulnificus in an iron-dextran-treated mouse model of V. vulnificus infection. Mice were injected subcutaneously with 10 times the lethal dose of V. vulnificus and injected intravenously, either simultaneously or at various times after infection, with phages. Treatment of mice with phages could prevent death; systemic disease, as measured by CFU per gram of liver and body temperature; and local disease, as measured by CFU per gram of lesion material and histopathologic analysis. Two different phages were effective against three different V. vulnificus strains with various degrees of virulence, while a third phage that required the presence of seawater to lyse bacteria in vitro was ineffective at treating mice. Optimum protection required that the phages be administered within 3 h of bacterial inoculation at doses as high as 108 PFU. One of the protective phages had a half-life in blood of over 2 h. These results demonstrate that bacteriophages have therapeutic potential for both localized and systemic infections caused by V. vulnificus in animals. This model should be useful in answering basic questions regarding phage therapy.

Virulence plasmids of Salmonella typhimurium and other salmonellae.

Related high molecular weight plasmids of several serotypes and species of Salmonella have been associated with virulence in a variety of animal models of infection. The primary virulence plasmid phenotype is in the ability of salmonellae to spread beyond the initial site of infection, the intestines. The mechanism of this plasmid-mediated invasive infection has not been identified, but may be a complex interaction in the host-pathogen relationship. A common region of the salmonella plasmids has been associated with virulence, and specific virulence genes and their products are now being identified; however, much is yet to be accomplished in this field. The combined analysis of pathogenesis and genetics associated with the salmonella virulence plasmids may identify new systems of bacterial virulence and the genetic basis for this virulence.

Pathogenesis of Infection by Clinical and Environmental Strains of Vibrio vulnificus in Iron-Dextran-Treated Mice

ABSTRACT Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality. V. vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six V. vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s.c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 108CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 105-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V. vulnificus.

Construction and Phenotypic Evaluation of a Vibrio vulnificus vvpE Mutant for Elastolytic Protease

ABSTRACT Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificusinfections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructingvvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.

Virulence Plasmid-Borne spvB and spvC Genes Can Replace the 90-Kilobase Plasmid in Conferring Virulence to Salmonella enterica Serovar Typhimurium in Subcutaneously Inoculated Mice

In a mouse model of systemic infection, the spv genes carried on the Salmonella enterica serovar Typhimurium virulence plasmid increase the replication rate of salmonellae in host cells of the reticuloendothelial system, most likely within macrophages. A nonpolar deletion in the spvB gene greatly decreased virulence but could not be complemented by spvB alone. However, a low-copy-number plasmid expressing spvBC from a constitutive lacUV5 promoter did complement the spvB deletion. By examining a series of spv mutations and cloned spv sequences, we deduced that spvB and spvC could be sufficient to confer plasmid-mediated virulence to S. enterica serovar Typhimurium. The spvBC-bearing plasmid was capable of replacing all of the spv genes, as well as the entire virulence plasmid, of serovar Typhimurium for causing systemic infection in BALB/c mice after subcutaneous, but not oral, inoculation. A point mutation in the spvBC plasmid preventing translation but not transcription of spvC eliminated the ability of the plasmid to confer virulence. Therefore, it appears that both spvB and spvC encode the principal effector factors for Spv- and plasmid-mediated virulence of serovar Typhimurium.

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid

The 90‐kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14‐kb virulence region to reduce this virulence region to 7.8kb. The 7.8‐kb virulence region subcloned into a low copy‐number vector conferred a wild‐type level of splenic infection to virulence plasmid‐cured S. typhimurium and conferred essentially a wild‐type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild‐type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho‐dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8‐kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid‐encoded virulence genes of S. typhimurium.

Hepcidin-Induced Hypoferremia Is a Critical Host Defense Mechanism Against the Siderophilic Bacterium Vibrio vulnificus

Hereditary hemochromatosis, an iron overload disease caused by a deficiency in the iron-regulatory hormone hepcidin, is associated with lethal infections by siderophilic bacteria. To elucidate the mechanisms of this susceptibility, we infected wild-type and hepcidin-deficient mice with the siderophilic bacterium Vibrio vulnificus and found that hepcidin deficiency results in increased bacteremia and decreased survival of infected mice, which can be partially ameliorated by dietary iron depletion. Additionally, timely administration of hepcidin agonists to hepcidin-deficient mice induces hypoferremia that decreases bacterial loads and rescues these mice from death, regardless of initial iron levels. Studies of Vibrio vulnificus growth ex vivo show that high iron sera from hepcidin-deficient mice support extraordinarily rapid bacterial growth and that this is inhibited in hypoferremic sera. Our findings demonstrate that hepcidin-mediated hypoferremia is a host defense mechanism against siderophilic pathogens and suggest that hepcidin agonists may improve infection outcomes in patients with hereditary hemochromatosis or thalassemia.

USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus

ABSTRACT Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Kochs postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.

Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers.

ABSTRACT Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.