Two tonB systems function in iron transport in Vibrio anguillarum, but only one is essential for virulence

We have identified two functional tonB systems in the marine fish pathogen Vibrio anguillarum, tonB1 and tonB2. Each of the tonB genes is transcribed in an operon with the cognate exbB and exbD genes in response to iron limitation. Only tonB2 is essential for transport of ferric anguibactin and virulence.     

Iron-vibriobactin transport system is not required for virulence of Vibrio cholerae.

The possible requirement of a functional siderophore (vibriobactin)-mediated iron transport system in the pathogenicity of Vibrio cholerae was determined. Two mutants of V. cholerae defective in the iron-vibriobactin transport system were examined for their ability to multiply and elicit diarrhea in infant mice. One mutant, 40130, was unable to synthesize vibriobactin. The second mutant, 1510, was able to synthesize, but not transport, the siderophore. Both mutants retained the ability to multiply and produce disease in the infant mouse, and virulence was indistinguishable from the parent strains. This indicates that a functional iron-vibriobactin transport system is not essential for cholera pathogenesis. These mutants, like the wild-type strains, were found to have a ferric citrate iron uptake system and could utilize citrate or asparagine for growth in low-iron medium. Compounds of this type may increase the availability of iron to V. cholerae in the host.     

Increased production of the siderophore anguibactin mediated by pJM1-like plasmids in Vibrio anguillarum.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.     

Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates

We have previously demonstrated the existence of Klebsiella pneumoniae clinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates.     

Detection and analysis of iron uptake components expressed by Acinetobacter baumannii clinical isolates

The Acinetobacter baumannii 19606 prototype strain produces a 78-kDa iron-regulated outer membrane protein immunologically related to FatA, which is required for iron acquisition by the fish pathogen Vibrio anguillarum via the anguibactin-mediated system. This A. baumannii strain also secretes histamine, a biosynthetic precursor of the siderophore anguibactin. In contrast, the A. baumannii 8399 clinical strain isolated in Oregon produces a siderophore and a putative 73-kDa iron-regulated outer membrane (OM73) receptor that are different from those produced by V. anguillarum and A. baumannii 19606. These observations suggest that different A. baumannii clinical isolates express unrelated iron uptake systems. This hypothesis is supported by differences in outer membrane protein profiles among A. baumannii isolates obtained from Oregon and Europe. The 19606 isolate and some European isolates expressed a FatA-like protein, while neither 19606 nor any of the European isolates expressed proteins related to OM73. Some European isolates failed to express FatA- and OM73-like proteins. All but one of the Oregon isolates expressed OM73-like proteins, while none of them contained a FatA-like protein. The presence of these proteins always correlated with the presence of the om73- and fatA-like genes in the cognate strains. While 19606 and a few European isolates produced histamine, none of the Oregon isolates had this capability. Interestingly, one strain each from the Oregon and European isolates did not express any of these products involved in iron acquisition, indicating that they could acquire iron through siderophore-mediated transport systems different from those expressed by the 19606 and 8399 clinical isolates.     

Characterization of a polysaccharide capsular antigen of septicemic Escherichia coli O115:K "V165" :F165 and evaluation of its role in pathogenicity.

Escherichia coli strains of serogroup O115:K(-):F165 have been associated with septicemia in calves and piglets. These strains express a capsular antigen referred to as K"V165" which inhibits agglutination of the O antigen by anti-O115 serum. We used hybrid transposon TnphoA mutants M48, 18b, and 2, and a spontaneous O-agglutinable mutant, 5131a, to evaluate the role of K"V165" in the pathogenicity of E. coli O115. Mutant M48 was as resistant to 90% rabbit serum and as virulent in day-old chickens as the parent strain 5131, mutants 18b and 5131a were less resistant to serum and less virulent in chickens, and mutant 2 was serum sensitive and avirulent. Analysis of outer membrane protein and lipopolysaccharide profiles failed to show any difference between the transposon mutants and the parent strain. In contrast, the spontaneous O-agglutinable mutant showed additional bands in the 16-kDa region of the polysaccharide ladder-like pattern. Mutants 2 and 5131a produced significantly less K"V165" capsular antigen than the parent strain, as demonstrated by a competitive enzyme-linked immunosorbent assay with adsorbed anti-K"V165" serum. In addition, electron microscopic analysis revealed that mutants 2 and 5131a had lost the capsular layer observed in the parent strain after fixation with glutaraldehyde-lysine. This capsule contained carbohydrate compounds and resembled an O-antigen capsule since it prevented O-antigen agglutination before the bacteria were heated at 100 degrees C and induced bacterial serum resistance. The capsule-defective mutants colonized the intestinal epithelium of experimentally infected gnotobiotic pigs but failed to induce clinical signs of septicemia. We concluded that E. coli strains of serogroup O115 expressed a polysaccharide capsular antigen which induced serum resistance and consequently contributed to the pathogenicity of the bacteria.     

Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity

Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.     

Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis

P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.     

Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes.

Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes. The localization of these epitopes on the O antigen is not known. We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V. anguillarum O2 (rfbVaO2) in Escherichia coli. E. coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V. ordalii and to V. anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V. anguillarum O2 O antigens. The recombinant strains were also protected from bactericidal killing by normal fish serum. Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V. ordalii O antigens. Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens. Similar results were obtained when pAM86 was transformed into E. coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E. coli rfb cluster. These data suggest that the epitope recognized by the MAb A16 is expressed by V. anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide. Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen. The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined.     

A K+ yptake protein, TrkA, is required for serum, protamine, and polymyxin B resistance in Vibrio vulnificus

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V. vulnificus and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium. A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.     

Four novel hemolysin genes of Vibrio anguillarum and their virulence to rainbow trout

Four nucleotide sequences showing homology to known hemolysin genes were cloned and sequenced from V. anguillarum strain H775-3. The four genes, vah2, vah3, vah4 and vah5, have open reading frames encoding polypeptides of 291, 690, 200 and 585 amino acid residues, respectively, with predicted molecular masses of 33, 75, 22 and 66KDa, respectively. VAH2 is most closely related to a putative hemolysin of Vibrio vulnificus YJ016 (89% identity). VAH3 is most closely related to a hemolysin-related protein in Vibrio cholerae O1 (68% identity). VAH4 is most closely related to a thermostable hemolysin in V. cholerae O1 (72% identity). VAH5 is most closely related to a putative hemolysin in V. cholerae O1 (73% identity). The purified hemolysin proteins showed hemolytic activities against erythrocyte of fish, sheep and rabbit. Four strains of V. anguillarum mutants were constructed, each deficient in one of the hemolysin genes. Each mutant was less virulent than V. anguillarum H775-3 to juvenile rainbow trout (Oncorhynchus mykiss), indicating that each hemolysin gene contributes to the virulence of V. anguillarum H775-3.     

Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum.

The fish pathogenic bacterium Vibrio anguillarum 775.17B was mutated by the use of transposon Tn5-132. Two hundred independent exconjugants were isolated and screened for a reduction of virulence in experimental infections of rainbow trout (Onchorhynchus mykiss). Two of these exconjugants, VAN20 and VAN70, showed a significant reduction in virulence after both intraperitoneal and immersion infections. The avirulent mutants showed no loss of any previously suggested virulence determinants of V. anguillarum. One of the mutants (VAN70) was further characterized. DNA sequence analysis revealed two open reading frames, the gene into which Tn5-132 had been inserted (virA) and a closely linked upstream gene (virB). A virB mutant of 775.17B, NQ706, was isolated and also shown to be avirulent. The deduced amino acid sequences of virA and virB correspond to proteins with molecular weights of 36,000 and 42,000, respectively. Insertional mutagenesis of the corresponding virA and virB genes of a clinical isolate of V. anguillarum, serotype O1, also resulted in avirulence. In immunoblot experiments, the total cell lysates of VAN70 (virA) and NQ706 (virB) did not respond to a rabbit polyclonal antiserum directed against whole cells of 775.17B (wild type). This suggests that virA and virB are involved in the biosynthesis of a major surface antigen important for the virulence of V. anguillarum. Immunogold electron microscopy showed that a constituent of the flagellar sheath was expressed by 775.17B (wild type) but not by VAN70 (virA) and NQ706 (virB), suggesting that the major surface antigen lacking in VAN70 and NQ706 is located on the outer sheath of the flagellum. Analysis of this major surface antigen revealed it likely to be lipopolysaccharide. Further analysis showed that the flagellum and the major surface antigen were expressed in vivo during fish infections.     

Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of V. cholerae iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in V. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source in vitro, we demonstrate that heme is not available to V. cholerae in the infant mouse intestine.     

Genetic and physiologic characterization of ferric/cupric reductase constitutive mutants of Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic yeast that causes meningitis in immunocompromised patients. Because iron acquisition is critical for growth of a pathogen in a host, we studied the regulation of the ferric reductase and ferrous uptake system of this organism. We isolated 18 mutants, representing four independent loci, with dysregulated ferric reductase. The mutant strains had >10-fold higher than wild-type WT reductase activity in the presence of iron. Two of the strains also had >7-fold higher than WT iron uptake in the presence of iron but were not markedly iron sensitive. Both were sensitive to the oxidative stresses associated with superoxide and hydrogen peroxide. One strain exhibited only 23% of the WT level of iron uptake in the absence of iron and grew poorly without iron supplementation of the medium, phenotypes consistent with an iron transport deficiency; it was sensitive to superoxide but not to hydrogen peroxide. The fourth strain had high reductase activity but normal iron uptake; it was not very sensitive to oxidative stress. We also demonstrated that the ferric reductase was regulated by copper and could act as a cupric reductase. Sensitivity to oxidants may be related to iron acquisition by a variety of mechanisms and may model the interaction of the yeast with the immune system.     

Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization

Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a approximately 30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.     

A Cluster of Genes Involved in Polysaccharide Biosynthesis from Enterococcus faecalis OG1RF

Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.     

Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems.

Vibrio cholerae iron transport mutants were tested for their ability to cause disease in an infant mouse model. The mice were challenged with either the wild-type strain, a vibriobactin synthesis mutant, a heme utilization mutant, or double mutants containing both the vibriobactin synthesis defect and the heme utilization defect. When mice were challenged with 10(7) bacteria, the ability of the double mutant to survive in the intestines was greatly reduced and that of the heme utilization mutant was slightly reduced compared with that of the wild type or the vibriobactin synthesis mutant. When the inoculum size was reduced 10-fold, all of the iron transport mutants failed to colonize the intestines and failed to cause diarrhea in the mice, whereas the wild-type strain was not cleared and elicited a diarrheal response. These data indicate that disruption of either the heme utilization or the vibriobactin uptake system reduces the ability of V. cholerae to cause disease. One of the heme utilization mutants, DHH1, was found to be defective also in utilization of vibriobactin and ferrichrome, mimicking the Escherichia coli TonB- phenotype. This mutant was the least virulent of the iron transport mutants tested. Transformation of DHH1 with the recombinant plasmid pHUT4 restored the abilities to use hemin, vibriobactin, and ferrichrome as iron sources, suggesting that pHUT4 encodes a gene(s) involved globally in the iron transport systems. Hybridization of Vibrio DNA with the V. cholerae heme utilization genes demonstrated the presence of DNA homologous to the genes encoding the outer membrane protein HutA and the inner membrane protein HutB in all the V. cholerae strains tested. The probe containing hutA, but not that containing hutB, also hybridized to DNA from Vibrio parahaemolyticus.