No abstract

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxRVacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5 end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-aminoacid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P Cl /P Na ‫؍‬ 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(⌬6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(⌬6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominantnegative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

Burkholderia cepacia complex (Bcc) is a group of phenotypically similar, genetically distinct bacteria that are beneficial to the environment but can also cause severe human infections. Bcc are being exploited for use as bioremediation agents and as a way to combat agricultural plant diseases. However, Bcc can cause lung infections in patients with chronic granulomatous disease or cystic fibrosis often resulting in mortality of these patients. Since it is unclear what bacterial components are necessary for causing human infections, studies of Bcc have focused on identifying putative virulence factors. As in other Gram-negative bacteria, the lipopolysaccharide (LPS) of Bcc induces a strong immune response that can contribute to host cell damage. The unusual structure of Bcc LPS lowers the anionic charge of the Bcc cell surface, which inhibits the binding and subsequent effects of cationic antibiotics. These distinguishing features include the substitution of a Ko for a Kdo residue in the inner core oligosaccharide and Ara4N residues bound to phosphates of the lipid A backbone. The structures of O antigen subunits and the consequent serotypes will also be discussed, with particular reference to the O antigen biosynthetic loci of two Bcc strains.

Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 immunoglobulin G1 anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.

Burkholderia cepacia complex (Bcc) is a group of phenotypically similar, genetically distinct bacteria that are beneficial to the environment but can also cause severe human infections. Bcc are being exploited for use as bioremediation agents and as a way to combat agricultural plant diseases. However, Bcc can cause lung infections in patients with chronic granulomatous disease or cystic fibrosis often resulting in mortality of these patients. Since it is unclear what bacterial components are necessary for causing human infections, studies of Bcc have focused on identifying putative virulence factors. As in other Gram-negative bacteria, the lipopolysaccharide (LPS) of Bcc induces a strong immune response that can contribute to host cell damage. The unusual structure of Bcc LPS lowers the anionic charge of the Bcc cell surface, which inhibits the binding and subsequent effects of cationic antibiotics. These distinguishing features include the substitution of a Ko for a Kdo residue in the inner core oligosaccharide and Ara4N residues bound to phosphates of the lipid A backbone. The structures of O antigen subunits and the consequent serotypes will also be discussed, with particular reference to the O antigen biosynthetic loci of two Bcc strains.

Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI. Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species. There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.