Expression of Bacteroides fragilis hemolysins in vivo and role of HlyBA in an intra-abdominal infection model.

Bacteroides fragilis is the most frequent opportunistic pathogen isolated from anaerobic infections. However, there is a paucity of information regarding the genetic and molecular aspects of gene expression of its virulence factors during extra-intestinal infections. A potential virulence factor that has received little attention is the ability of B. fragilis to produce hemolysins. In this study, an implanted perforated table tennis "ping-pong" ball was used as an intra-abdominal artificial abscess model in the rat. This procedure provided sufficient infected exudate for gene expression studies in vivo. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the relative expression of hlyA, hlyB, hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII mRNAs. The hlyA mRNA was induced approximately sixfold after 4 days postinfection compared with the mRNA levels in the inoculum culture prior to infection. The hlyB mRNA increased approximately sixfold after 4 days and 12-fold after 8 days postinfection. Expression of hlyC mRNA increased sixfold after 1 day, 45-fold after 4 days, and 16-fold after 8 days postinfection, respectively. The hlyD and hlyE mRNAs were induced approximately 40-fold and 30-fold, respectively, after 4-days postinfection. The hlyF expression increased approximately threefold after 4-days postinfection. hlyG was induced approximately fivefold after 4 and 8 days postinfection. The hlyIII mRNA levels had a steady increase of approximately four-, eight-, and 12-fold following 1, 4, and 8 days postinfection, respectively. These findings suggest that B. fragilis hemolysins are induced and differentially regulated in vivo. Both parent and hlyBA mutant strains reached levels of approximately 3-8 × 10(9) cfu/mL after 1 day postinfection. However, the hlyBA mutant strain lost 2 logs in viable cell counts compared with the parent strain after 8 days postinfection. This is the first study showing HlyBA is a virulence factor which plays a role in B. fragilis survival in an intra-abdominal abscess model.

Development of an IPTG inducible expression vector adapted for Bacteroides fragilis.

The genus Bacteroides are gram-negative, obligate anaerobes indigenous to the gastrointestinal tract of humans and animals. The Bacteroides and other members of the Bacteroidetes phylum have diverged from the Proteobacteria. These organisms evolved a unique promoter structure for the initiation of transcription, hence common genetic tools are of limited use in the Bacteroides. An expression vector that can control gene expression in the Bacteroides was constructed by engineering the lacO1,3 repressor binding sites into the promoter of the cfxA ß-lactamase gene. The gene for the LacI repressor was placed under control of the Bacteroides tetQ gene promoter for constitutive expression and inserted into the vector. Studies utilizing the xylosidase reporter gene, Xa, showed that the gene was induced by Isopropyl ß-d-1-thiogalactopyransoide (IPTG) in a time and concentration dependent manner from 10 to 250 µM over a 10-240 min time frame. The utility of the vector was demonstrated by insertion of the Bacteroides fragilis trxA gene into the plasmid. TrxA synthesis was monitored by Western hybridization and the results indicated that it was regulated by the presence of IPTG in the media. This is the first transcriptional regulatory system developed for the Bacteroides that has incorporated components from the Proteobacteria and demonstrates the feasibility of modifying existing genetic tools for use in these organisms.

The Bacteroides fragilis P20 scavengase homolog is important in the oxidative stress response but is not controlled by OxyR.

The oxidative stress response of obligate anaerobe, Bacteroides fragilis, is partially controlled by the redox regulator OxyR but an oxyR null mutant maintains a high level of aerotolerance. Studies using two-dimensional polyacrylamide gel electrophoresis showed that a thiol peroxidase-scavengase, Tps, was induced during oxygen exposure of an oxyR mutant. Tps is similar to 'atypical 2-cysteine peroxidases' such as scavengase p20 and it demonstrated catalytic activity against t-butyl hydroperoxide and H(2)O(2). A second gene, oim, encoding a putative membrane protein, was divergently transcribed from tps. Transcriptional analysis indicated that tps and oim were coordinately regulated by oxygen induction via an OxyR-independent mechanism. H(2)O(2) was a less potent inducer than oxygen exposure and in an oxyR mutant the mRNA levels were slightly reduced compared with the wild type. A null mutant of tps had increased sensitivity to killing by t-butyl hydroperoxide and oxygen but an oim mutant was similar to wild type. These data indicate that Tps is important for protection against some forms of oxidative stress.