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Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB.

ABSTRACT: This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.

SUBMITTER: Robertson KP

PROVIDER: S-EPMC1418898 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB.

Robertson Kirstin P KP Smith C Jeffrey CJ Gough Andrea M AM Rocha Edson R ER

Infection and immunity 20060401 4

This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in ...[more]

PMID: 16552061

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Project description: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.

Dataset Information

Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB.

Publications

Characterization of Bacteroides fragilis hemolysins and regulation and synergistic interactions of HlyA and HlyB.

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