Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)
Andrea M. Gough (Creator)
Kirstin P. Robertson (Creator)
Edson R. Rocha (Creator)
C. Jeffrey Smith (Creator)
East Carolina University (ECU )
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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 ironregulated 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. Originally published Infection and Immunity Vol. 74 No. 4 Apr 2006

Additional Information

Infection and Immunity. 74:4(April 2006) p. 2304-2316.
Language: English
Date: 2011
Bacteroides fragilis, hemolysins, synergistic activity

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