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Infection and Immunity, April 2006, p. 2304-2316, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2304-2316.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Kirstin P. Robertson,{dagger} C. Jeffrey Smith, Andrea M. Gough, and Edson R. Rocha*

Department of Microbiology and Immunology, East Carolina University Brody School of Medicine, Greenville, North Carolina

Received 3 November 2005/ Returned for modification 9 January 2006/ Accepted 27 January 2006

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, East Carolina University Brody School of Medicine, Biotechnology Bldg., Room 130, 600 Moye Blvd., Greenville, NC 27834. Phone: (252) 744-9563. Fax: (252) 744-3535. E-mail: rochae{at}ecu.edu.

Editor: F. C. Fang

{dagger} Present address: Department of Microbiology, School of Biological Sciences, University of Reading, Reading, United Kingdom.

Infection and Immunity, April 2006, p. 2304-2316, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2304-2316.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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