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. 2013 Apr;2(2):326-37.
doi: 10.1002/mbo3.76. Epub 2013 Feb 26.

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

Leandro A Lobo  1 , Audrey L Jenkins, C Jeffrey Smith, Edson R Rocha
Affiliations

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

Leandro A Lobo et al. Microbiologyopen. 2013 Apr.

Abstract

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.

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Figures

Figure 1
Figure 1
Intraperitoneal tissue cage rat model. A and B) A sterile perforated “tennis ping-pong” ball was implanted into the peritoneal cavity of Sprague Dawley rats (weight, ≥400 g). C) Exudate fluid aspirated from the implanted intraperitoneal perforated tissue cage ball. D) An exposure of the encapsulated perforated tissue cage removed from the peritoneal cavity at the time of necropsy. Panels do not show pictures to scale.
Figure 2
Figure 2
The genetic structure of the hlyA, hlyB hlyC, hlyD, hlyE, hlyF, hlyG, and hlyIII loci in the Bacteroides fragilis 638R genome. Hemolysins ORF and direction of transcription are depicted by a gray arrow. Open arrows depict ORFs and transcription orientation of the genes flanking each respective hemolysin chromosomal region. The locus tag for each gene and gene product are shown, respectively, below and above each ORF. The transcriptional products for the hlyBA operon were adapted from Robertson et al. (2006). The thin dark arrows indicate the direction and length of the hlyBA bicistronic mRNA and hlyA monocistronic mRNA. The dark bent arrow depicts the putative promoter region derived from Robertson et al. (2006). The bar below each hemolysin ORF represents the coding region and length of the PCR products amplified from cDNAs in the Real-Time RT-PCR experiments described in the Materials and Methods section.
Figure 3
Figure 3
Expression ratio of B. fragilis 6389R hemolysins in vivo. Real-time RT-PCR was carried out from total RNA isolated from the intra-abdominal infection tissue cage model implanted into rat peritoneal cavity. Real-time RT-PCR was performed for each hemolysin depicted in each respective panel using specific primers described in the Material and Methods section. hlyA expression levels include both hlyBA bicistronic mRNA and hlyA monocistronic mRNA combined. hlyB expression was measured as a component of the hlyBA bicistronic mRNA. Data were analyzed using the Relative Expression Software Tool (REST) V. 2.07 for group-wise comparison and statistical analysis of relative expression. In vivo expression of hemolysins was normalized to their respective levels of expression in culture media control (CM). 16S rRNA was used as housekeeping reference RNA. Data are from triplicated real-time RT-PCR reactions in two sets of pooled RNA from two groups of six animals.
Figure 4
Figure 4
Survival of B. fragilis strains inoculated into the encapsulated intraperitoneal implanted tissue cage as described in Figure 1. Fluid exudates were aspirated for viable cell counts (cfu) at time points indicated.formula image: B. Fragilis 638R parent strain (n = 6).formula image": BER-41 ΔhlyBA::teQ deletion mutant (n = 6).formula image: BER-45 ΔhlyIII::cfxA deletion mutant (n = 3).formula image: BER-46 ΔhlyBA::teQ ΔhlyIII::cfxA double mutant (n = 3).formula image: BER-47: ΔhlyBA::teQ pER-80 (n = 3).
Figure 5
Figure 5
Detection of Rat IgG antibodies reactive against rHlyA and rHlyB by enzyme-linked immunosorbent assay (ELISA). IgG levels reactive against rHlyA (panel A) or rHlyB (panel B) in the transudate preinfection (n = 3) and exudate fluids (n = 3) aspirated from the implanted tissue cage balls at 8 days and 22 days postinfection. Bf 638R: B. fragilis 638R parent strain. BER-41 ΔhlyBA::tetQ deletion mutant. Microsoft Excel T-TEST software was used to calculate the paired two-tailed Student t-test to determine the statistical significance between groups. Group differences were considered statistically significant for P-value <0.05. The numbers above horizontal brackets in panels A and B are the probability values associated with the Student t-test.
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