Expression of <i><scp>B</scp>acteroides fragilis</i> hemolysins in vivo and role of HlyBA in an intra‐abdominal infection model

Lobo, Leandro Araújo; Jenkins, Audrey L.; Smith, C. Jeffrey; Rocha, Edson R.

doi:10.1002/mbo3.76

Cited by 25 publications

(24 citation statements)

References 70 publications

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“…The tissue cage infection model has been described previously (27,28). Briefly, a perforated, sterilized ping pong ball was surgically implanted into the peritoneal cavity of an adult male SpragueDawley rat and allowed to encapsulate for 4-5 wk.…”

Section: Methodsmentioning

confidence: 99%

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Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

Cao

Rocha

Smith

2014

Proc. Natl. Acad. Sci. U.S.A.

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Bacteroides fragilis is the most common anaerobe isolated from clinical infections, and in this report we demonstrate a characteristic of the species that is critical to their success as an opportunistic pathogen. Among the Bacteroides spp. in the gut, B. fragilis has the unique ability of efficiently harvesting complex N-linked glycans from the glycoproteins common to serum and serous fluid. This activity is mediated by an outer membrane protein complex designated as Don. Using the abundant serum glycoprotein transferrin as a model, it has been shown that B. fragilis alone can rapidly and efficiently deglycosylate this protein in vitro and that transferrin glycans can provide the sole source of carbon and energy for growth in defined media. We then showed that transferrin deglycosylation occurs in vivo when B. fragilis is propagated in the rat tissue cage model of extraintestinal growth, and that this ability provides a competitive advantage in vivo over strains lacking the don locus.

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Section: Methodsmentioning

confidence: 99%

“…RNA extraction from in vivo samples was done as described previously with a few modifications (27). Fluid was aspirated and immediately mixed with RNAprotect Cell Reagent (Qiagen, Inc.) at a 1:2 ratio.…”

Section: Methodsmentioning

confidence: 99%

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

Cao

Rocha

Smith

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The inability of the host immune system to resolve the abscess can lead to bacteremia, sepsis, and in certain instances death (5, 6). B. fragilis is the most common anaerobe isolated from intra-abdominal abscesses, and it has been demonstrated to possess many factors that promote its survival outside the intestinal tract, such as capsular polysaccharides, proteases, neuraminidase, iron acquisition, hemolysins, and resistance to oxidative stress (1,2,7,8). Oxidative stress occurs immediately when B. fragilis translocates from the anaerobic intestine to the more oxygenated (6% O 2 ) peritoneal cavity, and there is additional oxidative stress resulting from the immune response and polymorphonucleocyte (PMN) recruitment to the site of infection (9-12).…”

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confidence: 99%

Dps and DpsL Mediate SurvivalIn VitroandIn Vivoduring the Prolonged Oxidative Stress Response in Bacteroides fragilis

2015

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Bacteroides fragilis is a Gram-negative anaerobe and member of the human intestinal tract microbiome, where it plays many beneficial roles. However, translocation of the organism to the peritoneal cavity can lead to peritonitis, intra-abdominal abscess formation, bacteremia, and sepsis. During translocation, B. fragilis is exposed to increased oxidative stress from the oxygenated tissues of the peritoneal cavity and the immune response. In order to survive, B. fragilis mounts a robust oxidative stress response consisting of an acute and a prolonged oxidative stress (POST) response. This report demonstrates that the ability to induce high levels of resistance to tert-butyl hydroperoxide (tBOOH) after extended exposure to air can be linked to the POST response. Disk diffusion assays comparing the wild type to a ⌬dps mutant and a ⌬dps ⌬bfr mutant showed greater sensitivity of the mutants to tBOOH after exposure to air, suggesting that Dps and DpsL play a role in the resistance phenotype. Complementation studies with dps or bfr (encoding DpsL) restored tBOOH resistance, suggesting a role for both of these ferritin-family proteins in the response. Additionally, cultures treated with the iron chelator dipyridyl were not killed by tBOOH, indicating Dps and DpsL function by sequestering iron to prevent cellular damage. An in vivo animal model showed that the ⌬dps ⌬bfr mutant was attenuated, indicating that management of iron is important for survival within the abscess. Together, these data demonstrate a role for Dps and DpsL in the POST response which mediates survival in vitro and in vivo. IMPORTANCEB. fragilis is the anaerobe most frequently isolated from extraintestinal opportunistic infections, but there is a paucity of information about the factors that allow this organism to survive outside its normal intestinal environment. This report demonstrates that the iron storage proteins Dps and DpsL protect against oxidative stress and that they contribute to survival both in vitro and in vivo. Additionally, this work demonstrates an important role for the POST response in B. fragilis survival and provides insight into the complex regulation of this response. Bacteroides spp. are members of the normal intestinal microbiome of humans. The intestine is a consistent and favorable environment that provides continuous access to nutrient sources for these strictly anaerobic organisms. Bacteroides spp. play many important roles in maintaining a healthy intestinal tract, such as polysaccharide degradation, protection of the gut epithelia from colonization by pathogenic bacteria, development of the intestinal tract, maturation of the mucosal and systemic immune systems, and transformation of toxic and mutagenic compounds (1-4). However, when the integrity of the intestinal wall is breached due to trauma, abdominal surgery, or diseases such as appendicitis, perforated ulcer, diverticulitis, and colon cancer, translocation of the normal flora into the peritoneal cavity can result in peritonitis and establishment of an intr...

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“…After the healing period, the balls became encapsulated in connective tissue, developed a blood supply, and were filled with sterile exudate with the appearance of serum and with characteristics similar to those already described (Bamberger et al, 2002;Lobo et al, 2013). Furthermore, the encapsulated tissue cage balls were inoculated with 1.0 mL B. fragilis ATCC 43859 (approximately 10 8 CFU/mL) in 0.85% NaCl.…”

Section: Experimental Infection Modelmentioning

confidence: 90%

“…This study focused on the evaluation of a murine model for in vivo transcriptional analysis of B. fragilis. The rat tissue cage model (Lobo et al, 2013), which consists of intraperitoneal implantation of a perforated table tennis "ping-pong" ball, was used to establish an intraperitoneal abscess infection and bacteria for RNA-seq were recovered from drained exudative secretions.…”

Section: Introductionmentioning

confidence: 99%

Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis

et al. 2015

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ABSTRACT. Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.

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

Cited by 25 publications

References 70 publications

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

Efficient utilization of complex N-linked glycans is a selective advantage for Bacteroides fragilis in extraintestinal infections

Dps and DpsL Mediate SurvivalIn VitroandIn Vivoduring the Prolonged Oxidative Stress Response in Bacteroides fragilis

Integrity of prokaryotic mRNA isolated from complex samples for in vivo bacterial transcriptome analysis

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