Edson R. Rocha

Regulation of Pseudomonas Quinolone Signal Synthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

The Bacteroides fragilis transcriptome response to oxygen and H2O2: the role of OxyR and its effect on survival and virulence

The intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H2O2. Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy‐generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase‐like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra‐abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra‐intestinal sites and promote opportunistic infections.

Genetic analysis of an important oxidative stress locus in the anaerobe Bacteroides fragilis

The obligate anaerobe, Bacteroides fragilis, is a highly aerotolerant intestinal tract organism that has evolved a complex oxidative stress response (OSR). The redox regulator OxyR controls several OSR genes (katB, dps, and ahpC), but there is little else known about other genes it regulates. To identify additional genes in the OxyR regulon, two-dimensional gel electrophoresis was used to isolate proteins from a mutant that constitutively expresses genes in the regulon. The 28,500 Da protein thioredoxin peroxidase (Tpx) was identified. Two additional genes induced during oxidative stress were identified adjacent to tpx, a putative RNA-binding protein (rbpA) and a cytochrome-c peroxidase (ccp). Transcriptional analyses showed that tpx and rbpA were transcribed as monocistronic mRNA species or as a bicistronic operon. Transcription of tpx was induced by exposure to air or H(2)O(2) from an OxyR-dependent promoter and to a lesser extent from a second OxyR-independent promoter. Expression of the rbpA gene during oxidative stress was regulated by the OxyR-dependent tpx promoter resulting in the bicistronic tpx/rbp mRNA. The ccp gene was expressed only as a monocistronic message and induction was only observed after exposure to H(2)O(2) in an OxyR-independent manner. Disruption of the tpx operon or ccp resulted in sensitivity to the organic peroxides cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP) but not to H(2)O(2). This work brings the total of oxyR-controlled genes in B. fragilis to five and suggests the existence of a second peroxide response regulator that controls ccp expression.

Flavin mononucleotide (FMN)‐based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies.

Thioredoxins in Redox Maintenance and Survival during Oxidative Stress of Bacteroides fragilis

The anaerobe Bacteroides fragilis is a gram-negative, opportunistic pathogen that is highly aerotolerant and can persist in aerobic environments for extended periods. In this study, the six B. fragilis thioredoxins (Trxs) were investigated to determine their role during oxidative stress. Phylogenetic analyses of Trx protein sequences indicated that four of the six Trxs (TrxA, TrxC, TrxD, and TrxF) belong to the M-type Trx class but were associated with two different M-type lineages. TrxE and TrxG were most closely associated to Y-type Trxs found primarily in cyanobacteria. Single and multiple trx gene deletions were generated to determine functional differences between the Trxs. The trxA gene was essential, but no anaerobic growth defects were observed for any other single trx deletion or for the DeltatrxC DeltatrxD::cfxA DeltatrxE DeltatrxF DeltatrxG quintuple mutant. Regulation of the trx genes was linked to the oxidative stress response, and all were induced by aerobic conditions. The DeltatrxC DeltatrxE DeltatrxF DeltatrxG and the DeltatrxC DeltatrxD::cfxA DeltatrxE DeltatrxF DeltatrxG multiple deletion strains were impaired during growth in oxidized media, but single trx gene mutants did not have a phenotype in this assay. TrxD was protective during exposure to the thiol oxidant diamide, and expression of trxD was induced by diamide. Diamide-induced expression of trxC, trxE, and trxF increased significantly in a trxD mutant strain, suggesting that there is some capacity for compensation in this complex Trx system. These data provide insight into the role of individual Trxs in the B. fragilis oxidative stress response.

The complex oxidative stress response of Bacteroides fragilis: the role of OxyR in control of gene expression

Gram-negative anaerobes in the genus Bacteroides are the predominant members of the GI-tract microflora where they play an important role in normal intestinal physiology. Bacteroides spp. also are significant opportunistic pathogens responsible for an array of intra-abdominal and other infections. Bacteroides fragilis is the most common anaerobic pathogen and it possesses virulence factors such as a capsule and neuraminidase that contribute to its success as a pathogen. Infection occurs when organisms escape from the anaerobic colon to aerobic sites such as the peritoneum where O(2) concentrations average 6%. Thus in addition to the classic virulence factors, resistance to oxidative stress is essential and may be involved in the initiation and persistence of infection. In fact, B. fragilis is highly O(2) tolerant, surviving extended periods (>24h) of O(2) exposure without a significant affect on viability. For protection against this oxidative stress B. fragilis mounts a complex physiological response that includes induction of >28 proteins involved in detoxification of oxygen radicals, protection of macromolecules, and adaptive physiology. One experimental strategy used to characterize this oxidative stress response is the direct detection of genes and proteins induced during exposure to O(2) or H(2)O(2). The methods employed have included RNA differential display to capture unique mRNA transcripts produced during oxidative stress, and native or 2D-gel electrophoresis to isolate and identify newly formed stress-induced proteins. Using these and other approaches a wide array of genes induced by oxidative stress have been discovered. These include genes for catalase, superoxide dismutase, thioredoxin-peroxidase, p20-peroxidase, cytochrome c peroxidase, Dps, alkyl hydroperoxidase, aerobic ribonucleotide reductase, ruberythrin, starch utilization, aspartate decarboxylase, and an RNA binding protein. The genes encoding these activities fall into three regulatory classes: (1) induced by O(2) only, (2) induced by H(2)O(2) only, and (3) induced by either O(2) or H(2)O(2). Such a complex regulatory response will likely involve multiple regulators. Thus far one regulator has been identified, OxyR, which controls a subset of the class 3 genes that are induced by either O(2) or H(2)O(2). OxyR responds rapidly to oxidative stress and transcriptional analyses have shown that OxyR-controlled genes are activated by as little as 0.5% O(2) or 10 microM H(2)O(2). Maximal expression of most OxyR regulon genes was reached at 50 microM H(2)O(2) and 2% O(2). These oxidant concentrations are similar to environmental levels that would be experienced by the organisms in tissues outside of the colon suggesting that the OxyR regulon would be induced during the course of an infection.

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.

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacteriums extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O(2) or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer.

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis

BACKGROUND Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear. METHODS A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole. RESULTS Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo. CONCLUSIONS Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

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

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 × 109 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.