<i>Burkholderia cenocepacia</i>
            Requires a Periplasmic HtrA Protease for Growth under Thermal and Osmotic Stress and for Survival In Vivo

Flannagan, Ronald S.; Aubert, Daniel; Kooi, Cora; Sokol, Pamela A.; Valvano, Miguel A.

doi:10.1128/iai.01581-06

Cited by 77 publications

(91 citation statements)

References 63 publications

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“…An orthologue of the hppD gene (Bmul_0223) that is required for the synthesis of a pigment that protects B. cenocepacia from in vitro and in vivo sources of oxidative stress was also downregulated (1.7-fold). One of the most downregulated genes within the dataset was Bmul_0682 (80-fold), which is an orthologue of the HtrA protease-encoding gene of B. cenocepacia that is required for in vivo survival within the rat agar bead model of chronic infection (Flannagan et al, 2007).…”

Section: Growth Of B Multivorans In Mannitol Results In Differentialmentioning

confidence: 99%

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner

et al. 2014

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In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitolrich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulenceassociated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23 % of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. Of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans.

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Section: Growth Of B Multivorans In Mannitol Results In Differentialmentioning

confidence: 99%

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner

et al. 2014

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“…Insertional inactivation of bceB was performed using the pGPVTp suicide vector, essentially as described previously (Flannagan et al, 2007). In brief, a 300 bp fragment internal to the bceB ORF of B. ambifaria AMMD and flanked by XbaI and EcoRI sites was PCRamplified and ligated into the corresponding sites in pGPVTp following appropriate restriction.…”

Section: Investigation Of Conserved Eps Gene Clusters In Bcc Speciesmentioning

confidence: 99%

“…Resulting plasmids were transformed into Escherichia coli GT115 competent cells (InvivoGen) and subsequently introduced into B. ambifaria AMMD by triparental mating. Resulting exconjugants were selected using gentamicin (50 mg l 21 ) and trimethoprim (100 mg l 21 ), and mutants identified by PCR using a chromosomal-specific primer in conjunction with the vector-specific primer RSF1300 (Flannagan et al, 2007).…”

Section: Investigation Of Conserved Eps Gene Clusters In Bcc Speciesmentioning

confidence: 99%

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex

et al. 2008

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The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

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“…The rpoE gene was cloned in-frame with and fused to the FLAG epitope coded for in pDA17. Expression of RpoE was confirmed by Western blot analysis of whole-cell lysates as described previously (Flannagan et al, 2007).RT-PCR. RT-PCR was performed as described previously (Ortega et al, 2005) to investigate the transcriptional organization of the rpoE operon.…”

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confidence: 99%

Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages

Flannagan

Valvano

2008

Microbiology

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Burkholderia cenocepacia is an opportunistic pathogen causing serious infections in patients with cystic fibrosis. The widespread distribution of this bacterium in the environment suggests that it must adapt to stress to be able to survive. We identified in B. cenocepacia K56-2 a gene predicted to encode RpoE, the extra-cytoplasmic stress response regulator. The rpoE gene is the first gene of a predicted operon encoding proteins homologous to RseA, RseB, MucD and a protein of unknown function. The genomic organization and the co-transcription of these genes were confirmed by PCR and RT-PCR. The mucD and rpoE genes were mutated, giving rise to B. cenocepacia RSF24 and RSF25, respectively. While mutant RSF24 did not demonstrate any growth defects under the conditions tested, RSF25 was compromised for growth under temperature (44 6C) and osmotic stress (426 mM NaCl). Expression of RpoE in trans could complement the osmotic growth defect but exacerbated temperature sensitivity in both RSF25 and wild-type K56-2. Inactivation of rpoE altered the bacterial cell surface, as indicated by increased binding of the fluorescent dye calcofluor white and by an altered outer-membrane protein profile. These cell surface changes were restored by complementation with a plasmid encoding rpoE. Macrophage infections in which bacterial colocalization with fluorescent dextran was examined demonstrated that the rpoE mutant could not delay the fusion of B. cenocepacia-containing vacuoles with lysosomes, in contrast to the parental strain K56-2. These data show that B. cenocepacia rpoE is required for bacterial growth under certain stress conditions and for the ability of intracellular bacteria to delay phagolysosomal fusion in macrophages. INTRODUCTIONThe Burkholderia cepacia complex (Bcc) comprises a group of nine closely related bacterial species that are phenotypically very similar but genetically distinct (Coenye et al., 2003;Mahenthiralingam et al., 2000). These bacteria are opportunistic pathogens that cause infections in immunocompromised individuals and often infect cystic fibrosis (CF) patients (Mahenthiralingam & Vandamme, 2005). Burkholderia cenocepacia, a member of the Bcc, is one of the species most often recovered from CF patients worldwide and is also associated with the most severe infections (Brisse et al., 2004;LiPuma, 2005;Manno et al., 2004;Reik et al., 2005; Speert et al., 2002). Furthermore, B. cenocepacia infections in CF patients are associated with an accelerated decline in lung function as compared to other CF-related pathogens such as Pseudomonas aeruginosa (Courtney et al., 2004;Jones et al., 2004). The treatment of infection is difficult because this bacterium is multi-drug resistant (Zhou et al., 2007) and can be transmitted from person to person (Govan et al., 1993;LiPuma et al., 1990;Smith et al., 1993). Infections are further complicated by the 'cepacia syndrome', a potentially fatal necrotizing pneumonia that occurs in a subset of patients (Isles et al., 1984;Thomassen et al., 1985).Although B. cenocepac...

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Burkholderia cenocepacia Requires a Periplasmic HtrA Protease for Growth under Thermal and Osmotic Stress and for Survival In Vivo

Cited by 77 publications

References 63 publications

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex

Burkholderia cenocepacia requires RpoE for growth under stress conditions and delay of phagolysosomal fusion in macrophages

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