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

Abstract

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...

A Decade of Burkholderia cenocepacia Virulence Determinant Research

“…RpoE is also required for the ability of B. cenocepacia to grow under conditions of high osmolarity and high temperature (53). In Gram-negative bacteria, RpoE mediates gene activation in response to extracytoplasmic stress (2), and individual components of the extracytoplasmic stress response are also likely to be important for the virulence of B. cenocepacia.…”

“…Alternative sigma factors are regulatory proteins that activate transcription of particular gene subsets by binding at sites within promoter regions and interacting with the RNA polymerase complex, to allow the initiation of transcription (2). Two alternative sigma factors, RpoE and RpoN, are required for the ability of engulfed B. cenocepacia to delay phagolysosomal fusion in murine macrophages (53,141).…”

“…However, a series of constantly improving mutagenesis systems specifically tailored for B. cenocepacia were developed over the last decade. Most of these systems employed a strategy in which nonreplicative plasmids are integrated into specific locations in the chromosome via homologous recombination, resulting in polar (51), nonpolar (53), or conditional (122) mutations. Recently, five systems for the creation of unmarked gene deletions have been published for Burkholderia spp., including B. cenocepacia (12,33,52,61,98).…”

At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1

“…In addition to the typical RpoD (LINAB1_0602) σ factor, L. iners has three alternative σ factors identified as RpoE (LINAB1_0946), RpoH (LINAB1_1028), and RpoN (LINAB1_0645). Interestingly, RpoE is used by the opportunistic intracellular pathogen Burkholderia cenocepacia for growth under stress (54). Another intracellular Gram-negative pathogen Brucella melitensis uses RpoH and RpoN to cope with stress from elevated temperature and exposure to H 2 O 2 (55).…”

Recognition of β-Strand Motifs by RseB Is Required for σ E Activity in Escherichia coli

“…This cohort of pathways allows the bacteria to specifically and appropriately respond to different types of damage, as well as plays a role in the virulence of pathogenic strains of bacteria (6,24,30,32). Specifically, the E envelope stress response pathway has been demonstrated to be important for the virulence and survival of pathogenic Salmonella spp., Burkholderia cenocepacia, and Vibrio vulnificus and for the formation of biofilms in Pseudomonas aeruginosa (4,6,12,16). Considering the importance of this stress response pathway to bacterial survival and pathogenesis, it is essential to gain a greater understanding of how the pathway is activated.…”