Date Published: January 3, 2017
Publisher: Public Library of Science
Author(s): Matthew M. Schaefers, Tiffany L. Liao, Nicole M. Boisvert, Damien Roux, Deborah Yoder-Himes, Gregory P. Priebe, Matthew R. Parsek.
Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.
Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of related Gram-negative bacilli that can be dangerous respiratory pathogens for patients with cystic fibrosis (CF) [1, 2]. BCC can also cause outbreaks of bacteremia or respiratory infection in hospitalized non-CF patients, including the recently discovered cluster of cases of BCC respiratory infection linked to contaminated stool softener (docusate) . BCC are also common pathogens for individuals with chronic granulomatous disease . Among CF patients in the United States colonized with BCC, the species most commonly seen are B. cenocepacia (45%), B. multivorans (35%), B. vietnamiensis (6%), B. cepacia (6%), and B. dolosa (3%), although there is significant variability based on geographic region and institution . A number of studies have shown an association between infection with BCC and clinical pulmonary deterioration of CF patients [6–10]. The so-called “cepacia syndrome” refers to the clinical presentation of fevers, leukocytosis, elevated erythrocyte sedimentation rate (ESR), and progressive severe pneumonia, sometimes with bacteremia, in CF patients occurring relatively soon after acquiring BCC, with a mortality rate initially reported at 62% . BCC infection in CF patients can be transmitted person-to-person, and multiple outbreaks have been described, including one of a highly antibiotic resistant strain of the BCC species B. dolosa among almost 40 CF patients at Boston Children’s Hospital . This outbreak has been associated with accelerated decline of lung function and decreased survival . BCC in general are commonly multidrug resistant (MDR) or extensively drug resistant (XDR) pathogens, highlighting the necessity for novel approaches for treatment of these bacteria .
A recent study using whole-genome sequencing of multiple strains from an outbreak of B. dolosa in CF patients at Boston Children’s Hospital discovered that the fixLJ genes show evidence of strong positive selection. These findings were recently corroborated to another BCC species, B. multivorans . In the current study, we report the functional significance of the fixLJ genes in B. dolosa, finding not only that the pathway is induced by low oxygen, but also that it regulates a large number of genes and is critical for pathogenicity in vivo and intracellular invasion in vitro.