Host adaptations in thefixLJpathway of theBurkholderia cepaciacomplex increase virulence

Abstract

The Burkholderia cepacia complex (BCC) is composed of multiple species, including B. multivorans and B. dolosa, that are significant pathogens for people with cystic fibrosis (CF) and are extensively resistant to many antibiotics. The fixL gene of the fixLJ 2component system (TCS) in these BCC species shows evidence of positive selection for nonsynonymous mutations during chronic lung infection in CF. Previous work showed that the B. dolosa fixLJ system regulates 11% of the genome and modulates biofilm formation, motility, persistence within macrophages, and virulence in a murine pneumonia model. Here, we assess the impacts of clinically observed FixL evolved variants in fixLJ pathway-mediated phenotypes in B. dolosa and B. multivorans. BCC carrying the ancestral fixL sequence are less pathogenic than constructs carrying evolved variants in both a macrophage infection model and a murine pneumonia model. In vitro phospho-transfer experiments demonstrate that the evolved B. dolosa FixL variants are able to reduce fixLJ pathway activity by either having lower levels of kinase activity or increased phosphatase activity. Notably, the ancestral fixL genotype has increased ability to survive within the soil compared to isogenic constructs with evolved fixL genotypes, demonstrating that increased virulence comes at an expense. Modulation of the FixLJ system has profound effects on many BCC phenotypes including full pathogenicity, and this modulation is critical for BCC adaptation to the host. MainThe Burkholderia cepacia complex (BCC) is a group of more than 20 species of closelyrelated Gram-negative bacilli that can be dangerous respiratory pathogens for people with cystic fibrosis (CF). 1,2 Among CF patients in the US colonized with BCC, the species most commonly seen are B. cenocepacia and B. multivorans, although there is significant variability based on geographic region and institution, 3-7 and B. multivorans has emerged as the most predominant BCC species infecting CF patients in some regions. 4-7 BCC members have caused several outbreaks within the CF community, 2 including one outbreak of a highly antibiotic-resistant strain of B. dolosa among almost 40 CF patients in Boston 8 and another of B. cenocepacia in Toronto. 9 BCC can also cause serious infections in individuals with chronic granulomatous disease (CGD). 10 Outbreaks of hospital-acquired BCC infections in non-CF and non-CGD patients have also been increasingly described, often associated with contaminated medications, 11-16 including a recent outbreak associated with contamination of the stool softener docusate with B. contaminans. 15,16 Analysis of genomic diversity arising during B. dolosa and B. multivorans chronic infections in CF identified the fixLJ two-component system (TCS) as a pathway that is under positive selection, evident from enrichment for non-synonymous mutations as opposed to synonymous mutations and genetic parallelism among many independent infections. [17][18][19] TCSs are one mechanism that bacteria use to sense and respond to thei...