Role of flagella in host cell invasion by Burkholderia cepacia

Abstract

Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of B. cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of B. cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of B. cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B. cepacia. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B. cepacia, contributing to establishment of infection and systemic spread of the organism.

FIG. 1.

Invasion of A549 cell monolayers by B. cepacia strain J2315 (wild type), mutant D9, and D9 complemented with fliG (strain CM340). (A) Invasion of A549 cell monolayers with centrifugation. Quantitative invasion assays were performed in triplicate as described in Materials and Methods. The invasion values were calculated by determining the percentages of the bacterial inocula that survived after 2 h of antibiotic treatment. The values were normalized to the value for wild-type strain J2315, which was arbitrarily set at 100%. The actual invasion frequency for strain J2315 was 0.67% ± 0.03%. The asterisk indicates that the level of invasion by mutant D9 was significantly less than the level of invasion by the parent strain (P < 0.000002). (B) Invasion of A549 cell monolayers without centrifugation. The actual invasion frequency for strain J2315 without centrifugation was 0.31% ± 0.01%. The double asterisks indicate that the P value was <0.0000002.

FIG. 2.

Physical map of the B. cepacia fli locus. The open box indicates the site of mini-Tn5Tc insertion in fliG mutant D9, and the cross-hatched box indicates the site of insertion of the cat cassette in fliI null strain CM58. The arrows indicate the directions of transcription. The numbers below the open reading frames indicate the levels of amino acid identity to the corresponding flagellar homologs in S. enterica serovar Typhimurium. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; X, XhoI; Xm, XmaI.

FIG. 3.

Phenotypes of B. cepacia mutants with impaired motility. Strains were stabbed into semisolid LB medium plates (0.25% agar) and incubated at 37°C for 24 h. (A) Wild-type strain J2315, mutant D9 (fliG::mini-Tn5Tc), and D9 complemented with fliG (strain CM340). (B) Wild-type strain J2315, mutant CM58 (fliI::cat), and CM58 complemented with fliI (strain CM100).

FIG. 4.

Immunoblot analysis of flagellin protein in wild-type and flagellar mutant strains. Equal amounts of protein from whole-cell or supernatant fractions were separated on SDS-12.5% PAGE gels and immunoblotted with anti-flagellin antibody as described in Materials and Methods. (A) Wild-type strain J2315, D9 (fliG::mini-Tn5Tc), and CM340 (D9 complemented with fliG). (B) Wild-type strain J2315, CM58 (fliI::cat), and CM100 (CM58 complemented with fliI). The molecular masses of the protein standards are indicated on the left. The position of the 45-kDa flagellin protein band is indicated by an arrow.

FIG. 5.

Invasion of A549 cell monolayers by B. cepacia strain J2315 (wild type), mutant CM58 (fliI::cat), and CM100 (CM58 complemented with fliI) with (A) or without (B) centrifugation. The invasion values were calculated by determining the percentages of the bacterial inocula that survived after 2 h of antibiotic treatment and were normalized to the value for wild-type strain J2315, which was arbitrarily set at 100%. The actual invasion frequencies for strain J2315 with and without centrifugation were 0.67% ± 0.03% and 0.26% ± 0.02%, respectively. The asterisk indicates that the P value was <0.0007, and the double asterisks indicate that the P value was <0.00004.