Identification of Burkholderia cenocepacia Strain H111 Virulence Factors Using Nonmammalian Infection Hosts

Abstract

c Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence. Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.

Variation ofBurkholderia cenocepaciavirulence potential during cystic fibrosis chronic lung infection

“…In fact, the divergence in the pathogenic potential of similar Burkholderia strains in the same infection model or of the same strain in different non-mammalian infection models was previously observed and proposed to be dependent on specific virulence factors. 23,24 Also, it was found that only a few B. cenocepacia virulence factors are required for full pathogenicity in multiple hosts, these including genes involved in quorum sensing signaling, siderophore production, lipopolysaccharide (LPS) biosynthesis, and purine, pyrimidine and shikimate biosynthesis. 23,24 Moreover, it was found that most of the B. cenocepacia mutants for virulence factors that were already tested in several infection models showed attenuation in only one infection host, similarly to what was described for P. aeruginosa.…”

“…23,24 Also, it was found that only a few B. cenocepacia virulence factors are required for full pathogenicity in multiple hosts, these including genes involved in quorum sensing signaling, siderophore production, lipopolysaccharide (LPS) biosynthesis, and purine, pyrimidine and shikimate biosynthesis. 23,24 Moreover, it was found that most of the B. cenocepacia mutants for virulence factors that were already tested in several infection models showed attenuation in only one infection host, similarly to what was described for P. aeruginosa. 37,38 However, only single mutants were tested for both species, which does not reflect the complex scenario that occurs in infected CF patients.…”

“…Results are the mean values of at least 3 independent experiments. Extracellular polysaccharides (EPS) production was visualized using mannitol agar plates supplemented with Congo red, as previously described, 24 after 24 h of incubation at 37 C and using 5 ml of B. cenocepacia cultures grown until the beginning of the stationary phase.…”

“…20 During the last decade, G. mellonella and C. elegans were established as infection models to assess both the virulence of different strains of Burkholderia cepacia complex (Bcc) species and the importance of specific bacterial genes in virulence, showing a good correlation with the pathogenicity observed in mice. [21][22][23][24] The isolates compared in the present study concerning the virulence potential, as well as a number of phenotypes that have been related with virulence, were the 11 sequential B. cenocepacia (recA lineage IIIA) clonal isolates retrieved from the onset of infection until patient s J death with the cepacia syndrome, including the 3 isolates examined before using human bronchial epithelial cells. [4][5][6]12 Given that this patient died after 3.5 y of chronic infection and thus the original strain could not have evolved toward a very long-term chronic infection, we have extended this study to 2 additional CF patients infected during 12.5 and 7.5 y with different strains also belonging to the species B. cenocepacia (recA lineage IIIA).…”

In-Frame and Unmarked Gene Deletions in Burkholderia cenocepacia via an Allelic Exchange System Compatible with Gateway Technology

“…Although a number of genes associated with virulence in B. cenocepacia have been identified (4,8,9) and tested in various infection models (10,11), it seems likely that the list of genes implicated in virulence is far from complete and will expand with genetic tools becoming available to manipulate B. cenocepacia strains. The deletion of genes potentially associated with virulence is a powerful way to investigate their function in bacterial physiology and pathogenesis.…”