The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection

Abstract

The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.

Genomic analyses of Burkholderia cenocepacia reveal multiple species with differential host-adaptation to plants and humans

“…To date, strains of B. cenocepacia s.s. and Burkholderia sp. nov. have been tested on nematodes, wax moth larvae and zebrafish and the strains respective virulence sometimes varied drastically depending on the tested host [51, 85, 86]. Tests that aim to study the human infection route through the lungs have only been performed with ET-12 representatives and we support that clinical research would greatly benefit if a wider diversity of strains including Burkholderia sp.…”

“…As the quality of genome sequences is heterogeneous for the strains used in this study, no comparison of the global genomic architecture was carried out. We screened the strains for the presence of the pC3 megaplasmid containing the virulence associated afc cluster [50, 51]. Although we cannot confirm its megaplasmid structure from the draft genomes, large genetic portions of the pC3 were detected in all strains but FL-5-3-30-S1-D7.…”

“…This replicon was shown to play an important role in the pathogenesis of various BCC strains, including B. cenocepacia , in diverse infection models [50]. Located on this pC3, the afc cluster and the adjacent transcriptional regulator shvR were recently demonstrated to be required for acute infection of B. cenocepacia in the zebrafish model [51]. However, the virulence levels of B. cenocepacia strains are strongly depending on the host model.…”

“…The specific core genomes of the two main clades, were screened for genes described as involved in host adaptation in the literature [50–54, 70, 71]. The complete sequence of the 875 kbp long virulence plasmid from B. cenocepacia J2315 and the 24 genes of the afc pathogenic cluster were also searched in all 31 genomes by BLAST [50, 51].…”

Presence of the Hmq system and production of 4-hydroxy-3-methyl-2-alkylquinolines is heterogeneously distributed betweenBurkholderia cepaciacomplex species and more prevalent among environmental than clinical isolates

“…Our results suggest that the Hmq system of Bcc is non-essential for pathogenicity but could be required for adaption to a particular environmental niche by, for example, influencing rhizosphere microbial community (Vial et al, 2008;Kilani-Feki et al, 2011Mahenthiralingam et al, 2011;Patten et al, 2012;Chapalain et al, 2013Chapalain et al, , 2017Gomes et al, 2018;Le Guillouzer, 2018;Jung et al, 2018;Mullins et al, 2019;Whalen et al, 2019;Piochon et al, 2020). It will be interesting to investigate a possible correlation between the presence of the hmqABCDEFG in Bcc species and their evolutionary trajectory.…”