Burkholderiacollagen-like protein 8, Bucl8, is a unique outer membrane component of a tetrapartite efflux pump inBurkholderia pseudomalleiandBurkholderia mallei
Authors: Grund Megan E , Choi Soo J , McNitt Dudley H , Barbier Mariette , Hu Gangqing , LaSala Paul R , Cote Christopher K. , Berisio Rita , Lukomski Slawomir 
Published: September 2020

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identifiedin silico Burkholderiacollagen-like protein 8 (Bucl8) in the Tier one select agentsBurkholderia pseudomalleiandBurkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of thebucl8gene locus revealed it resembles a classical efflux-pump operon. Thebucl8gene is co-localized with downstreamfusCDEgenes encoding fusaric acid (FA) resistance, and with an upstream gene, designated asfusR, encoding a LysR-type transcriptional regulator. Using RT-qPCR, we defined the boundaries and transcriptional organization of thefusR-bucl8-fusCDEoperon. We found exogenous FA inducedbucl8transcription over 80-fold inB. pseudomallei, while deletion of the entirebucl8locus decreased the MIC of FA 4-fold in its isogenic mutant. We furthermore showed that the Bucl8 pump expressed in the heterologousEscherichia colihost confers FA resistance. On the contrary, the Bucl8 pump did not confer resistance to a panel of clinically-relevant antimicrobials inBurkholderiaandE. coli. We finally demonstrated that deletion of thebucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

Author Summary

Burkholderia pseudomalleiandBurkholderia malleiare highly infectious and multidrug resistant bacteria that are classified by the National Institute of Allergy and Infectious Diseases as Tier one select agents partly due to the intrinsic multidrug resistance associated with expression of the efflux pumps. To date, only few efflux pumps predicted inBurkholderiaspp. have been studied in detail. In the current study we introduce Bucl8, an outer membrane component of an unreported putative efflux pump with a unique extended extracellular portion that forms a collagen triple helix and binds fibrinogen. We demonstrate Bucl8’s role in fusaric acid resistance by defining its operon via bioinformatic and transcriptional analyses, as well as by employing loss-of-function and gain-of-function genetic approaches. Our studies also implicate the Bucl8-associated pump in metabolic and physiologic homeostasis. Understanding how Bucl8 efflux pump contributes toBurkholderiapathology will foster development of pump inhibitors targeting transport mechanism or identifying potential surface-exposed vaccine targets.