The AcrAB efflux pump confers self-resistance to stilbenes in Photorhabdus laumondii.

The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus laumondii TT01 has emerged as a goldmine for novel anti-infective drug discovery. Outside plants, Photorhabdus is the only Gram-negative known to produce stilbene-derivatives including 3,5-dihydroxy-4-ethyl-trans-stilbene and 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS). IPS is a bioactive polyketide which received considerable attention, mainly because of its antimicrobial properties, and is currently in late-stage clinical development as a topical treatment for psoriasis and dermatitis. To date, little is known about how Photorhabdus survives in the presence of stilbenes. We combined genetic and biochemical approaches to assess whether AcrAB efflux pump exports stilbenes in P. laumondii. We demonstrated that the wild-type (WT) exerts an antagonistic activity against its derivative ΔacrA mutant, and that is able to outcompete it in a dual-strain co-culture assay. The ΔacrA mutant also showed high sensitivity to 3,5-dihydroxy-4-ethyl-trans-stilbene and IPS as well as decreased IPS concentrations in its supernatant comparing to the WT. We report here a mechanism of self-resistance against stilbene derivatives of P. laumondii TT01, which enables these bacteria to survive under high concentrations of stilbenes by extruding them out via the AcrAB efflux pump.

AcrAB, the major RND-type efflux pump of Photorhabdus laumondii, confers intrinsic multidrug-resistance and contributes to virulence in insects.

The resistance-nodulation-division (RND)-type efflux pumps AcrAB and MdtABC contribute to multidrug-resistance (MDR) in Gram-negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB-like (Plu0759-Plu0758). Only ΔacrA and ΔmdtAΔacrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue-triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis. Transcriptional analysis revealed intermediate levels of acrAB expression in vitro, in vivo and post-mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid-mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR-mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND-efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.

Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.