Multiple Quorum Quenching Enzymes Are Active in the Nosocomial Pathogen Acinetobacter baumannii ATCC17978

Abstract

Acinetobacter baumannii presents a typical luxI/luxR quorum sensing (QS) system (abaI/abaR) but the acyl-homoserine lactone (AHL) signal profile and factors controlling the production of QS signals in this species have not been determined yet. A very complex AHL profile was identified for A. baumannii ATCC17978 as well as for A. nosocomialis M2, but only when cultivated under static conditions, suggesting that surface or cell-to-cell contact is involved in the activation of the QS genes. The analysis of A. baumanni clinical isolates revealed a strain-specific AHL profile that was also affected by nutrient availability. The concentration of OHC12-HSL, the major AHL found in A. baumannii ATCC17978, peaked upon stationary-phase establishment and decreases steeply afterwards. Quorum quenching (QQ) activity was found in the cell extracts of A. baumannii ATCC17978, correlating with the disappearance of the AHLs from the culture media, indicating that AHL concentration may be self-regulated in this pathogen. Since QQ activity was observed in strains in which AidA, a novel α/β-hydrolase recently identified in A. baumannii, is not present, we have searched for additional QQ enzymes in A. baumannii ATCC17978. Seven putative AHL-lactonase sequences could be identified in the genome and the QQ activity of 3 of them could be confirmed. At least six of these lactonase sequences are also present in all clinical isolates as well as in A. nosocomialis M2. Surface-associated motility and biofilm formation could be blocked by the exogenous addition of the wide spectrum QQ enzyme Aii20J. The differential regulation of the QQ enzymes in A. baumannii ATCC17978 and the full dependence of important virulence factors on the QS system provides a strong evidence of the importance of the AHL-mediated QS/QQ network in this species.

Quorum Sensing as a Target for Controlling Surface Associated Motility and Biofilm Formation in Acinetobacter baumannii ATCC® 17978TM

“…The exogenous addition of the QQ enzyme Aii20J, mixed with the inoculum, had a similar effect that the mutation of the synthase, completely blocking motility (Figure 2) in LNLS-LB plates. On the contrary and in accordance with the higher production of AHLs that has been reported in low-salt LB liquid cultures in comparison with LNLS-LB (Mayer et al, 2018), in the case of the low-salt LB-plates, mixing the enzyme with the inoculum was not enough to block the motility, and it was necessary to mix the enzyme with all the culture media in order to fully abrogate this phenotype. At 37 • C the abaI:Km mutant of A. nosocomialis M2 presented the same spreading phenotype as the parental strain in all culture media (Supplementary Figure 2).…”

“…Deletion of the abaI gene completely abolished motility under permissive conditions in A. baumannii ATCC R 17978 TM while almost no effect could be observed in the A. nosocomialis M2 abaI:Km mutant at 37 • C (Figure 1). The exogenous addition of OHC12-HSL, the major AHL signal found in the supernatants of A. baumannii ATCC R 17978 TM and A. nosocomialis M2 cultures (Niu et al, 2008;Mayer et al, 2018) was able to restore this phenotype in the abaI mutant of ATCC R 17978 TM under permissive conditions, confirming that AHL-mediated QS is crucial for surface-associated motility in this species, while no significant effect was observed when added to the wild type (Figure 1).…”

“…Surface-associated motility assays were performed as described by Mayer et al (2018). Petri dishes were prepared with LB or low-nutrients low-salt LB (0.5% NaCl, 0.2% tryptone, and 0.1% yeast extract; LNLS-LB) media supplemented with 0.25% Difco (Bacto TM Agar) or Eiken agar (Eiken Chemical, Co. Ltd., Japan).…”

“…Several regulatory proteins have been described as involved in the control of surface-associated motility in Acinetobacter spp. (Mayer et al, 2018;Moubareck and Halat, 2020). The AHL-mediated QS system seems to be central in this regulatory network since an impairment in surface motility was recorded for QS defective mutants in A. nosocomialis, a phenotype that could be restored by the addition of OHC12-HSL (Clemmer et al, 2011;Oh and Choi, 2015).…”

“…The role of QS on motility in A. baumannii is less studied, but recently, important differences in motility characteristics have been reported for clinical isolates of A. baumannii differing in their antibiotic resistance (López et al, 2017). In A. baumannii ATCC R 17978 TM (Mayer et al, 2018) and in some but not all the clinical strains studied (López et al, 2017), the addition of the AHL-degrading enzyme Aii20J reduced or even blocked motility completely, supporting a role of AHL-mediated QS on this phenotype in A. baumannii, as previously described for A. nosocomialis. However, the effect of the mutation of the QS genes on motility in A. baumannii and its correlation with biofilm formation has not been studied so far.…”