Identification Of Lipidated Class D Oxa beta-lactamases And Packaging Into Outer Membrane Vesicles In Acinetobacter Baumannii

LÓPEZ, MARÍA CAROLINA; CAPODIMONTE, LUCIA; BONOMO, ROBERT A.; VILA, ALEJANDRO J.

Houston, Texas

Congreso; ASM MICROBE 2023; 2023

American Society for Microbiology

Background: Most β-lactamases are soluble periplasmic enzymes in Gram-negative bacteria. Exceptionally, NDM metallo-β-lactamases have been characterized as lipoproteins anchored to the inner leaflet of the outer membrane. Membrane association promotes packaging of active NDM-1 in outer membrane vesicles (OMVs), which are able to protect populations of susceptible bacteria. Lipidation can be predicted based on the presence of a lipobox sequence in the signal peptide. Here we report that lipidation and membrane anchoring are frequent in the OXA β-lactamases from Acinetobacter baumannii, favoring packaging into OMVs. Methods: Sequence analysis was performed with LipoP - 1.0 lipoprotein predictor. OXA-23 and OXA-24 were expressed in their full-length form fused to a C-terminal Strep-tag in A. baumanniiATCC 17978. We performed subcellular fractionation after lysozyme/EDTA treatment and a mild osmotic shock. Then, the proteins wereidentified by immunodetection in the periplasm, cytoplasm and membrane fractions using Strep-tag II monoclonal antibodies. Membrane fractions were treated with NaCl 1 M, Na2CO3 0.1 M pH 11.5 and Triton X-100 1% (v/v) to assess membrane association mode. OMVs were purified by ultracentrifugation, and OXA levels intoOMVs were determined by immunodetection. The protective effect of OXA-loaded vesicles was tested by monitoring the growth of β-lactam-susceptible bacteria withOMVs and the β-lactam piperacillin at 64 μg/ml (8-fold the A. baumannii MIC). Results: More than half of 1212 reported OXA enzymes contain a lipobox sequence intheir signal peptides, most of them frequently found in Acinetobacter spp. Among them, we selected OXA-23 and OXA-24. When expressed in A. baumannii, these enzymes were only detected in membrane fractions. OXA-23 and 24 were solubilized only by treatment with Triton, revealing that membrane anchoring occurs byhydrophobic interactions. Both enzymes were present at high levels into OMVs produced by A. baumannii, with OXA-24 showing higher amounts. The packagedenzymes were active, since OXA-loaded OMVs were able to protect A. baumannii, E. coli and Pseudomonas aeruginosa cells susceptible to piperacillin. Conclusions:We report that β-lactamases OXA-23 and OXA-24 are membrane-bound lipoproteins in A. baumannii. The frequent finding of a lipobox in the signal peptides of OXAs from Acinetobacter spp. suggests that membrane anchoring and vesicle packaging represent an evolutionary advantage for this pathogen, particularly in polymicrobial infections.