Metabolic versatility of Acinetobacter baumannii: distinct pathways for the utilization of short-, medium- and long-chain acyl-L-carnitines.

Acinetobacter baumannii is a multidrug-resistant pathogen capable of metabolizing host-derived acyl-L-carnitines, which contributes to its virulence. During A. baumannii growth on acetyl-L-carnitine, L-carnitine was initially released into the growth medium suggesting that acetate is metabolized first. The subsequent uptake of L-carnitine at the end of the exponential phase then facilitates the catabolism of L-carnitine. Involvement of the betaine/choline/carnitine transporter (BCCT) in the uptake of short- and medium-chain acyl-L-carnitines was confirmed through growth experiments using the Δbcct mutant strain. An uncharacterized hydrolase (HMPREF0010_01348) encoded by the L-carnitine degradation gene operon (HMPREF0010_01345 - HMPREF0010_01351) was biochemically identified as a short- and medium-chain acyl-L-carnitine hydrolase (ACH). Its cellular localization in the cytoplasm was demonstrated. The purified ACH exhibited no enzymatic activity toward long-chain acyl-L-carnitines. However, growth experiments revealed that both the wild type and the Δach mutant were able to grow clearly on these substrates, providing evidence for the involvement of an additional, as-yet-uncharacterized enzyme. The cell culture supernatant was used for the chromatographic enrichment of a protein fraction showing robust enzyme activity for the hydrolysis of long-chain acyl-L-carnitines. Proteomic analysis of a partially purified hydrolase fraction and of the A. baumannii secretome revealed the gene product of open reading frame HMPREF0010_02333 as a hydrolase candidate. The respective protein was recombinantly overproduced and purified and its ability to cleave various long-chain acyl-L-carnitines was demonstrated. Based on the substrate specificity of the characterized ACH from the cytosol and the acyl-L-carnitine hydrolase present in the culture supernatant (ACHS), a metabolic model for the versatile utilization of small- medium- and long-chain acyl-L-carnitines by A. baumannii was proposed.