The outer surface lipoprotein VolA mediates utilization of exogenous lipids by Vibrio cholerae

Abstract

Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling.

Importance: Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.

FIG 1

Growth of E. coli and Vibrio species on LPC or LCFA. (A) Chemical structures of PC (phosphatidylcholine), LPC (lysophosphatidylcholine), and an LCFA (long-chain fatty acid). (B) Both E. coli and V. cholerae can utilize LCFA as the sole carbon source for growth, while only V. cholerae can utilize LPC. (C) All Vibrio species tested were able to use LPC as the sole carbon source for growth, with the exception of V. fischeri.

FIG 2

Organization and expression of vca0862 and vca0863. (A) The genetic organization of vca0862 (FadL) and vca0863 (VolA) in V. cholerae, along with primer extension locations used in RT-PCR. (B) RT-PCR of vca0862 and vca0863 show that the genes are cotranscribed and basal levels of expression are independent of the presence of LPC. A genomic DNA template was used to confirm the amplified product sizes, and cDNA without reverse transcriptase (-RT) was used as the negative control verifying DNA-free RNA. (C) qRT-PCR data of the expression of vca0862 and vca0863 grown with glucose and/or LPC. When growth was in minimal medium containing LPC as the sole carbon source, expression of vca0862 and vca0863 increased 25- and 18-fold, respectively, compared to expression for a glucose-grown control.

FIG 3

Expression of vca0863 is required for utilization of LPC as the sole carbon source. (A) Growth of wild-type V. cholerae, vca0863 transposon mutant, and the complemented mutant on LPC. (B) Growth of wild-type E. coli and E. coli containing either pVcA0863 or pVcA0863-G20D on glucose, LCFA, or LPC.

FIG 4

Incorporation of LPC-derived fatty acids into membrane phospholipids of V. cholerae. (A) TLC of LCFA- and LPC-grown cultures of V. cholerae. V. cholerae strains, including the wild type, the vca0863 transposon mutant, and the vca0863-complemented mutant, were grown in the presence of 2 mM LCFA or LPC containing 18:2 and 18:3 unsaturated carbon chains that V. cholerae cannot synthesize de novo. All three strains show a shift in mobility for each of the major phospholipids when treated with LCFA, as expected. When treated with the LPC mix, only strains expressing vca0863 (wild type and complemented mutant) showed a similar shift, indicating that vca0863 is required for generating LPC-derived fatty acid. The dashed line has been included for comparison on mobility shifts. (B) Phosphatidylglycerol (PG) was isolated from wild-type, vca0863 mutant, and vca0863 mutant complement strains of V. cholerae and analyzed by liquid chromatography/ESI-mass spectrometry. Strains that express vca0863 showed a unique set of peaks (shown in red) corresponding to weights of PG that have acyl chains with unsaturations matching those in the LPC mix. These peaks were absent from the vca0863 mutant. (C) Tandem mass spectrometry (MS/MS) of the PG peak, with an m/z of 743.469 found only in vca0863-expressing strains. MS/MS showed that C_18:2 and C_18:3 acyl chains that originate with exogenous LPC are incorporated into the V. cholerae membrane.

FIG 5

Biotin labeling of surface-exposed VcA0863. (A) Antibiotin Western blot of soluble and insoluble protein fractions from either whole V. cholerae cells or cell lysates labeled with NHS-LC-LC-biotin. (B) SDS-PAGE and antibiotin Western blot of His-tagged VcA0863, β-lactamase, OmpT, and mislocalized VcA0863 affinity purified from cell extracts of biotin-labeled whole cells. Protein bands at ~81 kDa (VcA0863-His₆ and mislocalized VcA0863-His₆) and ~41 kDa (β-lactamase-His₆ and OmpT-His₆) in size were observed in SDS-PAGE, establishing that all proteins were running according to their molecular masses. The antibiotin Western blot showed two proteins, an 81-kDa band representing wild-type VcA0863 and a 41-kDa band representing OmpT. The fact that VcA0863 was labeled with biotin strongly indicates that VcA0863 is surface exposed. OmpT was also labeled due to its exposure on the surface of the cell. Little or no signal could be detected for either β-lactamase or mislocalized VcA0863 due to lack of surface exposure.

FIG 6

Immunogold electron micrographs of E. coli and V. cholerae strains expressing vca0863. Gold particles (examples are denoted with a red arrow) indicate the presence of surface-exposed VcA0863. Both E. coli expressing vca0863 from the plasmid and wild-type V. cholerae showed gold particles associated with the surface of the cell, indicating that VcA0863 is exposed to the surface in both strains. Wild-type E. coli or the V. cholerae vcA0863 mutant failed to display any associated gold particles on the bacterial surface. Mean gold particle counts are reported with standard errors (n > 10); statistical significance was observed between results for strains expressing and not expressing vca0863. *, P < 0.0005, Mann-Whitney U test.

FIG 7

Proposed model for VolA-dependent utilization of lysophospholipids. Exogenous LPC is initially cleaved by the surface-exposed lipoprotein VolA (VcA0863). One of the V. cholerae homologs of FadL (encoded by vc1042, vc1043, or vca0862) (see Fig. S4 in the supplemental material) can then transport the free fatty acid. Following transport across the periplasm and the inner membrane, the fatty acid is converted to an acyl-CoA by FadD. The activated fatty acid can be utilized for phospholipid biosynthesis or used as a carbon source for the bacterial cell.