Alterins Produced by Oyster-Associated Pseudoalteromonas Are Antibacterial Cyclolipopeptides with LPS-Binding Activity

Abstract

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins' mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

Keywords: Pseudoalteromonas; alterin; antibiotic; cyclolipopeptides.

Figure 1

Maximum likelihood tree using the Tamura-Nei model [16,17] indicating the phylogenetic relationships inferred from partial 16S rRNA gene sequences of alterin-producing Pseudoalteromonas strains (in blue) within the Pseudoalteromonas genus. Bootstrap values (expressed as percentage of 1000 replications) >50% are shown at branching point. A group of four bacteria was used as outgroup and tree was constructed using MEGA X [18].

Figure 2

RP-HPLC analyses of the cell-free supernatants of Pseudoalteromonas hCg-6 (blue) and hCg-42 (black) strains. (A) Analytic chromatograms of cell-free supernatants (150 µL) of Pseudoalteromonas hCg-6 (blue) and hCg-42 (black). The grey bar indicates antibacterial activity. (B) Semi-preparative chromatogram arising from the injection of the bioactive fraction (2 mL) resulting the solid phase extraction of Pseudoalteromonas hCg-6 cell-free supernatant. The values framed indicate the m/z values resulting from MS analyses.

Figure 3

Chemical structure of the alterins. from Pseudoalteromonas hCg-6 and hCg-42 strains. *: hydroxylation at C₃ position.

Figure 4

lipopolysaccharide (LPS)-binding properties of alterins. (A) Dose-dependent effects of alt_954 (■), alt_970 (▲), alt_980 (●) and Polymyxin B (PMB) (◆) on LPS detection using the Limulus Amoebocyte Lysate (LAL) assay. (B) LPS-binding of alt_954 (■), alt_970 (■) and PMB (■) expressed as resonant units (RU).

Figure 5

LPS-bound structure of alt_980. (A) Overlay of the 20 lowest energy structures with no violations >0.3 Å from the 100 structures calculated with AMBER software for alt_980 in presence of LPS micelles. Overlay was performed using the backbone atoms of the peptidic cycle. (B) Detail of the lowest energy structure displaying the hydrogen bonds between carbonyl of 7-OH-Dab and amide protons of 3-Leu and 4-Arg. Aliphatic protons are omitted for clarity.

Figure 6

Flow cytometry analyses of Vibrio tasmaniensis LGP32 after a 4h-hour-long incubation with alterins (20 µM). (A) without antibacterial peptide in reconstituted seawater (negative control), (B) with PMB (20 µM) (positive control), (C) with alt_952, (D) with alt_954, (E): with alt_970 and (F) with alt_980.

Figure 7

Physiological impact of alterins at 20 µM on Vibrio tasmaniensis LGP32 after a 4h-long incubation in sea salts (3%): (A) viable cells expressed as CFU.mL⁻¹ (●) and cells exhibiting a depolarized membrane (histograms (■)), (B) intact cells () and cells exhibiting an altered membrane with low () and high () nucleic acid content.