Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification - PubMed (original) (raw)

Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification

Kiyoshi Kawasaki et al. J Bacteriol. 2005 Apr.

Abstract

Salmonella enterica serovar Typhimurium remodels the lipid A component of lipopolysaccharide, a major component of the outer membrane, to survive within animals. The activation of the sensor kinase PhoQ in host environments increases the synthesis of enzymes that deacylate, palmitoylate, hydroxylate, and attach aminoarabinose to lipid A, also known as endotoxin. These modifications promote bacterial resistance to antimicrobial peptides and reduce the host recognition of lipid A by Toll-like receptor 4. The Salmonella lipid A 3-O-deacylase, PagL, is an outer membrane protein whose expression is regulated by PhoQ. In S. enterica serovar Typhimurium strains that had the ability to add aminoarabinose to lipid A, 3-O-deacylated lipid A species were not detected, despite the PhoQ induction of PagL protein expression. In contrast, strains defective for the aminoarabinose modification of lipid A demonstrated in vivo PagL activity, indicating that this membrane modification inhibited PagL's enzymatic activity. Since not all lipid A molecules are modified with aminoarabinose upon PhoQ activation, these results cannot be ascribed to the substrate specificity of PagL. PagL-dependent deacylation was detected in sonically disrupted membranes and membranes treated with the nonionic detergent n-octyl-beta-d-glucopyranoside, suggesting that perturbation of the intact outer membrane releases PagL from posttranslational inhibition by aminoarabinose-containing membranes. Taken together, these results suggest that PagL enzymatic deacylation is posttranslationally inhibited by membrane environments, which either sequester PagL from its substrate or alter its conformation.

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Figures

FIG. 1.

PhoP-PhoQ- and PmrA-PmrB-regulated lipid A modifications in S. enterica serovar Typhimurium. The phosphate residues and acyl chains of lipid A in S. enterica serovar Typhimurium can be derivatized in a PhoP-PhoQ- and PmrA-PmrB-regulated manner (reviewed in reference 5). Phosphate residues can be attached with

-Ara4N and/or phosphoethanolamine groups (shown in blue), both of which are under the control of PmrA-PmrB (11, 44). Minor species were present in which the locations of the

-Ara4N and phosphoethanolamine groups were reversed or in which both phosphates were modified with the same substituent (44). pmrE (also known as ugd) is predicted to encode a UDP-glucose dehydrogenase. The pmrF locus is an operon (pmrHFIJKLM) that carries seven open reading frames which are predicted to encode other enzymes involved in

-Ara4N synthesis and

-Ara4N transfer to lipid A (12, 40). Both the pmrE and pmrF loci are necessary for the PmrA-PmrB-regulated

-Ara4N attachment to lipid A (11). The addition of the palmitate chain is catalyzed by PagP (3, 15), the formation of the 2-hydroxymyristate group requires LpxO (8), and deacylation at the 3 position of lipid A is catalyzed by PagL (39) (shown in red). The pagL and pagP genes and lipid A hydroxylation are regulated by PhoP-PhoQ (2, 8). PhoP-PhoQ also activates PmrA-PmrB; therefore, the

-Ara4N and phosphoethanolamine modifications occur under PhoP-PhoQ-activating conditions (14, 43).

FIG. 2.

MALDI-TOF mass spectrometry of lipid A purified from mutant S. enterica serovar Typhimurium strains. Strains were cultivated in growth medium containing 10 mM MgCl2 (A) or 10 μM MgCl2 (B to L). Lipid A species prepared from CS019 (A and B), KCS039 (C), KCS040 (D), KCS042 (E), KCS043 (F), KCS044 (G), KCS045 (H), KCS049 (I), KCS050 (J), KCS044 transformed with pWKS30 (K), and KCS044 transformed with pWKS30-pmrE (L) were analyzed by use of a Bruker BiflexIII mass spectrometer. For the cultivation of transformants, 10 μg of ampicillin/ml was used (K and L). m/z values of lipid A species are shown, and those that represent deacylated lipid A species are marked with asterisks. Structural interpretations of lipid A species are summarized in Table 2.

FIG. 3.

Thin-layer chromatography analysis of 32P-labeled lipid A species purified from mutant S. enterica serovar Typhimurium strains. Strains were cultivated in growth medium containing 10 μM MgCl2. [32P]orthophosphate (10 μCi/ml) was added to the growth medium for the labeling of lipid A species. Lipid A species purified from CS019 (lane 1), KCS039 (lanes 2 and 4), KCS040 (lanes 3 and 5), KCS044 (lane 6), KCS045 (lane 7), KCS049 (lane 8), and KCS050 (lane 9) were spotted onto HPTLC plates and developed. To identify lipid A species, we prepared unlabeled lipid A species from KCS039 and KCS040 and cultivated them as described above, without [32P]orthophosphate. Unlabeled lipid A species were developed by HPTLC, and lipid A species were visualized by spraying water on the plate. Lipid A species were extracted from the plate and analyzed by use of a Bruker BiflexIII mass spectrometer (data not shown). Major m/z values of lipid A species are shown, and those that represent deacylated lipid A species are marked with asterisks. Structural interpretations of lipid A species are summarized in Table 2.

FIG. 4.

Expression of recombinant PagL in a _pmrA_-null mutant strain, but not in a wild-type pmrA strain, induced lipid A deacylation. (A) Lipid A species prepared from KCS040 transformed with pWKS30-_pagL_-His6 (a), CS283 transformed with pWKS30-_pagL_-His6 (b), and KCS040 transformed with pWKS30 (c) were analyzed by use of a Bruker BiflexIII mass spectrometer. Transformants were cultivated in growth medium containing 10 μM MgCl2 and 10 μg of ampicillin/ml. m/z values of lipid A species are shown, and those that represent deacylated lipid A species are marked with asterisks. Structural interpretations of lipid A species are summarized in Table 2. (B) Fifty-microgram samples of membrane proteins prepared from strains cultivated as described for panel A were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by staining (lanes 1 to 3) or by Western blotting (lanes 4 to 6). Lanes 1 and 4, KCS040 transformed with pWKS30-_pagL_-His6; lanes 2 and 5, CS283 transformed with pWKS30-_pagL_-His6; lanes 3 and 6, KCS040 transformed with pWKS30.

FIG. 5.

Sonic disruption of Salmonella membranes promotes PagL-dependent lipid A deacylation. Strains CS401 and CS586 were cultivated in growth medium containing 10 μM MgCl2. Cells collected from 25-ml cultures were suspended in 200 μl of 50 mM HEPES containing 250 mM NaCl, pH 7.5. Disruptions of cells were performed with a Virsonic 450 sonicator. Sonically disrupted and intact cells were incubated at 37°C for 1 h, and their lipid A species were analyzed by use of a Bruker BiflexIII mass spectrometer. (A) Intact CS401; (B) sonically disrupted CS401; (C) intact CS586; (D) sonically disrupted CS586. m/z values of lipid A species are shown, and those that represent deacylated lipid A species are marked with asterisks. Structural interpretations of lipid A species are summarized in Table 2.

FIG. 6.

Treatment of Salmonella membranes with _n_-octyl-β-

-glucopyranoside promotes PagL-dependent lipid A deacylation. Strains CS401 and CS586 were cultivated in growth medium containing 10 μM MgCl2. Collected cells were treated with _n_-octyl-β-

-glucopyranoside or Triton X-100 at a final concentration of 1%. Additionally, sonic disruptions of Triton X-100-treated cells were performed. The cells were incubated at 37°C for 1 h, and their lipid A species were analyzed by use of a Voyager-DE STR mass spectrometer. (A) Intact CS401; (B) 1% _n_-octyl-β-

-glucopyranoside (OG)-treated CS401; (C) 1% _n_-octyl-β-

-glucopyranoside-treated CS586; (D) 1% Triton X-100-treated CS401; (E) 1% Triton X-100-treated and sonically disrupted CS401; (F) 1% Triton X-100-treated and sonically disrupted CS586. m/z values of lipid A species are shown, and those that represent deacylated lipid A species are marked with asterisks. Structural interpretations of lipid A species are summarized in Table 2.

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Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification - PubMed (original) (raw)