Takayuki Manabe and Kiyoshi Kawasaki* Faculty of Pharmaceutical Sciences, Doshisha Women s College, Kyotanabe, Kyoto , Japan

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 2008, p Vol. 190, No /08/$ doi: /jb Copyright 2008, American Society for Microbiology. All Rights Reserved. Extracellular Loops of Lipid A 3-O-Deacylase PagL Are Involved in Recognition of Aminoarabinose-Based Membrane Modifications in Salmonella enterica Serovar Typhimurium Takayuki Manabe and Kiyoshi Kawasaki* Faculty of Pharmaceutical Sciences, Doshisha Women s College, Kyotanabe, Kyoto , Japan Received 29 April 2008/Accepted 12 June 2008 Salmonella enterica serovar Typhimurium modifies its lipopolysaccharide (LPS), including the lipid A portion, in response to changes in its environment including host tissues. The lipid A 3-O-deacylase PagL, the expression of which is promoted under a host-mimetic environment, exhibits latency in S. enterica; deacylation of lipid A is not usually observed in vivo, despite the expression of the outer membrane protein PagL. In contrast, PagL does not exhibit latency in S. enterica pmra and pmre mutants, both of which are deficient in the aminoarabinose-based modification of lipid A, indicating that aminoarabinose-modified LPS species were involved in the latency. In order to analyze the machinery for PagL s repression, we generated PagL mutants in which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipid A 3-O deacylation was observed in S. enterica expressing the recombinant mutants PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A), but not in S. enterica expressing wild-type PagL, suggesting that the point mutations released PagL from the latency. In addition, mutations at Arg-43, Arg-44, Cys-85, and Arg-135 did not affect lipid A 3-O-deacylase activity in an S. enterica pmra mutant or in Escherichia coli BL21(DE3). These results, taken together, indicate that specific amino acid residues located at extracellular loops of PagL are involved in the recognition of aminoarabinose-modified LPS. Furthermore, S. enterica expressing the recombinant PagL(R43A) or PagL(R135A) mutant showed apparent growth arrest at 43 C compared with S. enterica expressing wild-type PagL, indicating that the latency of PagL is important for bacterial growth. * Corresponding author. Mailing address: Faculty of Pharmaceutical Sciences, Doshisha Women s College, Kodo, Kyotanabe, Kyoto , Japan. Phone: Fax: kkawasak@dwc.doshisha.ac.jp. Published ahead of print on 20 June Exclusive areas of the outer leaflet of the outer membrane are occupied by lipopolysaccharide (LPS) molecules in gramnegative bacteria (reviewed in reference 33). LPS consists of a hydrophobic membrane anchor portion known as lipid A and a nonrepeating core oligosaccharide coupled to a distal polysaccharide (O antigen) that extends from the bacterial surface (reviewed in reference 37). The outer membrane of pathogenic gram-negative bacteria, including Salmonella enterica serovar Typhimurium, functions as a barrier to harmful host-derived compounds, such as antimicrobial peptides and reactive oxygen. The protection is a consequence of strong lateral interaction between LPS molecules (reviewed in reference 33). In response to environmental signals, including those from the host, S. enterica covalently modifies its lipid A through palmitoylation, deacylation, formation of a 2-hydroxymyristate group (hydroxylation), and addition of 4-amino-4-deoxy-L-arabinose (aminoarabinose) or phosphoethanolamine (Fig. 1) (reviewed in references 9, 36, and 37). These modifications are implicated in the virulence of pathogenic gram-negative bacteria in that they increase resistance to cationic antimicrobial peptides (17, 29, 34). On the other hand, the lipid A portion of LPS, which is known as endotoxin, induces inflammatory responses through recognition by Toll-like receptor 4 (TLR4) MD2 complex (reviewed in reference 32). The modifications of lipid A in S. enterica were implicated in a reduction of recognition through the TLR4-MD2 complex (26, 44), leading to the evasion of host immune responses. A two-component regulatory system, PhoP-PhoQ, which is essential for the pathogenesis of S. enterica (11, 16, 31), promotes the expression of genes involved in lipid A modifications (20). PhoQ is a sensor histidine kinase that responds to environmental conditions, including those within mammalian tissues, which are mimicked by magnesium-limited growth medium (11, 12, 16, 31). In response to specific environmental factors, such as magnesium limitation (12), low ph (3, 35), and antimicrobial peptides (1, 2), PhoQ phosphorylates PhoP, leading to the activation of pagl and pagp encoding lipid A 3-O-deacylase and lipid A palmitoyltransferase, respectively (4, 6, 21, 45). Since 3-O deacylation decreases the endotoxic activity of lipid A, the modification is thought to help S. enterica evade immunosurveillance (26). Activation of PhoP-PhoQ leads to the activation of a second two-component regulatory system, PmrA-PmrB (18, 24), which promotes the expression of genes involved in the attachment of aminoarabinose and phosphoethanolamine to phosphate groups on lipid A (Fig. 1). The modification with aminoarabinose is essential for PhoP- PhoQ-dependent resistance to cationic antimicrobial peptides, including polymyxin B (17, 18, 29, 34). In addition to gene expression, the posttranslational regulation of outer membrane enzymes, including lipid A 3-O-deacylase PagL, is suggested to be involved in the regulation of lipid A s modifications. Previous studies found that lipid A was not deacylated despite the induction of PagL expression in S. enterica (27, 45); therefore, PagL is thought to be latent in the outer membrane (27). In contrast, PagL-dependent deacylation of lipid A was detected in pmra, pmre, and pmrf mutants 5597

2 5598 MANABE AND KAWASAKI J. BACTERIOL. Downloaded from FIG. 1. PhoP-PhoQ-regulated lipid A modifications in S. enterica. (A) Prototype lipid A of S. enterica serovar Typhimurium. (B) Modified lipid A of S. enterica serovar Typhimurium. The phosphate residues and acyl chains of lipid A of S. enterica can be derivatized in a PhoP-PhoQ- or PmrA-PmrB-regulated manner (reviewed in reference 9). Aminoarabinose and/or phosphoethanolamine groups can be attached to phosphate residues, under the control of PmrA-PmrB (20, 49). Minor species were present in which the locations of the aminoarabinose and phosphoethanolamine groups were reversed or in which both phosphates were modified with the same substituent (49). Both the pmrf operon and pmre are necessary for the PmrA-PmrB-regulated attachment of aminoarabinose to lipid A (17, 49). The pmrc gene mediates the PmrA-PmrB-regulated attachment of phosphoethanolamine to lipid A (29). The addition of the palmitate chain is catalyzed by PagP (6, 21), the formation of the 2-hydroxymyristate group requires LpxO (15), and the deacylation at position 3 of lipid A is catalyzed by PagL (45). The pagl and pagp genes are regulated by PhoP-PhoQ (4), and the lpxo gene is partly regulated by PhoP-PhoQ (14, 15). PhoP-PhoQ also activates PmrA-PmrB; therefore, the aminoarabinose and phosphoethanolamine modifications occur under PhoP-PhoQ-activating conditions (20, 48). deficient in the aminoarabinose-based modification of lipid A (27). In addition, the release of PagL from latency in the S. enterica pmra mutant partly compensated for susceptibility to polymyxin B, which was caused by a lack of modification of lipid A (25). These results, taken together, suggest that aminoarabinose-containing outer membranes directly inactivate the lipid A 3-O-deacylase activity of PagL. Alternatively, modification of lipid A with aminoarabinose inhibits the physical interaction of LPS with PagL. In this study, we generated PagL mutants in which an amino acid residue located at four extracellular loops was replaced with alanine and examined the effect on the latency. Several mutations apparently released PagL from its latency, indicating the extracellular loops are involved in the recognition of aminoarabinose-modified lipid A. Furthermore, S. enterica expressing PagL mutants that lost the latency exhibited growth arrest at 43 C, implying the physiological importance of the latency for cell growth under specific conditions. MATERIALS AND METHODS Materials. All chemicals were of reagent grade or better. Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs (Beverly, MA) and Takara Bio (Ohtsu, Japan). Oligonucleotides were prepared commercially by Greiner Japan (Tokyo, Japan). Prestained molecular weight standards were from Apro Science (Naruto, Japan). 2, 5-Dihydroxybenzoic acid was from Sigma-Aldrich (St. Louis, MO), and 5-chloro-2-mercaptobenzothiazole was from Wako Chemicals (Osaka, Japan). Proteinase K was from Roche Diagnosis (Basel, Switzerland). Bacterial strains and growth conditions. CS283 (14028s phon2 zxx::6251 Tn10d-Cam pagl1::tnphoa) (27, 31) and KCS040 (14028s phon2 zxx::6251 Tn10d-Cam pagl1::tnphoa pmra::tn10d) (27), which are derivatives of S. enterica serovar Typhimurium strain 14028s (American Type Culture Collection, Manassas, VA), were used in this study. Unless otherwise indicated, S. enterica cells were grown at 37 C with aeration in N-minimal medium [5 mm KCl, 7.5 on August 26, 2018 by guest

3 VOL. 190, 2008 RELEASE OF PagL FROM LATENCY BY POINT MUTATIONS 5599 mm (NH 4 ) 2 SO 4, 0.5 mm K 2 SO 4,1mMKH 2 PO 4, 0.28% glycerol (vol/vol), 0.1% (wt/vol) Casamino Acids, 0.2 mg of thiamine/liter, and 0.1 M Tris-HCl (ph 7.4)] supplemented with 10 M MgCl 2. Ampicillin (10 g/ml) was used for the cultivation of strains transformed with the low-copy vector pwks30 (46) or its derivatives. S. enterica colonies were picked out and grown overnight at 37 C in 1 to 10 ml of growth medium. Unless otherwise indicated, the overnight cultures were diluted 1:10 with fresh growth medium and then grown at 37 C for 12 h. After the cultivation, stationary-phase cells were used for further analysis. E. coli BL21(DE3) (Invitrogen) cells were grown at 37 C with aeration in LB medium. Ampicillin (100 g/ml) was used for the cultivation of strains transformed with pbluescript II KS( ) (Stratagene, La Jolla, CA) or its derivatives. E. coli colonies were picked out and grown overnight at 37 C in 3 ml of growth medium. The overnight cultures were diluted to an optical density at 600 nm of 0.1 and then grown at 37 C for 16 h. After the cultivation, stationary-phase cells were used for further analysis. Bacterial genetic and molecular biology techniques. Plasmid DNA was introduced into bacterial strains by electroporation using a Gene Pulser (Bio-Rad, Hercules, CA) following the manufacturer s instructions. Recombinant DNA techniques were performed according to standard protocols (41). PagL mutant plasmid constructs. S. enterica serovar Typhimurium PagL mutants were generated by PCR-based overlap extension with Pfu Turbo DNA polymerase (Stratagene). The sequences of the PCR primers are available upon request. The expression construct pwks30-pagl-his 6 (27) was used as a template, and every mutant pagl gene bears a His 6 epitope at the C terminus and the 79-bp upstream region of pagl. The mutants were cloned into EcoRI/BamHI sites of the low-copy vector pwks30 (46) or pbluescript II KS( ). The name of each mutant construct includes the wild-type residue (single-letter amino acid designation) followed by the codon number and mutant residue (typically alanine). The amplified insert in the plasmid constructs was verified by DNA sequencing. Preparation of lipid A. The lipid A used for mass spectrometry was purified as described previously (47). In brief, cells collected from 25 ml of culture were resuspended in 500 l of Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH). After incubation for 15 min at room temperature, 100 l of chloroform was added. After 30 min, the mixture was centrifuged, and the aqueous phase was recovered. LPS was extracted three times by the addition of 500 l of water to the organic phase, and the aqueous phase containing LPS was dried with a vacuum concentrator. Five hundred microliters of 10 mm sodium acetate buffer (ph 4.5) containing 1% sodium dodecyl sulfate (SDS) was add to the dried LPS, and then the LPS was hydrolyzed to remove sugar chains from lipid A by incubation at 95 C for 1 h (39) followed by drying. The dried lipid A was washed twice with 0.02 N HCl in 99.5% ethanol and three times with 99.5% ethanol. The washed lipid A was dried with a vacuum concentrator and then used for mass spectrometric analysis. Alternatively, the lipid A used for the detection of aminoarabinose-modified lipid A species was prepared as described previously (7, 25). In brief, LPS purified from 25 ml of cell culture using an LPS extraction kit (intron Biotechnologies, Inc., Seongnam-Si, Korea) was hydrolyzed for 3hat95 C in 150 l of 100 mm sodium acetate buffer (ph 4.5). Then, 600 l of a chloroformmethanol mixture (1:2 [vol/vol]), 200 l of chloroform, and 100 l of phosphatebuffered saline were added in succession, and the lipid A fraction (chloroform phase) was dried under a stream of nitrogen gas. Mass spectrometry. Dried lipid A was dissolved in 20 mg/ml of 5-chloro-2- mercaptobenzothiazole matrices in chloroform-methanol (1:1 [vol/vol]). Alternatively, it was dissolved in chloroform-methanol (1:2 [vol/vol]) for the detection of aminoarabinose-modified lipid A species and then mixed with 77 mg/ml of 2,5-dihydroxybenzoic acid matrices in methanol at a ratio of 1:1. The mixtures were allowed to dry at room temperature on the sample plate prior to analysis. Spectra were obtained in the negative-reflection mode using a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) Voyager-DE STR mass spectrometer (Applied Biosystems Japan, Tokyo, Japan). Each spectrum was the average of 200 shots. Structural interpretations of lipid A species detected by mass spectrometry in this study are summarized in Table 1. Membrane preparation. All steps were carried out at 4 C or on ice. Cells collected from 20 ml of bacterial culture were suspended in 300 l of phosphatebuffered saline and then sonically disrupted three times for 10 s each at 1-min intervals at setting 1 with a Branson sonifier model S-150D. The crude lysate was cleared by centrifugation at 1,000 g for 5 min. Membranes were precipitated by centrifugation at 100,000 g for 30 min and were resuspended in 100 l of phosphate-buffered saline. Protein concentrations were determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) using bovine serum albumin as a standard. TABLE 1. Structural interpretations of lipid A species detected by mass spectrometry in this study m/z Lipid A modification(s) a 1,572...Deacylation 1,588...Deacylation and hydroxylation 1,711...Deacylation, hydroxylation, and phosphoethanolamine attachment 1,798...Unmodified 1,810...Deacylation and palmitoylation 1,814...Hydroxylation 1,826...Deacylation, palmitoylation, and hydroxylation 1,906...Deacylation, palmitoylation and hydroxylation (pyrophosphate lipid A) b 1,937...Hydroxylation and phosphoethanolamine attachment 1,945...Aminoarabinose attachment and hydroxylation 1,957...Aminoarabinose attachment, deacylation, palmitoylation, and hydroxylation 2,036...Palmitoylation 2,052...Palmitoylation and hydroxylation 2,132...Hydroxylation and palmitoylation (pyrophosphate lipid A) b 2,175...Palmitoylation, hydroxylation, and phosphoethanolamine attachment a Interpretations were based on previous works (19, 25, 27, 45, 48). b Pyrophosphate lipid A has a pyrophosphate residue instead of a monophosphate residue at position 1 of the disaccharide glucosamine of lipid A. Regulation of pyrophosphate lipid A synthesis is not well understood (48). SDS-polyacrylamide gel electrophoresis and Western blotting. Proteins were fractionated by SDS 12.5% polyacrylamide gel electrophoresis under reducing conditions (28). Proteins separated on the gel were stained with Coomassie blue. For the Western blot analysis, proteins separated on the gel were electroblotted onto a nitrocellulose membrane in 25 mm Tris 192 mm glycine 0.02% SDS 20% methanol at 22 V/cm for 60 min. Then the blot was incubated with antitetra-his antibodies (Qiagen, Valencia, CA) and subsequently incubated with anti-mouse immunoglobulin G linked to horseradish peroxidase (GE Healthcare Bio-Sciences, Piscataway, NJ). Cross-reactive proteins were detected with ECL enhanced chemiluminescence Western blotting detection reagents (GE Healthcare Bio-Sciences). Analysis of LPS by Tricine-SDS-polyacrylamide gel electrophoresis. LPS from E. coli BL21(DE3) strains was prepared as described previously (13, 22, 43) with slight modifications. In brief, bacterial cells from 1.0 ml of culture, diluted to an optical density at 600 nm of 1.0, were collected, and suspended in 100 l of sample buffer (100 mm Tris-HCl [ph 6.8], 20% glycerol [vol/vol], 4% [wt/vol] SDS, % [wt/vol] bromophenol blue, 5% [vol/vol] 2-mercaptoethanol). The samples were boiled for 10 min prior to digestion with proteinase K at a final concentration of 1 mg/ml for 16 h at 55 C, followed by boiling for 5 min to inactivate proteinase K. The LPS samples were subjected to Tricine-SDS-polyacrylamide gel electrophoresis (30). The separating gel was prepared at a final concentration of 18% acrylamide, 1 M Tris-HCl (ph 8.45), 5% glycerol, and 0.05% SDS. The stacking gel was prepared at a final concentration of 4% acrylamide, 1.67 M Tris-HCl (ph 8.45), and 0.083% SDS. The samples were loaded under electrophoresis buffer (0.1 M Tris-HCl [ph 8.3], 0.1 M Tricine, 0.1% SDS) and allowed to run at 30 V for 50 min and then at 105 V for 215 min. The gels were fixed overnight in 11:8:1 (vol/vol/vol) water-ethanol-acetic acid and subsequently stained with a silver staining kit from Daiichi Pure Chemicals, Tokyo, Japan, according to the manufacturer s instructions.

4 5600 MANABE AND KAWASAKI J. BACTERIOL. FIG. 2. Topology model for S. enterica PagL. A model for the topology of S. enterica serovar Typhimurium PagL was constructed based on the sequence similarity to P. aeruginosa PagL (13). The proposed model consists of an eight-stranded -barrel with four loops (L1 to L4) extending into the external environment. Residues in the postulated -strands are shown in squares. Numbers refer to the position of residues in the precursor sequence. Asn-21 was identified as the N-terminal amino acid residue of S. enterica PagL after cleavage of the signal peptide (13). Downloaded from RESULTS Alanine-scanning mutagenesis of extracellular loops of S. enterica PagL. The outer membrane protein PagL was predicted to consist of an eight-stranded -barrel with four loops extending into the external environment (13, 40). The PagL of S. enterica serovar Typhimurium is unique in that it is latent in aminoarabinose-containing outer membranes (27). We speculated that the extracellular loops (L1 L4) of this PagL (Fig. 2) sense aminoarabinose-containing outer membranes. Previously, Kawasaki et al. demonstrated that the introduction of a low-copy-vector-based expression construct containing a recombinant PagL into a S. enterica pmra pagl mutant, which is deficient in the aminoarabinose-based modification of lipid A, induced lipid A deacylation, but introduction into an S. enterica pmra pagl strain did not (27). These results prompted us to screen for PagL mutants that are no longer latent in S. enterica pmra strains. We generated low-copy expression constructs containing mutant PagL, in which an amino acid residue located at four loops (L1 L4) extending into the external environment (Fig. 2) was replaced with alanine (Table 2). The expression constructs were introduced into an S. enterica pmra pagl strain, and the structure of lipid A prepared from the resultant transformants cultivated in magnesium-limited growth medium, which activates the PhoP-PhoQ two-component regulatory system, was analyzed by MALDI-TOF mass spectrometry. Introduction of expression constructs containing most PagL mutants as well as wild-type PagL induced the production of a negligible or undetectable amount of 3-O-deacylated lipid A species (Fig. 3A and Table 2). In contrast, the introduction of several expression constructs containing PagL mutants, such as PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A), induced apparent lipid A 3-O deacylation (Fig. 3B to E and Table 2). Since aminoarabinose-modified lipid A species were not observed well under standard detection conditions for MALDI-TOF mass spectrometry, as described previously (25), the existence of aminoarabinose-modified lipid A species in the transformants was confirmed by using 2,5-dihydroxybenzoic acid as a matrix (inset of Fig. 3). The expression levels of recombinant PagL proteins in the transformants were confirmed to be similar by Western blot analysis of the membrane preparations (Fig. 4). These results, taken together, suggest that PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A) lost the ability to be latent in vivo in the presence of aminoarabinose-modified lipid A species. In addition, the introduction of the PagL(S41A), PagL(I42A), PagL(D83A), PagL(D133A), PagL(V136A), PagL(N137A), and PagL(K172A) mutants into the S. enterica pmra pagl strain induced moderate levels of lipid A 3-O deacylation, suggesting that these mutants also lost the ability to be latent in vivo in the presence of aminoarabinose-modified lipid A species (Table 2). Arg-43, Arg-44, and Arg-135 are essential for the latency of S. enterica pagl. Aminoarabinose-modification of lipid A decreases the net anionic charge at this position and the electrostatic on August 26, 2018 by guest

5 VOL. 190, 2008 RELEASE OF PagL FROM LATENCY BY POINT MUTATIONS 5601 TABLE 2. MALDI-TOF mass spectrometry of lipid A prepared from S. enterica Typhimurium strain CS283 transformed with low-copy expression vector pwks30 containing wild-type or mutant PagL Lipid A PagL form or mutation deacylation level a Wild type...1 Mutants Replacement of amino acid residue with alanine Loop 1 S41A...2 I42A...2 R43A...4 R44A...3 Loop 2 G76A...1 F77A...1 K78A...1 K80A...1 G81A...1 S82A...1 D83A...2 D84A...1 C85A...3 S86A...1 K87A...1 Loop 3 I126A...1 K127A...1 S128A...1 K129A...1 S130A...1 R131A...1 D132A...1 D133A...2 M134A...1 R135A...4 V136A...2 N137A...2 S138A...1 F140A...1 T141A...1 F142A...1 Loop 4 N166A...1 G167A...1 S168A...1 L169A...1 T170A...1 D171A...1 K172A...2 N173A...1 S174A...1 G175A...1 H176A...1 N177A...1 Replacement of amino acid residue with other than alanine R43K...3 R43H...3 R43Q...4 R44K...2 C85M...3 C85V...3 C85S...3 D133E...1 R135K...3 R135H...3 R135Q...3 a Lipid A deacylation levels were determined by MALDI-TOF mass spectrometry and are defined as follows: 1, lipid A deacylation was not observed or a negligible peak was detected (Fig. 3A); 2, small but apparent peaks that correspond to deacylated lipid A species are observed (Fig. 5B); 3, approximately half of the peaks corresponded to deacylated lipid A (Fig. 3C and D and Fig. 5A and C); 4, almost all peaks corresponded to deacylated lipid A species (Fig. 3B and E). repulsion between neighboring LPS molecules (33). It is plausible that the positive charges of the Arg-43, Arg-44, and Arg-135 residues of S. enterica PagL are involved in the direct interaction with aminoarabinose-modified lipid A species. In order to exam- FIG. 3. Introduction of PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A) mutants, but not wild-type PagL, induced LPS deacylation in an S. enterica pmra strain. Lipid A prepared from S. enterica serovar Typhimurium pmra pagl strain CS283 transformed with pwks30 containing wild-type PagL (A), PagL(R43A) (B), PagL(R44A) (C), PagL(C85A) (D), or PagL(R135A) (E) was analyzed by MALDI-TOF mass spectrometry. Insets in panels show results of MALDI-TOF mass spectrometry of lipid A using 2, 5-dihydroxybenzoic acid matrices. The m/z values of lipid A species are shown, and those that represent deacylated lipid A species are denoted by asterisks. The structural interpretations of lipid A species are summarized in Table 1. The results are representative of at least two independent experiments.

6 5602 MANABE AND KAWASAKI J. BACTERIOL. FIG. 4. Expression levels of recombinant PagL proteins were similar among S. enterica pmra strains transformed with expression constructs containing wild-type PagL, PagL(R43A), PagL(R44A), PagL(C85A), or PagL(R135A). Ten-microgram samples of membrane proteins prepared from S. enterica serovar Typhimurium pmra pagl strain CS283 transformed with the pwks30 vector (vector) or pwks30 containing wildtype PagL, PagL(R43A), PagL(R44A), PagL(C85A), or PagL(R135A) were subjected to SDS 12.5% polyacrylamide gel electrophoresis and analyzed by staining with Coomassie blue (A) or by Western-blotting using anti-tetra-his antibody (B). ine whether these positive charges are essential for the latency or arginine residues at the positions are essential, we generated PagL(R43K), PagL(R44K), and PagL(R135K) mutants in which a cationic arginine residue was replaced with a cationic lysine residue. The PagL mutants were introduced into the S. enterica pmra pagl strain, and the structures of lipid A species in the transformants were analyzed by MALDI-TOF mass spectrometry. Apparent 3-O-deacylated lipid A species were observed in lipid A prepared from the strains transformed with the PagL (R43K), PagL(R44K), and PagL(R135K) mutants (Fig. 5), suggesting that replacement of arginine with lysine at position 43, 44, or 135 resulted in PagL losing the ability to be latent. In addition, apparent 3-O deacylation was also induced by introduction of PagL(R43H), PagL(R43Q), PagL(R135H), or PagL(R135Q), indicating that these mutants also lost the ability to be latent (Table 2). Furthermore, PagL(R43K), PagL(R44K), and PagL(R135K) were introduced into the S. enterica pmra pagl double-mutant strain, and the lipid A prepared from each transformant was analyzed. The introduction of the expression construct containing PagL(R43K), PagL(R44K), or PagL(R135K) induced levels of deacylation similar to those in the S. enterica pmra pagl strain transformed with the expression construct containing wild-type PagL, suggesting that the replacement of arginine with lysine at position 43, 44, or 135 did not affect the lipid A 3-O-deacylase activity (Fig. 6). The levels of recombinant PagL in the strains transformed with the expression constructs containing wild-type PagL, PagL(R43K), PagL(R44K), and PagL(R135K) were confirmed to be similar by Western blotting (data not shown). Previously, the lipid A 3-O-deacylase activity of S. enterica PagL was examined by heterologous expression in E. coli (13). Therefore, E. coli BL21(DE3) was transformed with the expression construct containing wild-type PagL, PagL(R43A), PagL(R44A), or FIG. 5. Introduction of PagL(R43K), PagL(R44K), and PagL (R135K) induced LPS deacylation in an S. enterica pmra strain. Lipid A prepared from S. enterica serovar Typhimurium pmra pagl strain CS283 transformed with pwks30 containing PagL(R43K) (A), PagL(R44K) (B), or PagL(R135K) (C) was analyzed by MALDI-TOF mass spectrometry. The m/z values of lipid A species are shown, and those that represent deacylated lipid A species are denoted by asterisks. The structural interpretations of lipid A species are summarized in Table 1. The results are representative of at least two independent experiments. PagL(R135A), and LPS prepared from the resultant transformants was analyzed by Tricine-SDS-polyacrylamide gel electrophoresis. Introduction of the PagL(R43A), PagL(R44A), or PagL(R135A) mutant into E. coli BL21(DE3) induced levels of LPS modification similar to that in the E. coli strain transformed with wild-type PagL (Fig. 7). These results, taken together, indicate that Arg-43, Arg-44, and Arg-135of S. enterica PagL were essential for the latency, and mutations at these positions did not affect the lipid A 3-O-deacylase activity. The importance of the arginine residues could not simply be attributed to their positive charge. In addition, to evaluate the importance of Cys-85 for latency, PagL(C85M), PagL(C85V), and PagL(C85S) mutants were generated. Apparent 3-O-deacylated lipid A species were observed in S. enterica transformed with the expression construct containing the PagL(C85M), PagL(C85V), or PagL(C85S) mutant (Table 2). In addition, introduction of PagL(C85A) into E. coli BL21(DE3) induced the modification of LPS to levels similar to that in E. coli transformed with wild-type PagL (Fig. 7). These results, taken together, indicate that Cys-85 is involved in latency. Asn-173 and Asn-177 of PagL were essential for lipid A 3-O-deacylase activity. The low-copy expression constructs containing mutants, in which an amino acid residue located at the extracellular loops (L1 L4) was replaced with alanine, were introduced into the S. enterica pmra pagl strain, in which lipid A is not modified by aminoarabinose. Lipid A was pre-

7 VOL. 190, 2008 RELEASE OF PagL FROM LATENCY BY POINT MUTATIONS 5603 FIG. 6. Introduction of PagL(R43K), PagL(R44K), and PagL (R135K) mutants into an S. enterica pmra mutant strain induced LPS deacylation to similar levels as those induced by introduction of wild-type PagL. Lipid A prepared from S. enterica serovar Typhimurium pmra pagl double-mutant strain KCS040 transformed with pwks30 containing wildtype PagL (A), PagL(R43K) (B), PagL(R44K) (C), or PagL(R135K) (D) was analyzed by MALDI-TOF mass spectrometry. The m/z values of lipid A species are shown, and those that represent deacylated lipid A species are denoted by asterisks. The structural interpretations of lipid A species are summarized in Table 1. The results are representative of at least two independent experiments. pared from the transformants, and its structure was analyzed by MALDI-TOF mass spectrometry. The analysis revealed that the PagL(N173A) and PagL(N177A) mutants did not induce the deacylation of lipid A (Fig. 8). Expression levels of the mutant PagL proteins were similar to that of wild-type recombinant PagL protein (data not shown), suggesting that the PagL mutants lost lipid A 3-O-deacylase activity. In addition, introduction of expression constructs containing PagL (N173A) or PagL(N177A) into E. coli BL21(DE3) did not induce modification of LPS (Fig. 7). These results, taken together, indicate that Asn-173 and Asn-177 were essential for the lipid A 3-O-deacylase activity of PagL. Asn-173 and Asn- 177 of S. enterica PagL correspond to Asn-159 (136 from predicted N terminus) and Glu-163 (140 from predicted N terminus) of P. aeruginosa PagL, which were previously demonstrated to be important for the lipid A 3-O-deacylase activity of P. aeruginosa PagL (40). In addition, previous reports demonstrated that FIG. 7. Analysis of LPS modification in E. coli transformed with PagL mutant by Tricine-SDS-polyacrylamide gel electrophoresis. LPS prepared from E. coli BL21(DE3) transformed with pbluescript II KS( ) (vector) or pbluescript II KS( ) containing wild-type PagL, PagL(R43A), PagL (R44A), PagL(C85A), PagL(R135A), PagL(N166A), PagL(N173A), or PagL(N177A) was analyzed by Tricine-SDS-polyacrylamide gel electrophoresis. The results are representative of at least two independent experiments. P. aeruginosa PagL Asn-152 (129 from predicted N terminus), which corresponds to Asn-166 of S. enterica PagL, was important for PagL activity (40). However, the introduction of expression constructs containing S. enterica PagL(N166A) into the S. enterica pmra pagl or E. coli BL21(DE3) strain induced the modification of LPS (Fig. 6 and 7), suggesting that Asn-166 is not crucial for the lipid A 3-O-deacylase activity of S. enterica PagL. Introduction of other low-copy expression constructs containing mutant PagL, in which an amino acid residue located in the four extracellular loops (Fig. 2) was replaced with alanine, induced lipid A deacylation in the S. enterica pmra strain (data not shown), suggesting other amino acid residues located at extracellular loops of PagL not to be essential for the 3-O-deacylase activity. S. enterica strains expressing PagL mutants that lost the latency showed growth arrest at 43 C. In order to examine the physiological significance of PagL s latency, we analyzed the growth rates of S. enterica strains expressing PagL mutants that had lost latency. S. enterica pmra pagl cells transformed with low-copy expression constructs containing PagL(R43A) or PagL(R135A), which lost latency as described above, had a growth rate at 37 C similar to that of S. enterica transformed with low-copy expression constructs containing wild-type PagL (Fig. 9A). In contrast, S. enterica pmra pagl cells transformed with the expression construct containing PagL(R43A) or PagL(R135A) showed apparent growth arrest at 43 C compared with that of S. enterica transformed with the expression construct containing wild-type PagL (Fig. 9B). In addition, the S. enterica pmra pagl strain transformed with the expression construct containing PagL(R131A), which retains the ability to be latent (Table 2), did not show growth arrest at 43 C (Fig. 9B). These results, taken together, suggest that the aminoarabinose modification-dependent latency of PagL is important for cell growth at 43 C. DISCUSSION We demonstrated that mutations of several amino acid residues located at extracellular loops in the PagL of S. enterica

8 5604 MANABE AND KAWASAKI J. BACTERIOL. FIG. 8. Asn-173 and Asn-177 are essential for lipid A 3-O-deacylase activity of PagL in an S. enterica pmra mutant strain. Lipid A prepared from S. enterica serovar Typhimurium pmra pagl doublemutant strain KCS040 transformed with the pwks30 vector (A) or pwks30 containing PagL(N166A) (B), PagL(N173A) (C), or PagL(N177A) (D) was analyzed by MALDI-TOF mass spectrometry. The m/z values of lipid A species are shown, and those that represent deacylated lipid A species are denoted by asterisks. The structural interpretations of lipid A species are summarized in Table 1. The results are representative of at least two independent experiments. FIG. 9. Introduction of PagL(R43A) and PagL(R135A) mutants into S. enterica induced growth arrest at 43 C. S. enterica serovar Typhimurium pmra pagl strain CS283 transformed with pwks30 (vector) or pwks30 containing wild-type PagL, PagL(R43A), PagL(R131A), or PagL(R135A) was grown overnight at 37 C. Then the cells were diluted to an optical density at 600 nm of 0.05 and grown at 37 C (A) or 43 C (B). Cell growth was measured by monitoring optical density at 600 nm. The results are representative of two (A) or three (B) independent experiments. serovar Typhimurium, including Arg-43, Arg-44, Cys-85, and Arg-135, released PagL from the aminoarabinose modification-dependent latency. These mutations were distinct from those that affected the lipid A 3-O-deacylase activity of PagL. Furthermore, S. enterica cells expressing mutant PagL that lost latency exhibited growth arrest at 43 C. These observations indicate that the extracellular loops of S. enterica PagL are involved in recognition of aminoarabinose-modified outer membranes, and this recognition is important for bacterial growth at 43 C. The modification of lipid A by aminoarabinose changes the cell surface membrane charge, since the primary amine of aminoarabinose possesses a positive charge (Fig. 1). The changes are known to increase bacterial resistance to cationic antimicrobial peptides, including polymyxin B (17, 18, 29, 34). The involvement of cationic amino acid residues, such as Arg- 43, Arg-44, and Arg-135, in latency suggests some direct electrostatic interaction between the positively charged domain of PagL s extracellular loops and cell surface aminoarabinosemodified lipid A, although the replacement of Arg-43, Arg-44, and Arg-135 of PagL with cationic lysine residues did not sustain the latency. PagL was latent in S. enterica (27, 45) but not in Pseudomonas aeruginosa (8, 10). Consistent with our observations, the Arg-43, Arg-44, Cys-85, and Arg-135 residues of S. enterica PagL, which were essential for latency, were not conserved in P. aeruginosa PagL (13, 45). The crystal structure of P. aeruginosa PagL reveals that its active site faces the outer surface of the outer membrane (40). The potential of PagL to form a dimer at the interface within the active sites suggests a possible mechanism to inhibit the activity of S. enterica PagL in the outer membrane (40). Modification of the outer membrane by aminoarabinose as well as amino acid residues located at extracellular loops of PagL, such as Arg-43, Arg-44, Cys-85, and Arg-135, might be involved in the structural changes of PagL. The structural changes, which make PagL silent in the outer membrane, remain to be elucidated. In addition to the amino acid residues involved in the sensing of aminoarabinose-containing membranes, we showed that Asn-173 and Asn-177 located at extracellular loops of S. enterica PagL were essential for the lipid A 3-O-deacylase activity. These results were consistent with previous findings on P. aeruginosa PagL (13). On the other hand, the replacement of Asn-166 of S. enterica PagL with alanine did not abolish the deacylase activity, a result not consistent with the report that the replacement of Asn-152 (129 from predicted N terminus) of P. aeruginosa PagL, which corresponds to Asn-166 of S.

9 VOL. 190, 2008 RELEASE OF PagL FROM LATENCY BY POINT MUTATIONS 5605 enterica PagL, with alanine abolished the lipid A 3-O-deacylase activity (13). The discrepancy suggests some structural difference in the active sites between S. enterica PagL and P. aeruginosa PagL. In this study, we observed that S. enterica expressing the PagL-R43A or PagL-R135A mutant showed growth arrest at 43 C. These results suggest PagL s latency to help S. enterica to grow under specific conditions, including in the tissues of a host who has a fever, and are the first observations to suggest the physiological importance of PagL s latency. Several other outer membrane enzymes involved in the modification of lipid A, such as S. enterica LpxR and E. coli PagP, also display latency (5); LpxR-dependent lipid A deacylation in S. enterica (38) and PagP-dependent lipid A palmitoylation in E. coli (23, 42) were not usually observed under normal culture conditions. These observations suggest that the latency of outer membrane enzymes is generally conserved for regulation of lipid A modifications in gram-negative bacteria. Although little is known about the physiological functions of the repression of outer membrane enzymes involved in the modification of lipid A, our results regarding PagL suggest that the latency of these enzymes is involved in bacterial pathogenesis. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. REFERENCES 1. Bader, M. W., W. W. Navarre, W. Shiau, H. Nikaido, J. G. Frye, M. McClelland, F. C. Fang, and S. I. Miller Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides. Mol. Microbiol. 50: Bader, M. W., S. Sanowar, M. E. Daley, A. R. Schneider, U. Cho, W. Xu, R. E. Klevit, H. Le Moual, and S. I. Miller Recognition of antimicrobial peptides by a bacterial sensor kinase. Cell 122: Bearson, B. 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10 5606 MANABE AND KAWASAKI J. BACTERIOL. Chemical degradations and identification of products. J. Biol. Chem. 254: Rutten, L., J. Geurtsen, W. Lambert, J. J. Smolenaers, A. M. Bonvin, A. de Haan, P. van der Ley, M. R. Egmond, P. Gros, and J. Tommassen Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL from Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 103: Sambrook, J., E. F. Fritsch, and T. Maniatis Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 42. Smith, A. E., S. H. Kim, F. Liu, W. Jia, E. Vinogradov, C. L. Gyles, and R. E. Bishop PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7. J. Biol. Chem. 283: Steiner, K., R. Novotny, K. Patel, E. Vinogradov, C. Whitfield, M. A. Valvano, P. Messner, and C. Schaffer Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a. J. Bacteriol. 189: Tanamoto, K., and S. Azumi Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells. J. Immunol. 164: Trent, M. S., W. Pabich, C. R. Raetz, and S. I. Miller A PhoP/ PhoQ-induced lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium. J. Biol. Chem. 276: Wang, R. F., and S. R. Kushner Construction of versatile low-copynumber vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100: Yi, E. C., and M. Hackett Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria. Analyst 125: Zhou, Z., S. Lin, R. J. Cotter, and C. R. Raetz Lipid A modifications characteristic of Salmonella typhimurium are induced by NH 4 VO 3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate. J. Biol. Chem. 274: Zhou, Z., A. A. Ribeiro, S. Lin, R. J. Cotter, S. I. Miller, and C. R. Raetz Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRAdependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation. J. Biol. Chem. 276: Downloaded from on August 26, 2018 by guest