Unique lipid anchor attaches Vi antigen capsule to the surface of Salmonella enterica serovar Typhi Sean D. Listona, Olga G. Ovchinnikovaa, and Chris Whitfielda,1 a

Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada, N1G2W1

Edited by Hiroshi Nikaido, University of California, Berkeley, CA, and approved April 29, 2016 (received for review December 14, 2015)

polysaccharide capsule polysaccharide export

| Vi antigen | Salmonella | glycolipid |

M

any bacteria produce high-molecular-weight cell-surface polysaccharides that form a hydrated layer known as a “capsule.” There is enormous diversity in capsular polysaccharide (CPS) structures resulting from variations in sugar residue composition, linkage(s), and the addition of nonsugar substituents (1). The capsule is often the outermost structure of a bacterial cell and therefore is critical for interactions with the environment. Depending on the organism, capsules assist bacteria in resisting desiccation, forming biofilms, colonizing host tissues, resisting bacteriophages, and reducing opsonophagocytosis and complement-mediated killing (2). In Salmonella enterica, the virulence capsular polysaccharide, known as “Vi antigen,” is produced by human-restricted serovar Typhi (hereafter S. Typhi, the etiological agent of typhoid fever) and serovar Paratyphi C, but it is absent in other serovars commonly associated with gastroenteritis. Vi antigen capsule is implicated in the evasion of the innate immune system (reviewed in ref. 3). The production of Vi antigen reduces serum complement binding/killing and promotes intracellular replication; Vi antigen-deficient mutants are 10,000-fold less virulent in a mouse model of infection (4). Purified Vi antigen is currently used in parenteral vaccines (5). Despite the structural diversity of Gram-negative CPS, most are synthesized by one of two widely distributed mechanisms, with model systems provided by Escherichia coli K antigens (reviewed in ref. 1). The two pathways are differentiated by the mechanism and location of the polymerization reaction and by the machinery that exports the nascent glycan (or its biosynthetic www.pnas.org/cgi/doi/10.1073/pnas.1524665113

intermediates) across the cytoplasmic membrane. One of these systems requires a pathway-defining ATP-binding cassette (ABC) transporter to export CPS that is fully polymerized in the cytoplasm. This mechanism is shared by extraintestinal pathogenic E. coli (i.e., group 2 CPS), Neisseria meningitidis, Haemophilus influenzae, Campylobacter jejuni, and other pathogens of humans and livestock. In these bacteria, the CPS glycans are attached to a (lyso)phoshatidylglycerol moiety via an oligosaccharide of five to nine β-linked 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues (6). The Kdocontaining glycolipid is synthesized by conserved β-Kdo transferases (known as “KpsS” and “KpsC” in E. coli) (7). The CPS glycan is built on the nonreducing end of the β-Kdo oligosaccharide linker by serotype-specific glycosyltransferases. The lipidated terminus is essential for CPS export (6) and is likely recognized by the pathway-defining ABC transporters, which are interchangeable among CPS serotypes and species (reviewed in refs. 1 and 8). CPS translocation to the cell surface is believed to involve an envelope-spanning complex composed of the ABC transporter and members of the polysaccharide co-polymerase (PCP) and outer membrane polysaccharide export (OPX) protein families (1, 8). Vi antigen is a linear polymer of GalNAcA residues nonstoichiometrically O-acetylated at C-3 (9). The Vi antigen biosynthesis (viaB) operon encodes enzymes implicated in Vi antigen biosynthesis (TviA–E) as well as a characteristic ABC transporter (VexBC) and OPX (VexA) and PCP (VexD) homologs (Fig. 1A) (10, 11). Loci similar to S. Typhi viaB are found in the opportunistic pathogens Citrobacter freundii (12), Bordetella petrii (GenBank accession no. AM902716.1), and Achromobacter species (Fig. 1A), although the glycan product has not been investigated in either Bordetella or Achromobacter. The chromosomes of the Vi antigen-producing bacteria lack homologs of kpsS or kpsC that are found in all other currently known Gram-negative bacteria with ABC transporter-dependent CPS assembly pathways (8). Here we Significance Polysaccharide capsules are protective surface layers that enhance virulence of many pathogenic bacteria. Salmonella enterica serovar Typhi is the causative agent of typhoid fever, and it produces the virulence capsular polysaccharide known as “Vi antigen.” This glycan is part of some current vaccines. In some Gram-negative bacteria, capsular polysaccharides are attached to a conserved glycolipid that anchors the polysaccharide to the cell surface and is required for its transport across the cell envelope. S. enterica Typhi follows a different strategy; this work identifies a reducing terminal lipid structure unique to the Vi antigen that is required for attachment of the capsular surface layer. This lipid is structurally (and potentially biosynthetically) related to the conserved lipid A component of bacterial lipopolysaccharides. Author contributions: S.D.L., O.G.O., and C.W. designed research; S.D.L. performed research; S.D.L., O.G.O., and C.W. analyzed data; and S.D.L. and C.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. KT99772). 1

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1524665113/-/DCSupplemental.

PNAS | June 14, 2016 | vol. 113 | no. 24 | 6719–6724

MICROBIOLOGY

Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. It produces a capsular polysaccharide known as “Vi antigen,” which is composed of nonstoichiometrically O-acetylated α-1,4-linked N-acetylgalactosaminuronic acid residues. This glycan is a component of currently available vaccines. The genetic locus for Vi antigen production is also present in soil bacteria belonging to the genus Achromobacter. Vi antigen assembly follows a widespread general strategy with a characteristic glycan export step involving an ATP-binding cassette transporter. However, Vi antigen producers lack the enzymes that build the conserved terminal glycolipid characterizing other capsules using this method. Achromobacter species possess a Vi antigen-specific depolymerase enzyme missing in S. enterica Typhi, and we exploited this enzyme to isolate acylated Vi antigen termini. Mass spectrometry analysis revealed a reducing terminal N-acetylhexosamine residue modified with two β-hydroxyl acyl chains. This terminal structure resembles one half of lipid A, the hydrophobic portion of bacterial lipopolysaccharides. The VexE protein encoded in the Vi antigen biosynthesis locus shares similarity with LpxL, an acyltransferase from lipid A biosynthesis. In the absence of VexE, Vi antigen is produced, but its physical properties are altered, its export is impaired, and a Vi capsule structure is not assembled on the cell surface. The structure of the lipidated terminus dictates a unique assembly mechanism and has potential implications in pathogenesis and vaccine production.

A

D

E

B C

Fig. 1. S. Typhi wild-type and ΔvexE mutant Vi antigens possess altered physical properties. (A) Shared organization and gene content in viaB loci. The viaB loci from Achromobacter sp. include an additional gene (vexL) encoding a Vi antigen lyase enzyme. The A. denitrificans sequence is deposited at GenBank (accession no. KT997721), but the same locus is found in other sequenced genomes of Achromobacter sp. [A. xylosoxidans (GenBank accession no. CP012046.1), A. arsenitoxydans (GenBank accession no. NZ_AGUF01000055.1), A. spanius (GenBank accession no. NZ_LGVG01000001.1), and A. piechaudii (GenBank accession no. ADMS01000020.1)]. (B) VexE contains a predicted N-terminal region of tetratricopeptide repeats (TPR), which form α-helical superstructures implicated in protein–protein interactions (25), and a C-terminal lysophospholipid acyltransferase (LPLAT) domain. (C) Multiple sequence alignment of the LPLAT domain of VexE from S. Typhi, A. denitrificans, and E. coli LpxL. Motifs characteristic of LPLAT are highlighted in yellow, and the putative role of particular residues is noted (17). Residues that were mutated are boxed in blue. (D) In immunoblots, Vi antigen produced by S. Typhi bound PVDF and positively charged nylon membranes, whereas Vi antigen from the ΔvexE mutant bound only nylon. The panels show immunoblots of proteinase K-digested whole-cell lysates probed with anti-Vi antigen antibody. PVDF binding was restored when the ΔvexE mutant was complemented with either S. Typhi vexE or A. denitrificans vexE. The corresponding putative catalytic mutants of VexE from either S. Typhi (H487A) or A. denitrificans (H466A) failed to restore PVDF binding. A Y471F mutation in the A. denitrificans enzyme (at position 6 of HX4(D/E) motif in VexE) had no discernible effect on its activity. VexE expression was monitored by Western blotting of hexahistidine-tagged VexE constructs from identical cell cultures. (E) Vi antigen is produced in E. coli Top10 and its ΔwecA mutant.

identify the structure of a glycolipid terminus unique to the Vi antigen and propose a biosynthetic origin that takes advantage of the conserved lipid A machinery in Gram-negative bacteria. Results Vi Antigen from a ΔvexE Mutant Has Altered Physical Properties.

Despite the absence of kpsS and kpsC from the chromosomes of viaB-positive bacteria, we speculated that a glycolipid terminus of some form may be a unifying feature for all ABC transporterdependent CPS biosynthesis pathways. Other bacterial surface glycoconjugates exported by ABC transporters frequently use undecaprenyl diphosphate carrier lipids. To test the possibility that these lipids participated in Vi antigen production, the viaB locus was introduced into E. coli CWG1214 ΔwecA, which is unable to make undecaprenyl diphospho-N-acetylglucosamine in the obligatory first step in biosynthesis of E. coli LPS O antigens and enterobacterial common antigen (13). E. coli also lacks wbaP, whose gene product produces undecaprenyl diphospho-galactose in the corresponding initiation step for most Salmonella O antigens (13). E. coli CWG1214, transformed with the viaB locus, displayed robust Vi antigen production, evident in immunoblots (Fig. 1E), ruling out the logical candidates for undecaprenyl-active enzymes in Vi antigen assembly. All the viaB loci encode a predicted VexE protein containing a potential C-terminal lysophospholipid acyltransferase (LPLAT) motif (Fig. 1B). This motif is also found in E. coli LpxL (Fig. 1C), an acyl carrier protein (ACP)-dependent secondary acyltransferase involved in the biosynthesis of LPS lipid A, a conserved glycolipid essential for viability of almost all Gramnegative bacteria (reviewed in refs. 13 and 14) (see Fig. 4). LpxL and VexE share only 18% identity overall (e-value: 1.08 × 10−3), but the conserved LPLAT domain (cd07984) shares higher similarity 6720 | www.pnas.org/cgi/doi/10.1073/pnas.1524665113

(e-value: 1.67 × 10−8). The putative LPLAT motif in VexE led to the hypothesis that this protein is an acyltransferase that creates a different type of lipid terminus on Vi antigen chains. Previous analyses of Vi antigen phenotypes in viaB gene mutants were performed using recombinant E. coli transformed with plasmid-encoded viaB, but the possibility of a lipid terminus and the precise role of VexE has not been examined (10, 11). To avoid complications arising from multicopy gene expression and the unnatural host background, we examined the role of VexE using chromosomal mutations in S. Typhi. In Western immunoblots, Vi antigen in cell lysates of the parent strain bound to both hydrophobic PVDF and positively charged nylon membranes (Fig. 1D). In contrast, Vi antigen in lysates from the ΔvexE mutant bound only to nylon, indicating a change in the physical properties of Vi antigen produced by the mutant. Wildtype binding properties were restored in the mutant by the expression of VexE homologs from S. Typhi or Achromobacter denitrificans (Fig. 1D). LPLAT enzymes possess a conserved HX4D/E motif, which contains the essential catalytic His/Asp pair (Fig. 1C) (15). The corresponding H→A mutant in E. coli LpxL results in a >1,000-fold reduction in lauroyltransferase activity (16). The putative catalytic His residue was mutated in the VexE homologs from S. Typhi and A. denitrificans, and these proteins were expressed in S. Typhi ΔvexE. Vi antigen from these transformants did not bind to PVDF despite robust expression of the enzymes (Fig. 1D). Proper folding of the mutant VexE was confirmed by comparing circular dichroism spectra of purified wild-type and mutant proteins (Fig. S1). The catalytic activity of VexE therefore is linked to alterations in the physical properties of Vi antigens, resulting in differential binding to membranes with varying chemistries. Liston et al.

shared sequence similarity with known pectate lyase enzymes (Fig. S3A); the corresponding gene is renamed “vexL.” Pectate lyases degrade acidic polymers, such as pectin, from plant tissues (18), and the α-1,4-linked polygalacturonic acid backbone structure of pectin superficially resembles Vi antigen. Purified A. denitrificans VexL enzyme depolymerized Vi antigen (Fig. S3B). Hydrophobic products released in these reactions were collected by solid-phase extraction and were analyzed by LC-MS. LC-MS of these molecules revealed a series of species that differed by 217.059 m/z, representing increments of one GalNAcA residue (Fig. 2A and Table S2). Oligosaccharides modified by one or more O-acetyl groups (δ m/z = 42.011) were also present. Lyase enzymes cleave polysaccharides through an eliminative mechanism and create a characteristic (anhydro) 4-deoxyα-D-galact-4-enuronosyl residue at the nonreducing end of the oligosaccharide (18). This modification was evident in MS/MS fragmentation products (Fig. 2B). The Vi antigen oligosaccharides were linked to a reducing terminal N-acetylhexosamine (HexNAc) residue modified with either two β-hydroxymyristate chains or one β-hydroxymyristate and one β-hydroxypalmitate chain. Fragmentation of the [M-H]− ion at 1333.666 m/z produced products (including cross-ring cleavages) consistent with a structure comprising three GalNAcA residues linked to a single reducing terminal HexNAc possessing β-hydroxymyristate and β-hydroxypalmitate modifications (Fig. 2B). As confirmation, the isotopic distribution of the [M-H]− ion at 1333.666 m/z agreed with that predicted for the glycolipid (Fig. S4A), and fragmentation of the [M-2H]2- ion at m/z = 774.858 revealed the same structure extended with an additional GalNAcA residue (Fig. S4B). No ions corresponding to this glycolipid were identified when the procedure was repeated for Vi antigen purified from a ΔvexE mutant of S. Typhi.

To determine whether the altered binding properties reflected differences in the repeat-unit structure of the parental and mutant Vi antigens, such as alterations in O-acetylation, CPS was purified, and its structure was examined by NMR. In the initial preparations we were unable to obtain wild-type Vi antigen free from LPS contamination, and substantial amounts of wild-type Vi antigen sedimented with LPS micelles in centrifugation. Deletion of vexE abrogated this property (Fig. S2B), adding weight to the contention that VexE influenced the physical properties of Vi antigen. To obtain Vi antigen free of LPS, we created ΔwaaG mutants generating truncated LPS molecules (resulting from the loss of most of the core oligosaccharide and O antigen; reviewed in ref. 15) that could be separated from Vi antigen by gel filtration chromatography (Fig. S2 A–C). 13C NMR spectra of Vi antigens from ΔwaaG and ΔwaaG ΔvexE double mutants were identical (Fig. S2 D and E and Table S1) and were comparable to those previously published (17). The altered properties of the Vi antigen produced by the vexE mutant therefore were not caused by changes in the polysaccharide backbone structure but could be explained by alterations in a putative acylated terminus. Vi Antigen Has a Unique Glycolipid at Its Reducing Terminus. Structural investigation of the termini of high-molecular-weight polysaccharides requires a method that reduces the degree of polymerization while preserving linkages between terminal modification(s) and the remaining glycan. Previously, we exploited endoglycanase enzymes from capsule-specific bacteriophages to identify the terminal glycolipid structure from the CPS of E. coli K1 and K5 and meningococcal serotype b (6). The viaB locus of Achromobacter sp. and B. petrii contain an additional ORF located downstream of vexE in the otherwise similar locus (Fig. 1A). The predicted gene product

1334.678

HexNAcA

1150

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OH

NH O

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500

-H2O X

881.544 (.537) 899.547 (.548)

N

P

632.145 (.158) 650.167 (.168) 682.480 (.489)

550.120 (.128)

371.109 (.109)

J K

332.060 (.062)

I

O

415.095 (.099) 433.106 (.109) 475.121 (.116)

H

1750

*

1232.618 (.618)

M L

M (.849) 1985.852

HO

1098.589 (.596) 1116.600 (.607)

227.201 (.201)

115.003 (.003)

154.050 (.050)

EF D

*[M-H] m/z = 1333.6659 *[M-2H]2- m/z = 666.3290 -

180.030 (.030) 198.038 (.040) 216.050 (.051) 255.232 (.232) 258.059 (.061)

112.041 (.040)

A

+Ac

-CH2

Mass to Charge (m/z)

BC G

+Ac

+CH2

+Ac

-CH2

1200

+Ac

-CH2

1315.656 (.655)

Intensity (a.u.)

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+Ac

+Ac -H2O

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M (.615)

HexNAcA

-C2H4

+Ac +Ac

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-C2H4

M (.790) 1768.786

HexNAcA

-C2H4 +Na-H

+CH2

1100

Intensity (a.u.)

HexNAcA

-C2H4 +Na-H

+CH2

B

M (.732) 1551.730

M (.673)

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NH O

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700

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Mass to Charge (m/z)

900

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CH 2(CH 2) 9CH3

CH 2(CH 2)11 CH3

Fig. 2. Glycolipid terminus of the Vi antigen as determined by MS. (A) Charge deconvoluted LC-electrospray ionization (ESI)-QTOF-MS spectrum in negative mode for Vi antigen termini purified from S. Typhi. All ions correspond to a di-β-hydroxyacylated HexNAc residue linked to two or more variably O-acetylated HexNAcA residues. (B) LC-ESI-QTOF-MS/MS data for the singly charged (blue) and doubly charged (red) ions corresponding to a GalNAcA3 oligosaccharide attached to a reducing terminal diacyl-HexNAc. Overlapping signals are colored purple. Fragmentations are illustrated in green.

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PNAS | June 14, 2016 | vol. 113 | no. 24 | 6721

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A

VexE Is Required for Efficient Export and Cell-Surface Retention of Vi Antigen. The role of the glycolipid terminus in Vi antigen export

Discussion Vi antigen has a lipid terminus that differs from those of any other known CPS assembled in an ABC transporter-dependent pathway. It is composed of a reducing terminal HexNAc residue modified with two β-hydroxy fatty acids and resembles one half of the structure of lipid A (Fig. 4). This structure, together with the similarity shared by VexE and LpxL, a secondary acyl-ACP– dependent acyltransferase from lipid A biosynthesis, is consistent with the proposal that VexE is an acyltransferase that transfers a β-hydroxymyristate or β-hydroxypalmitate chain to the terminus of Vi antigen. The action of VexE would be comparable to that of the secondary acyltransferases in lipid A biosynthesis, although LpxL and LpxM transfer nonhydroxylated fatty acids (Fig. 4) (16). VexE is the only acyltransferase encoded by the viaB locus. There is no precedent for such enzymes being able to transfer both acyl chains, and doing so would require radically different acceptor specificities in a single catalytic site. A logical origin of this terminal moiety involves secondary acylation of the UDP-activated 6722 | www.pnas.org/cgi/doi/10.1073/pnas.1524665113

A

whole cells S.Typhi

VexL

B

-

-

+

vexE

vexC -

+

anti-Vi-antigen vexC vexC + vexC

-

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vexC -

+

anti-Vi-antigen vexE vexE + vexE

Live

S. Typhi

+

lysed cells

vexE

Fixed + permeabilized

and surface assembly of the Vi capsule was investigated in S. Typhi by its susceptibility to degradation by VexL. VexL degraded almost all detectable Vi antigen in the wild type, indicating minimal amounts of intracellular (untransported) glycan. In contrast, Vi antigen profiles from the ΔvexC (lacking the ABC transporter ATPase) or ΔvexE mutant were unaffected by the presence of the enzyme (Fig. 3A). Vi antigen stability in whole cells was caused solely by inaccessibility to the lyase, as permeabilization of the mutant cells facilitated complete digestion of the glycan. Furthermore, a Vi antigen-specific bacteriophage infected the wild type but formed no plaques on the ΔvexC or ΔvexE mutants, confirming that no phage receptor is available on the mutant cell surfaces (Fig. S5B). Complementation of the mutations with the respective genes restored phage sensitivity. Wildtype S. Typhi possessed a Vi antigen capsule on the cell surface that was detectable by immunofluorescence microscopy (Fig. 3B), and complementation of the ΔvexC and ΔvexE mutants with the respective genes restored the Vi antigen capsule. Both the ΔvexC and ΔvexE mutants possessed inclusion bodies (Fig. 3B, Insets), which were labeled with Vi antigen-specific antibodies in immunofluorescence microscopy of permeabilized cells. Electron microscopy revealed that the inclusions were cytosolic in both the ΔvexC and ΔvexE mutants (Fig. S5C). To examine possible deleterious effects of these inclusions on cell physiology, the Cpx envelope stress response was assessed in S. Typhi and mutant derivatives (Fig. S5D). Surprisingly, the Cpx response was up-regulated significantly only in the ΔvexC mutant, in which no export occurs, and this increase was eliminated in a ΔvexC ΔvexE mutant, indicating that activation of a stress response by the inclusions was dependent on acylation. These results are consistent with the intracellular accumulation of Vi antigen in the ΔvexC and ΔvexE mutants, suggesting that both mutations resulted in export defects. However, published mutant phenotypes indicated extensive Vi antigen export in a vexE mutant of an E. coli recombinant containing viaB (11). Because lyase treatment and immunofluorescence microscopy of whole cells cannot account for Vi antigen released into the growth medium, we examined the cell-free supernatants from early exponential-phase cultures of S. Typhi and its mutant derivatives for Vi antigen release (Fig. 3C). Wild-type cells released some Vi antigen into the medium, as expected with any encapsulated bacterium and as is consistent with published observations (10, 11). The ΔvexC mutant released only a trace of Vi antigen; release could be explained by small amounts of lysis during growth and is consistent with the release of cytosolic RNA polymerase in the same cultures (Fig. 3C). Export and release of Vi antigen was restored when the ΔvexC mutation was complemented with vexC. In contrast, ΔvexE cells released large quantities of Vi antigen. This material was eliminated in a ΔvexCE double mutant (Fig. 3C and Fig. S5E), indicating an active process involving the ABC transporter rather than elevated leakage resulting from the vexE defect.

C

vexCE vexE vexC S. Typhi

vexC +vexC vexE +vexE vexE +vexE H466A vexCE +vexC

anti-Vi-antigen 5036-

anti-RNApol

Fig. 3. VexE is required for efficient export and surface retention of Vi antigen. (A) The Vi antigen depolymerase was unable to access Vi antigen within intact cells of the ΔvexE mutant. Whole or lysed cells of S. Typhi and mutants were incubated with purified VexL, collected, digested with proteinase K, and probed for Vi antigen. VexL was able to degrade the Vi antigen in wild-type S. Typhi but not in S. Typhi ΔvexC, providing positive and negative controls for export, respectively. (B) Immunofluorescence microscopy of cells probed with anti-Vi antigen antibodies illustrated that the ΔvexE mutant possessed no Vi antigen on its surface but accumulated intracellular Vi antigen in inclusion bodies, which became accessible to antibody in permeabilized cells. (Scale bars, 10 μm.) Insets are enlarged to show a representative cell. (C) S. Typhi ΔvexE was able to export Vi antigen in a transporter-dependent manner. Growth medium from early exponential-phase cultures was collected and probed for Vi antigen and (cytosolic) RNA polymerase by Western immunoblotting.

Liston et al.

β-hydroxymyristoyl-GlcNAc product of LpxA in lipid A biosynthesis (Fig. 4) (19). Such a reaction could divert this intermediate for use in Vi antigen biosynthesis and, to our knowledge, would represent the first off-pathway use of an Lpx-pathway intermediate. We investigated the possibility that VexE interfered with normal lipid A biosynthesis in E. coli. Expression of VexE slowed the growth of E. coli slightly, but this effect was likely caused by protein overexpression and was independent of VexE catalytic activity (Fig. S6A). In addition, VexE was unable to modify lipid A (Fig. S6B). The inability of VexE activity to influence lipid A biosynthesis is perhaps not surprising, given the regulation of the essential Raetz pathway process. The LpxA reaction equilibrium favors the reverse reaction, and the first committed step of lipid A biosynthesis (LpxC) is tightly regulated (15, 16, 20), so pathway flow is regulated according to lipid A requirement. We pursued the possibility that ΔvexE Vi antigen possesses a single acyl chain (the product of LpxA) at its reducing terminus. However, we were unable to detect either diacyl- or monoacyl-HexNAc in either extracellular or intracellular (accumulated) Vi antigen from the ΔvexE mutant. This negative result could reflect an absolute requirement for diacylated UDP-GlcNAc, offering an additional means of separation from the lipid A pathway. However, we cannot rule out technical issues in which monoacylated Vi antigen termini lack sufficient hydrophobicity for separation protocols (consistent with altered PVDF binding). The apparent ability to synthesize Vi antigen in the absence the acylated terminus could reflect the mutations creating conditions that facilitate polymer synthesis on nonphysiological acceptors, as is the case in the E. coli and N. meningitidis kpsS and kpsC mutants (6). However, this assumption requires that the diacyl-HexNAc actually serves as an acceptor, but the identity and mechanism of the Vi antigen polymerase is unknown. Vi antigen potentially could be synthesized by growth at the reducing terminus in a process similar to class I hyaluronan synthases. These enzymes use UDP-GlcNAc or UDP-glucuronic acid (GlcA) as acceptors and the nascent [3)-GlcNAc-β-(1→4)GlcA-β-(1→]n-UDP chain as the donor during chain extension (21). It is unknown how the terminal UDP moiety is removed in the final glycan product. In such a scenario, the addition of diacyl-HexNAc could represent the last step in Vi antigen biosynthesis before export, explaining the ability to synthesize Vi antigen in the absence of VexE. Biochemical characterization of the Vi antigen polymerase(s) is required to resolve this question. In the E. coli group 2 CPS assembly, export is dependent on the presence of the glycolipid terminus (6). In contrast, defective acylation of the Vi antigen in the ΔvexE mutant does not prevent export, but accumulation of intracellular Vi antigen (which is not seen in the wild type) also occurs. Accumulation of Vi antigen

OH

O

HO O

Vi antigen Biosynthesis

O

O HO

?

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NH O UDP

O HO

UDP-GlcNAc

Raetz Pathway

O O

OH

O

NH O UDP

3-OH-C14/16 -ACP

Acetate

VexE?

LpxC

14

Primers. Oligonucleotide primers used to amplify genes from S. Typhi, A. denitrificans, and E. coli genomic DNA were obtained from Sigma-Aldrich and are described in Table S4.

3-OH-C14-ACP OH

HO

Strains and Plasmids. The bacterial strains used in this study are listed in Table S3. The background for the generation of viaB mutants was S. Typhi H251.1 (aroC); clean mutations were generated by recombineering using the λ-red system (see SI Methods for details). Strains and transformants were grown at 37 °C in lysogeny broth (LB) medium supplemented with 100 μg/mL 2,3dihydroxybenzoic acid and antibiotics where appropriate. Complementation of mutations was performed using L-arabinose–inducible pBAD-based vectors described in Table S3.

NH O O UDP

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Kdo2– O O O O H2O4P O HO O O NH NH O O O PO4H2 O O O HO HO O O

OH O HO O NH O O O UDP HO HO

UDP-acyl-GlcN

UDP-diacyl-GlcN

LpxM14 14 14 14 14 LpxL12 Kdo –lipid A 2

Fig. 4. Proposed model for the biosynthesis of the Vi antigen glycolipid terminus. The diacyl-HexNAc residue at the Vi antigen terminus originates from the secondary acylation of the UDP-activated acyl-GlcNAc product produced by LpxA in the conserved lipid A biosynthesis Raetz pathway (14).

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PNAS | June 14, 2016 | vol. 113 | no. 24 | 6723

MICROBIOLOGY

HO HO

could reflect altered recognition by the export machinery. For example, the LPS ABC transporter MsbA from E. coli is highly selective for completed (hexaacylated) LPS molecules. Export of tetraacylated precursors occurs only at low levels (reviewed in ref. 13). Alternatively, the export defect in the ΔvexE mutant could reflect alteration of essential interactions that couple synthesis to export in a multiprotein complex. However, altered interactions are unlikely because the phenotype resulting from the VexE catalytic-site mutation (which should preserve protein– protein interactions) is indistinguishable from the vexE deletion (Fig. 3C). Interestingly, intracellular Vi antigen in transportdefective mutants showed an increase in the average chain length (Fig. 3C and Fig. S5A), whereas overexpression of VexE caused a reduction. Lowering chain length requires VexE catalytic activity rather than a simple structural requirement for the protein, because the size reduction was not evident in ΔvexE cells expressing VexEH466A. Altered chain lengths can be explained by an elongation phase differing from the normal assembly process occurring with molecules with a complete glycolipid terminus. There is precedent for the modulation of glycan chain length by competition between export and extension in other bacterial systems with ABC transporters (22, 23). The use of a conserved intermediate from the lipid A– biosynthesis pathway to create the lipid terminus potentially facilitates Vi antigen production in diverse Gram-negative bacteria by horizontal transfer of the viaB locus with a limited gene complement. This diversity is evident in the possession of the locus by Achromobacter, Bordetella, and Citrobacter sp. and expression in E. coli, but why some Vi antigen producers possess the additional VexL component is unknown. It is also unknown whether the terminal lipid itself is important in the interaction of Vi antigen with the host immune system. In the context of Vi antigen-based vaccines, a production strain lacking vexE may offer advantages because it exports Vi antigen with altered micellar properties and a reduced association with LPS.

Purification of Vi Antigen. Vi antigen was purified from the supernatant of a hot aqueous phenol extract of lyophilized S. Typhi ΔwaaG and mutant derivatives (24). Secreted Vi antigen was precipitated from culture supernatants using hexadecyltrimethylammonium bromide (11). The polysaccharide preparations were digested with DNase, RNase, and Proteinase K and were separated from residual LPS by gel filtration chromatography in the presence of detergent (see SI Methods for details). Isolation of the Vi Antigen Glycolipid Terminus. Twenty milligrams of purified Vi antigen were resuspended at 1 mg/mL in 50 mM sodium bicarbonate, 0.1 mM CaCl2 (pH 7.5). Purified VexL-His6 was added to a final concentration of 100 μg/mL; the reaction mixture was incubated at 37 °C for 5 h and then was loaded into a SepPak C18 cartridge. The column was washed with 10 mL of water, and bound hydrophobic material was eluted in 70% (vol/vol) acetonitrile. Eluted material was dried by SpeedVac and was resuspended in 100 μL 25% (vol/vol) acetonitrile in water. MS. LC-MS analyses of the glycolipid terminus were performed on an Agilent 1200 high-performance liquid chromatograph interfaced with an Agilent ultra-high-definition (UHD) 6530 quadrupole TOF (QTOF) mass spectrometer. A C18 column was used for chromatographic separation. Conditions for LC and MS are described in SI Methods.

antibody P2B1G2/A9, which is specific for Vi antigen (20). To ensure that the undigested Vi antigen resulted only from its inaccessibility, aliquots of cells were lysed by French press, unbroken cells were removed by centrifugation, and VexL-His6 was added, incubated, and analyzed as above. Detection of Cell-Free Vi Antigen in Culture Supernatants. LB cultures (50 mL) were grown at 37 °C until an OD600 of 0.5 was reached. Cells then were collected by centrifugation at 5,000 × g for 15 min. The supernatant was dialyzed against water for 2 d, using a dialysis membrane with a 3,500 molecular weight cutoff (MWCO). The dialyzed supernatant was lyophilized, resuspended in 1 mL of water, and examined by Western immunoblotting. Immunofluorescence Microscopy. Live and fixed/permeabilized cells were probed with Vi antigen-specific monoclonal antibody (P2B1G2/A9) (20) and were labeled with rhodamine red-conjugated goat anti-mouse IgG. See SI Methods for details.

Digestion of Cell Surface and Intracellular VI Antigen with VexL Lyase. Cultures were grown until OD600 = 0.5 was reached. Cells equivalent to one OD600 unit were collected by centrifugation and were resuspended in PBS supplemented with 0.1 mM CaCl2, with and without VexL-His6 (100 μg/mL final concentration). Cell suspensions were incubated at 37 °C for 1 h and were collected by centrifugation. The cells were solubilized in SDS/PAGE buffer and were analyzed by Western immunoblotting using mouse monoclonal

ACKNOWLEDGMENTS. Plasmids pGVXN158 containing the viaB locus, pNLP15 containing the spy promoter fused to the luxCDABE cassette, and E. coli BKT09 were generous gifts from Dr. Michael Wetter, Dr. Tracy Raivio, and Dr. Russell Bishop, respectively. We thank Prof. Ayub Qadri for the gift of monoclonal antibodies raised to Vi antigen; Drs. Dyanne Brewer and Armen Charchoglyan for technical assistance with MS; Mrs. Valerie Robertson and Dr. Andy Lo for technical support with NMR spectroscopy; Dr. Michaela Strüder-Kypke and Mr. Robert Harris for freeze substitution and electron microscopy; Dr. Colin Cooper for sequencing A. denitrificans; and Dr. Iain Mainprize for the generation of a wecA mutant in E. coli. This work was supported by funding from Canadian Institutes of Health Research. C.W. holds a Canada Research Chair, and S.D.L. is the recipient of a Canada Graduate Scholarship from the National Sciences and Engineering Research Council of Canada.

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Liston et al.

Unique lipid anchor attaches Vi antigen capsule to the surface of Salmonella enterica serovar Typhi.

Polysaccharide capsules are surface structures that are critical for the virulence of many Gram-negative pathogenic bacteria. Salmonella enterica sero...
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