Identification of the Pseudomonas aeruginosa O17 and O15 O-Specific Antigen Biosynthesis Loci Reveals an ABC Transporter-Dependent Synthesis Pathway and Mechanisms of Genetic Diversity
ABSTRACT
Many bacterial cell surface glycans, such as the O antigen component of lipopolysaccharide (LPS), are produced via the so-called Wzx/Wzy- or ABC transporter-dependent pathways. O antigens are highly diverse polysaccharides that protect bacteria from their environment and engage in important host-pathogen interactions. The specific structure and composition of O antigens are the basis of classifying bacteria into O serotypes. In the opportunistic pathogen Pseudomonas aeruginosa, there are currently 20 known O-specific antigen (OSA) structures. The clusters of genes responsible for 18 of these O antigens have been identified, all of which follow the Wzx/Wzy-dependent pathway and are located at a common locus. In this study, we located the two unidentified O antigen biosynthesis clusters responsible for the synthesis of the O15 and the O17 OSA structures by analyzing published whole-genome sequence data. Intriguingly, these clusters were found outside the conserved OSA biosynthesis locus and were likely acquired through multiple horizontal gene transfer events. Based on data from knockout and overexpression studies, we determined that the synthesis of these O antigens follows an ABC transporter-dependent rather than a Wzx/Wzy-dependent pathway. In addition, we collected evidence to show that the O15 and O17 polysaccharide chain lengths are regulated by molecular rulers with distinct and variable domain architectures. The findings in this report are critical for a comprehensive understanding of O antigen biosynthesis in P. aeruginosa and provide a framework for future studies.
IMPORTANCE P. aeruginosa is a problematic opportunistic pathogen that causes diseases in those with compromised host defenses, such as those suffering from cystic fibrosis. This bacterium produces a number of virulence factors, including a serotype-specific O antigen. Here, we identified and characterized the gene clusters that produce the O15 and O17 O antigens and show that they utilize a pathway for synthesis that is distinct from that of the 18 other known serotypes. We also provide evidence that these clusters have acquired mutations in specific biosynthesis genes and have undergone extensive horizontal gene transfer within the P. aeruginosa population. These findings expand on our understanding of O antigen biosynthesis in Gram-negative bacteria and the mechanisms that drive O antigen diversity.
INTRODUCTION
The lipopolysaccharide (LPS) in the Gram-negative bacterial outer membrane is an important virulence factor of many pathogens. By virtue of its localization, this glycolipid interacts with the surrounding environment and plays a role in protecting the bacterium from external stresses. The structure of LPS is comprised of up to three domains. Lipid A is the anchor that holds the macromolecule in the outer membrane and is the endotoxic component of LPS. This is covalently linked to a core oligosaccharide (OS) that can then be substituted, or “capped,” with a long polymer of sugars termed the O antigen. The O antigen is heterogeneous in both the chemical makeup and the lengths of the polysaccharides that are displayed on the surface of various bacterial species. Functionally, O antigens of specific lengths confer protection to Gram-negative bacteria against the killing effects of phage (1), bacteriocins (2), complement (3, 4), and bile acid (5, 6). The length of the polysaccharide is also an important factor when designing effective glycoconjugate vaccines against bacterial pathogens (7–12).
In the literature, the synthesis of O antigens generally follows one of two working models: the ABC transporter-dependent or Wzx/Wzy-dependent pathway. These model pathways differ in both the site of polymer synthesis and the molecular machinery used to regulate the O antigen chain length. The ABC transporter-dependent pathway begins at the cytoplasmic face of the inner membrane (IM), where synthesis is initiated by the addition of a short sugar adapter to the lipid carrier, undecaprenyl phosphate (Und-P). Glycosyltransferases (GTs) then polymerize the long polysaccharide, which is exported to the periplasmic face of the IM by the concerted action of Wzm and Wzt, the membrane and cytosolic components of the O antigen ABC transporter, respectively (13). In contrast, O antigen that follows the Wzx/Wzy route of synthesis is polymerized from a pool of undecaprenyl-linked oligosaccharides termed O-units. The O-units are synthesized in the cytoplasm but flipped to the periplasmic face of the IM by Wzx, where Wzy then polymerizes them into long chains (14). After the synthesis of O antigen by either route, the polysaccharide follows common steps that ligate it to lipid A-core and export the completed molecule to the outer membrane (15–17). Importantly, the synthesis of O antigen via the Wzx/Wzy-dependent system can also yield a so-called “core+1” moiety, which is the consequence of an O-unit bypassing polymerization and proceeding directly to ligation. Two other less common models of O antigen biosynthesis are the synthase-dependent pathway and the Wzk-dependent pathway, but to date, they have been identified only in Salmonella enterica serovar Borreze and Helicobacter pylori, respectively (18, 19).
The ABC transporter and Wzx/Wzy pathways have evolved separate strategies for controlling the length of the polysaccharide polymer. During the synthesis of O antigen by ABC transporter-dependent pathways, the chain length of the polysaccharide may be determined by several means. One approach is to establish equilibrium between the glycosyltransferases that extend the polymer and the ABC transporter that exports it. In such systems, the experimental overexpression of glycosyltransferases results in longer polysaccharides, while the overexpression of the ABC transporter results in shorter chains due to the premature export of the polymer (20). The second strategy is to terminate synthesis after a specific chain length has been achieved. The Escherichia coli O8/O9/O9a and Klebsiella pneumoniae O12 O antigen systems are well-studied prototypes for this particular strategy. A termination protein or domain is generally separated from the membrane and growing polymer by a coiled-coil domain, ensuring that synthesis is halted only when the polysaccharide achieves a given length (21, 22). The termination moiety is specifically recognized by the carbohydrate-binding module (CBM) of Wzt and signals its export (23). Since the length of the coiled-coil domain determines the chain length, these proteins effectively measure the polysaccharide, and hence, they are described as “molecular rulers” (21, 22). In E. coli O8/O9/O9a and K. pneumoniae O12, polymerization is terminated at the nonreducing end by the addition of a methyl group (O8) (24), methylphosphate (O9/O9a) (24, 25), or a β-3-deoxy-d-manno-octulosonic acid (β-Kdo) moiety (O12) (26, 27). WbdD is a terminator protein that has methyltransferase (MT) and dual kinase/methyltransferase activities in O8 and O9a, respectively, and is encoded separately from the chain-extending glycosyltransferase WbdA (24, 28). In contrast, K. pneumoniae O12 encodes a unique multidomain protein, WbbB, which is a fusion of the two glycosyltransferases that extend the polymer, a coiled coil, and the terminating β-Kdo transferase. This integrated approach to polymerization, chain length determination, and termination appears to be used by many bacterial species that utilize molecular rulers (22). In the Wzx/Wzy-dependent pathway, polysaccharide copolymerase class 1a (PCP1a) proteins, which are more commonly known as Wzz, are the dedicated chain length regulators of the O antigen. These proteins form a large bell-shaped oligomer that determines polymer length via an unknown mechanism but is likely dependent on the stability of Wzz and interactions with both Wzy and the polysaccharide (29–31). We point the reader to a recent review by Whitfield et al. for excellent diagrammatic representations of these two pathways (32).
The opportunistic pathogen Pseudomonas aeruginosa is known to be remarkably drug resistant and can cause acute or chronic infections in individuals with compromised host defenses, such as those suffering from cystic fibrosis. The need for new antibiotics targeting this organism has been classified as “critical” by the Centers for Disease Control and Prevention. P. aeruginosa uses both the ABC transporter- and Wzx/Wzy-dependent systems for the synthesis of two distinct chemotypes of O antigen that may be simultaneously produced on the cell surface: common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA is a homopolymer of d-rhamnose that is produced through an ABC transporter-dependent pathway that is encoded by a conserved gene cluster (33, 34). In contrast, OSA is a heteropolymer of O-units that follows a Wzx/Wzy-dependent pathway. The sugar identities and specific linkages of the O-units, and, thus, the genes in the biosynthesis clusters, are highly variable (35). This diversity forms the basis of categorizing P. aeruginosa into 20 unique serotypes (O1 to O20) according to the International Antigen Typing Scheme (IATS) (34). The OSA biosynthesis clusters of 18 of these serotypes have been identified, which has facilitated the systematic identification of genes essential for OSA production and their function and, recently, the advent of an in silico method to rapidly determine the serotype of P. aeruginosa isolates from whole-genome sequencing data (36). The chromosomal locus of each of the clusters of these 18 serotypes is characteristically localized to a region that is flanked by himD (also known as ihfB [integration host factor subunit beta]) at the 5′ end and by wbpM at the 3′ end of the P. aeruginosa chromosome (34, 35). The biosynthesis clusters of O15 and O17 have yet to be identified due to genetic anomalies at this usual OSA locus. In IATS O17, an O11 cluster with two inactivating insertions is found between himD and wbpM (35, 37). Similarly, two O15 strains investigated previously by Raymond et al. contained either an inactivated O3 cluster or a nearly complete deletion of an O11 cluster (35). The O3 and O11 OSA structures are unrelated to those of O15 and O17, and therefore, genes from these clusters are not likely to contribute to the synthesis of these O antigens (38). Taken together, these observations led to the assumption that the O15 and O17 clusters are found elsewhere in the genome and may have been acquired by horizontal gene transfer (HGT) (35, 37). The identification of these clusters would facilitate the discovery of new OSA biosynthesis proteins and allow the accurate serotyping of isolates by in silico methods. In this work, we took advantage of the data generated from one of our recent whole-genome sequencing studies of IATS O1 to O20 to identify the O15 and O17 OSA biosynthesis clusters (39). We found that unlike the other serotypes studied to date, O15 and O17 follow an ABC transporter-dependent pathway. We then set out to characterize the chain length-determining strategies in these serotypes. We provide evidence that O17 utilizes a modular, three-domain protein that is distinct from WbbB for the synthesis and termination of OSA, while O15 chain length is controlled by a WbdD-like putative methyltransferase. The dissemination of these clusters in the P. aeruginosa population and their various genomic contexts were also investigated, which confirmed that they have undergone extensive horizontal gene transfer.
RESULTS
Identification of putative O17 and O15 OSA biosynthesis clusters containing ABC transporter and chain length-determining genes.
O17 is a unique serotype because it lacks the core+1 glycoform (40). This led us to hypothesize that the biosynthesis of this O antigen is ABC transporter dependent rather than Wzx/Wzy dependent. Therefore, we used the sequence of the CPA Wzm protein as a query in a BLAST search of the genome of IATS O17, which identified two homologs of WzmCPA: one was localized to the locus of a type II secretion cluster that was also present in PAO1, and the other was found within an O17-specific cluster of genes similar to those known to be involved in O antigen biosynthesis (Fig. 1; Table 1). We first investigated this cluster for genes that might be involved in the synthesis of the O17→4)-l-Rha-(α1→3)-d-ManNAc-(β1→ repeat unit (40). Accordingly, a homolog of WecB (orf10) (Fig. 1; Table 1), an enzyme that catalyzes the epimerization of UDP-GlcNAc to UDP-ManNAc (41), was identified. Furthermore, two copies of the rmlBDAC operon were present in the opposite orientations (Fig. 1). The rml operon is essential for the synthesis of dTDP-l-Rha (42). However, several mutations were discovered in the duplicated operon compared to the “native” rmlBDAC operon, suggesting that one or more of the genes may be nonfunctional. Since the addition of l-Rha to Glc-I of the core OS by the rhamnosyltransferase WapR is essential to cap it with O antigen (43, 44), at least one of these operons is expected to be functional. Also encoded in this cluster are several glycosyltransferases (GTs) (products of orf1, orf11, and orf12), a second epimerase (product of orf13), and a WbpL homolog (product of orf14) (Table 1). WbpL was reported by our group to be essential for the initiation of O antigen biosynthesis by the addition of a monosaccharide to Und-P (45). Importantly, as expected, downstream of wzm is a homolog of wzt, the cognate gene that encodes the ATPase of O antigen ABC transporters. The protein sequence of Wzt has an extended C-terminal domain and a predicted CBM, suggesting that it recognizes a sugar moiety at the terminus of its substrate. Downstream of wzt is an open reading frame (ORF) (orf8) that encodes a large 1,003-amino-acid protein. Evidence of specific roles played by Wzm/Wzt and Orf8 in the biosynthesis of O antigen is presented below.
FIG 1
TABLE 1
| Gene | Conserved domain(s) (identifier), E value(s) | Representative homolog (species, % coverage, % identity)a | Predicted function(s)b |
|---|---|---|---|
| hipA | HipA_C (pfam07804), 1.90e−30; HipA_N (pfam07805), 7.50e−24; Couple_HipA (pfam13657), 4.06e−20 | HipA (E. coli K-12 substrain MG1655, 81, 31) | Bacterial persistence protein |
| orf1 | GT2-like domain (cl11394), 1.55e−85 | NA | Glycosyltransferase |
| rmlB | RfbB (COG1088), 0e+0 | RmlB (P. aeruginosa PAO1, 98, 83) | dTDP-d-glucose 4,6-dehydratase |
| rmlD | NADB_Rossmann superfamily (cl21454), 1.76e−159 | RmlD (P. aeruginosa PAO1, 96, 58) | dTDP-6-deoxy-l-xylo-4-hexulose reductase |
| rmlA | GCD1 superfamily (cl28239), 2.16e−174 | RmlA (P. aeruginosa PAO1, 100, 87) | Glucose-1-phosphate thymidylyltransferase |
| rmlC | dTDP_sugar_isomerase (pfam00908), 1.30e−113 | RmlC (P. aeruginosa PAO1, 99, 74) | dTDP-6-deoxy-d-xylo-4-hexulose reductase |
| wzm | ABC2 membrane superfamily (cl21474), 2.46e−48 | Wzm (P. aeruginosa PAO1, 95, 45) | Membrane component of ABC transporter |
| wzt | ABC_KpsT_Wzt (cd03220), 2.46e−115 | Wzt (P. aeruginosa PAO1, 56, 50) | Cytosolic ATP-binding component of ABC transporter |
| orf8 | Methyltransf_25 (pfam13649), 3.27e−9; GT1_like (cd04950), 4.43e−103; GT_2_like_c (cd04186), 2.11e−46 | NA | GT1 and GT2 glycosyltransferase domains, methyltransferase domain |
| orf9 | Ado_MTase superfamily (cl10013), 1.36e−6 | NA | Methyltransferase |
| orf10 | WecB (COG1216), 1.34e−171 | WecB (E. coli K-12, 97, 56) | UDP-GlcNAc epimerase |
| orf11 | GTB_type superfamily (cl10013), 9.71e−50 | NA | Glycosyltransferase |
| orf12 | GT2 (COG1216), 2.44e−64 | NA | Glycosyltransferase |
| orf13 | UDP_G4_E_SDR_e (CD05232), 2.05e−15 | NA | UDP-glucose-4-epimerase |
| orf14 | GT_WbpL_WbcO_like (cd06854), 2.25e−65 | WbpL (P. aeruginosa PAO1, 91, 41) | Transfer of FucNAc to Und-P, initiation of O antigen biosynthesis |
| wbpM | FlaA1 (COG1086), 0e+0 | WbpM (P. aeruginosa PAO1, 96, 49) | UDP-d-GlcNAc 4,6-dehydratase |
| orf16 | COG4584, 7.91e−22 | NA | Transposase |
| orf17 | IstB_IS21 (pfam01695), 3.61e−75 | NA | P-loop ATPase associated with IS21 insertion sequences, pseudogene (frameshifted) |
| orf18 | FlaA1 (COG1086), 5.80e−12 | WbpM (P. aeruginosa PAO1, 94, 48) | C-terminal end of WbpM |
| orf19 | NA | NA | Hypothetical protein |
a
NA, not applicable.
b
Predicted based on the function of the closest characterized homolog or the CDD prediction when no characterized homolog was available.
Serotype O15 similarly lacks the core+1 glycoform; hence, we hypothesized that this OSA is also synthesized via an ABC transporter-dependent pathway. Using the same approach as the one used for O17, we identified a cluster of genes in O15 that contained a homolog of wzmCPA (Fig. 1). As expected, a homolog of wztCPA was located downstream of, and partially overlapping, the wzm ORF. This cluster was analyzed for genes that might be essential for the synthesis of the O15 O antigen, which is a repeat structure of →2)-d-Ribf-(β1→4)-d-GalNAc-(α1→ (40) (Table 2). Accordingly, a homolog of GalE (orf1), an epimerase that catalyzes the reversible conversion of UDP-GlcNAc to UDP-GalNAc, and/or UDP-Glc to UDP-Gal (46), was identified; however, genes for the synthesis of the ribofuranose sugar nucleotide were not discerned within the cluster, possibly because this sugar donor is acquired from common pools (47). Similar to O17, this cluster also contained a homolog of WbpL (orf10). Interestingly, these two WbpL homologs are 95% identical and 97% similar to each other but approximately 38% identical and 53% similar to the previously characterized WbpL proteins of serotypes O5 and O6. This suggests that the initiating sugars of O15 and O17 are the same (or similar) but are different from those of O5 and O6. Several predicted glycosyltransferases (orf6 and orf8), a hypothetical protein with unknown function (orf5), and a homolog of WbpM (orf11) were also present in this gene cluster (Table 2). Interestingly, we did not find a protein with a similar domain arrangement or homology to Orf8O17, but we identified a putative methyltransferase with a predicted C-terminal coiled-coil domain that was encoded by orf4 downstream of wzt. Based on these observations, we proposed that the biosynthesis of the O15 OSA also follows an ABC transporter-dependent pathway and that the putative methyltransferase gene would encode the terminator/chain length regulator of this polysaccharide.
TABLE 2
| Gene | Conserved domain(s) (identifier), E value(s) | Representative homolog (species, % coverage, % identity) | Predicted function(s)a |
|---|---|---|---|
| orf1 | GalE (COG1087), 0e+0 | GalE (P. aeruginosa PAO1, 99, 59) | UDP-glucose-4-epimerase |
| wzm | TagG (COG1682), 2.19e−54 | Wzm (P. aeruginosa PAO1, 95, 43) | Membrane component of ABC transporter |
| wzt | TagH (COG1134), 3.42e−108; Wzt_C-like (CD10147), 9.47e−27 | Wzt (P. aeruginosa PAO1, 93, 36) | Cytosolic ATP-binding component of ABC transporter |
| orf4 | Methyltransf_25 (pfam13649), 4.47e−14; DUF390 superfamily (cl25642), 1.14e−5; SMC_N superfamily (cl25732), 7.77e−5 | NA | Methyltransferase |
| orf5 | NA | NA | NA |
| orf6 | GTB_type superfamily (cl10013), 1.89e−10 | NA | Glycosyltransferase (GTB superfamily) |
| orf7 | HAD_like superfamily (cl21460), 1.51e−18 | NA | Hydrolase |
| orf8 | GT2 (COG1216), 1.48e−61 | WbbL (K. pneumoniae, 79, 28) | Rhamnosyltransferase |
| orf9 | WcaG (COG0451), 1.85e−15 | NA | UDP-glucose-4-epimerase |
| orf10 | GT_WbpL_WbcO_like (cd06854), 8.02e−67 | WbpL (P. aeruginosa PAO1, 88, 42) | Transfer of FucNAc to Und-P; initiation of O antigen biosynthesis |
| wbpM | FlaA1 (COG1086), 0e+0 | WbpM (P. aeruginosa PAO1, 98, 49) | UDP-d-GlcNAc 4,6-dehydratase |
| orf12 | NA | NA | Hypothetical protein |
a
Predicted based on the function of the closest characterized homolog or the CDD prediction when no characterized homolog was available.
Evidence that an O17 wzm-wzt deletion abrogates OSA transport.
To determine if Wzm/Wzt encoded in the putative O17 cluster was involved in OSA biosynthesis, we constructed a simultaneous clean deletion of wzmO17 and wztO17. When LPS prepared from this mutant (ΔwzmO17 ΔwztO17) was analyzed by SDS-PAGE and silver staining, there was a significant reduction in the amount of OSA that could be detected compared to that of the wild-type control, implicating Wzm and Wzt in the production of OSA (Fig. 2A). By expressing the wzm and wzt genes in trans on the arabinose-inducible plasmid pHERD20T (designated wzm-t-20T), the level of O17 OSA production was restored, which confirmed that the mutant phenotype was due specifically to the loss of wzm and wzt. OSA detection was restored to an amount that was comparable to that of the wild type in the complemented strain with and without induction (0.1% l-arabinose). When the same samples were analyzed by Western immunoblotting using polyclonal anti-O17 antibodies, OSA was detected in LPS samples from both the ΔwzmO17 ΔwztO17 strain and the ΔwzmO17 ΔwztO17 strain complemented with wzm-t-20T. Since Und-P-linked polysaccharides are detected poorly by silver staining but are detectable by Western blotting (48), this suggests that in the ΔwzmO17 ΔwztO17 mutant, OSA is not ligated to lipid A-core. This phenotype is consistent with a defect in OSA transport. Interestingly, the average chain length of the polysaccharide detected by Western blotting was shorter in the ΔwzmO17 ΔwztO17 mutant but was rescued by providing wzm-t-20T in trans. This apparent shift may be a bona fide reduction in chain length or simply a difference in the electrophoretic mobility of lipid A-linked versus Und-P-linked polysaccharide. To confirm that OSA was not expressed on the cell surface of the ΔwzmO17 ΔwztO17 strain, we used anti-O17 antibodies to perform immunofluorescence analysis of whole P. aeruginosa cells. As expected, the O17 strain or the ΔwzmO17 ΔwztO17 strain complemented with wzm-t-20T exhibited a strong fluorescence signal, indicating that O antigen was presented on the cell surface. In contrast, the ΔwzmO17 wztO17 strain or the ΔwzmO17 ΔwztO17 strain containing the empty vector was not fluorescent (see Fig. S1 in the supplemental material). Together, these results implicate the wzmO17 and wztO17 genes in the transport of O17 OSA.
FIG 2
Orf8O17 is involved in the extension and chain length determination of O17 OSA.
We next investigated the role of the large three-domain protein encoded by orf8 in O17 OSA biosynthesis. The deletion of orf8 abrogated OSA biosynthesis, and no LPS banding pattern could be discerned when LPS samples from this mutant were analyzed by SDS-PAGE and silver staining (Fig. 3A). In contrast to the observations made in the ΔwzmO17 ΔwztO17 strain, only a few faint O antigen bands were detected by Western immunoblotting in these preparations. These bands may indeed be OSA, which could indicate that Orf8 is important but not essential for OSA synthesis or that both Orf8-dependent and Orf8-independent OSA synthesis mechanisms exist simultaneously. However, since the antibodies used in this experiment are polyclonal (i.e., raised against wild-type O17), the more likely explanation is that this material is CPA and not OSA. Although we were unable to detect CPA in O17 or the Δorf8O17 strain using the CPA-specific monoclonal antibody (mAb) N1F10, the banding pattern in the Δorf8O17 mutant is consistent with the size of CPA. We also determined that the O17 polyclonal antibodies cross-reacted with LPS that is consistent with the size of CPA in LPS preparations from PAO1 (PAO1 is a serotype O5 strain that produces CPA) (see Fig. S2 in the supplemental material). We suggest that in O17, CPA might not be produced in amounts that permit immunochemical detection of this LPS antigen with mAb N1F10. Alternatively, the O17 CPA may contain unidentified modifications that block the binding of N1F10 antibodies. Overall, the results presented in Fig. 3A indicate that the Δorf8O17 mutant is deficient in the synthesis of the O17 OSA polysaccharide. The production of OSA was restored when the Δorf8 mutant was supplied with pHERD20T::orf8 (designated orf8-20T) in trans (Fig. 3A). Interestingly, without the induction of gene expression (i.e., at a basal expression level), the complemented strain seemed to produce more OSA chains of a shorter length than the wild type. In contrast, only wild-type chain lengths were displayed when orf8 expression was induced with 0.1% l-arabinose, which suggests that the O17 OSA chain length may be influenced by the amount of Orf8 produced. The increased expression of orf8 in the presence of arabinose was confirmed by repeating these experiments with a FLAG-tagged variant of Orf8 and measuring protein production by Western blotting (see Fig. S3 and S4 in the supplemental material).
FIG 3
Due to the large size of Orf8, we reasoned that this protein could play multiple roles in OSA biosynthesis, particularly in elongation and chain length determination. Indeed, subjecting the sequence of Orf8 to a conserved domain search revealed three predicted domains: a methyltransferase (MT)-like domain followed by the glycosyltransferase family 1 (GT1) and GT2 domains. The methyltransferase domain is part of the “methyltransferase 25” conserved protein domain family (pfam13649), which also includes WbdD as a member. We therefore analyzed OSA production in the Δorf8 strain expressing either the MT domain or the two GT domains (Fig. 3A). The Δorf8 strain expressing either of the constructs did not produce any OSA when the LPS from this strain was analyzed by SDS-PAGE and silver staining. However, when the same samples were analyzed by Western immunoblotting, a high-molecular-weight polysaccharide “smear” that was longer than the longest OSA chains produced by wild-type O17 was detected in the Δorf8 strain expressing only the GT domains (Fig. 3A, arrow). Taken together, these data suggest a role for the GT domains in the synthesis of the polysaccharide chain and the MT domain in both chain length determination and export. To demonstrate that the phenotypes of the truncated orf8(MT) or orf8(GT) constructs were not a result of impaired expression, we added a FLAG tag to the N terminus of each construct, expressed the constructs in the Δorf8 strain, and analyzed protein levels by Western blotting. Bands consistent with the expected molecular masses of both FLAG-Orf8(MT) and FLAG-Orf8(GT) were detected in induced whole-cell protein samples. The intensity of the bands corresponding to both constructs was greater than that of the full-length FLAG-Orf8 construct, indicating that FLAG-orf8(MT) and FLAG-orf8(GT) were robustly expressed (see Fig. S3 in the supplemental material). The LPS phenotypes of the tagged and untagged constructs were similar (see Fig. S4 in the supplemental material).
To determine whether or not the putative methyltransferase domain in Orf8 is in fact a determinant of chain length, we overexpressed it in the wild-type O17 background. The controlled induction of orf8(MT) expression by increasing the arabinose concentration resulted in the bacteria exhibiting reduced OSA chain lengths, most notably in the highest-molecular-weight band (Fig. 3B). We note that this decrease in chain length is subtle, which is expected since the orf8(MT) construct must compete with the wild-type biosynthetic machinery when expressed in O17. In fact, FLAG-orf8(MT) could not be detected by Western blotting when expressed in the O17 background (data not shown). The phenotype conferred by FLAG-orf8(MT) on O17 was not as strong as that of the untagged variant but still resulted in a reduction of the chain length (see Fig. S4 in the supplemental material). Given the apparent role of Orf8 in the regulation of the chain length of O17 OSA, we examined the protein sequence for similarities to WbdD or WbbB. We first analyzed the sequence for potential coiled coils using the NPS@ coiled-coil prediction server (49), but this exercise did not yield any significant predictions. Furthermore, a BLAST search did not identify WbdD or WbbB as a homolog of either the full-length Orf8 protein sequence or the methyltransferase domain alone. Similarly, the alignment of the methyltransferase domain of Orf8 to that of WbdD was poor (15% identity and 27% similarity). These results indicate that although Orf8 appears to terminate and regulate the chain length of O17 OSA, it is unique compared to the previously studied molecular rulers of E. coli and K. pneumoniae.
The putative O15 OSA biosynthesis cluster is recalcitrant to gene deletions.
To test the role of wzm-wzt and orf4 in O15, we attempted to knock out these genes using pEX18Gm-based deletion constructs, as we did for serotype O17. The pEX18 series of plasmids are suicide vectors allowing gene modification by two-step allelic exchange. Briefly, the gene deletion plasmid is inserted at the targeted locus by homologous recombination, resulting in a merodiploid strain (containing both the gene deletion and the wild-type gene). Sucrose counterselection mediated by pEX18Gm-borne sacB is then used to select for a second recombination event that removes all plasmid sequences along with either the wild-type or modified gene. Repeated attempts to resolve the ΔwzmO15 ΔwztO15 or Δorf4O15 merodiploid by sucrose counterselection consistently resulted in the detection of the wild-type gene but not the deletion when potential mutants were screened by colony PCR (data not shown). These results suggested that the deletion of wzm-wzt and orf4 might be lethal and that only colonies harboring secondary mutations are viable.
To alleviate any potential toxicity that the construction of this deletion had on O15, we isolated a ΔwzmO15 ΔwztO15 merodiploid (designated ΔwzmO15 ΔwztO15-M) and transformed this strain with wzm-tO15-pHERD20T before sucrose counterselection, resulting in the ΔwzmO15 ΔwztO15-M(wzm-t-20T) strain. Our group has previously used a similar methodology to study an essential core OS kinase, waaP, and the lipid A-core ABC transporter MsbA in P. aeruginosa (50, 51). Using this approach, we were able to isolate several deletions of wzm and wzt. When the LPS of these mutants containing the complementing plasmid was extracted and visualized by SDS-PAGE and silver staining, the LPS profile was comparable to that of wild-type O15 (Fig. 2B). Using a similar strategy for orf4O15, we were still unable to isolate a deletion of this gene.
We reasoned that another gene in the cluster might be more amenable to deletion, so we targeted a gene in the cluster that is a putative homolog of wbpL (wbpLO15), which presumably initiates O antigen synthesis by the addition of a monosaccharide to Und-P. A deletion of wbpL is not expected to have lethal consequences because Und-P will not be sequestered by a blocked O antigen synthesis pathway. Accordingly, we were able to isolate a deletion of this gene, and the resulting strain was devoid of O antigen (see Fig. S5 in the supplemental material). Surprisingly, however, O antigen could not be restored in the ΔwbpLO15 strain by providing the gene in trans (data not shown). We hypothesized that the ΔwbpLO15 strain may have acquired secondary mutations during the passage and culturing necessary to transform it with the complementing plasmid. Amazingly, PCR analysis of three colonies isolated after the transformation of ΔwbpLO15 with either wbpL-20T or the pHERD20T empty vector revealed several gross mutations in wbpL and wzm-wzt compared to the parent strain: deletions of wbpL, deletions of wzm-wzt, and an insertion in wzm-wzt. Sequencing and subsequent BLAST analysis of the insertion in wzm-wzt revealed 99% identity to a transposon found in the O15 genome (see Fig. S5 in the supplemental material). Overall, these results indicate that gene deletions in this cluster rapidly drive other mutations within the cluster, including deletions and transposon insertions.
The putative methyltransferase of O15 has a role in OSA chain length regulation.
Although orf4O15 could not be deleted, we wanted to test if it had a role in OSA biosynthesis. orf4 is a predicted methyltransferase containing the methyltransferase 25 conserved domain according to the CDD and contains a predicted C-terminal coiled coil. Thus, although Orf4 has low identity to WbdD (11% identity and 18% similarity), the domain architectures of the two proteins are similar (methyltransferase domain and coiled-coil domain). Therefore, we hypothesized that Orf4 is the terminator/chain length determinant of the O15 OSA polysaccharide. We note that the O15 OSA has an unusual length distribution that is concentrated around a few bands, which necessitates its investigation (see reference 14 for a comparison of all P. aeruginosa LPS profiles). Previous reports have indicated that the expression level of the polysaccharide terminator can modulate the length of the O antigen chain (24), so we reasoned that the overproduction of full-length or truncated versions of Orf4 might affect the OSA chain length. Consistent with this hypothesis, we found that when this gene was overexpressed, there was an OSA species that migrated faster in the SDS-PAGE gel than the main population of O15 OSA (Fig. 3C, arrow). In an attempt to further perturb the wild-type chain length-determining system, we constructed a recombinant protein lacking the coiled-coil domain. When this truncated version of orf4 was overexpressed in O15, the average chain length was increased (Fig. 3C, sixth lane). We tested whether the overexpression of orf4 corresponded to the overproduction of Orf4 or Orf4(MT) by measuring the protein levels of FLAG-tagged variants of these constructs when overexpressed in the O15 background (see Fig. S3 in the supplemental material). Although the FLAG-tagged variant of Orf4 produced the same phenotype as the untagged protein, the tagged variant of Orf4(MT) did not confer a change in chain length to O15 OSA, suggesting an interference of the tag in the function of the protein. Overall, the production of shorter or longer chains when variations of orf4 are expressed is consistent with the role of a terminator/chain length regulator of this polysaccharide.
Genetic analysis of strains containing the O15 and O17 clusters reveals mechanisms of horizontal gene transfer.
Since the O15 and O17 OSA biosynthesis clusters have not been identified previously, we sought to examine their dissemination in the P. aeruginosa population by determining the overall relatedness of strains carrying these loci. This was motivated by a previous study by our group that discovered that strains belonging to the same serotype tended to be more related to each other than to those of other serotypes. However, some exceptions to this relationship were due to the phenomenon of “serotype switching,” whereby the horizontal gene transfer of an OSA cluster from one strain had replaced the native cluster of another (39). Importantly, O15 and O17 isolates could not be accurately examined in that study, and this was therefore warranted here. We first performed BLAST searches using the WztO15 and WztO17 protein sequences as queries to identify strains carrying the O15 and O17 clusters. We then constructed a maximum likelihood phylogeny based on single nucleotide polymorphisms (SNPs) in the core genomes of these isolates and the 20 IATS reference strains, as described previously (39). The phylogeny contained two major groups (A and B) and two minor groups (C and D), as expected (Fig. 4). Interestingly, the O15 isolates were found in the branches of both the A and B groups of the phylogeny, even though IATS O15, O11, and O3 were localized to group A (recall that the two O15 strains originally sequenced contained inactivated O11 or O3 clusters). Furthermore, the O15 isolates did not necessarily cluster together; i.e., they were found distributed throughout both groups on many different branches. In contrast, the O17 isolates were all grouped on one branch of group B, which contains IATS O17. These results suggest that the O15 cluster has disseminated throughout the P. aeruginosa population, whereas the O17 cluster is found in a group of closely related strains.
FIG 4
The presence of O15 strains in both phylogenetic groups prompted us to test if there was a divergence in the identity of the OSA clusters at the himD-wbpM locus. Indeed, by comparing the sequences of himD and wbpM to those reported previously by Raymond et al. (35), we found significant heterogeneity in the clusters at this location (Table 3). For example, we found several strains where the O11 cluster remained largely intact but appeared to be inactivated by transposon insertions at various sites. One O11-containing strain (MRSN20176) had a significant deletion of this cluster, with only the wzz, wbjF, wbpL, and wbpM genes remaining. Other isolates had disrupted O2/O5/O16/O18/O20 (O2 serogroup), O6, or O10/O19 (O10 serogroup) clusters that were similarly inactivated by transposons (Table 3). This observed diversity indicates that multiple events have occurred that resulted in both the acquisition of OSA clusters and their inactivation. Furthermore, some clusters did not have any obviously disruptive mutations, which raises the interesting possibility that these bacteria simultaneously produce two glycoforms of OSA, which is commonly observed in bacteria (52). In contrast to the O15 strains, we found that the O17 cluster was always associated with an inactivated O11 locus, but this may be due to a relatively low number of sequenced genomes containing the O17 cluster; six out of the eight strains that we could identify as O17 strains were from a pair of studies that examined a set of P. aeruginosa strains from cystic fibrosis clinical isolates compared to those isolated from dental unit waterlines (53, 54). These O17 strains (Chir_D-144, PPF-1, PPF-21, PPF-2, 146-assisstante, and PPF-7) were reported to contain an O11 biosynthesis locus with two transposon insertions that had disrupted the essential biosynthesis gene wzx (O-unit flippase) (54). Notably, the orientation and location of these insertions differ from those found in the O11 locus of IATS O17 (54), again indicating that multiple inactivation events have occurred.
TABLE 3
| Straina | Cluster (himD-wbpM) | Evidence of cluster inactivation | Source |
|---|---|---|---|
| Env_58 | O15 (O11 deletion) | Yes—deletion | River water |
| AZPAE13853 | O11 | No | India |
| PABL056 | O11 | No | Blood, USA (Chicago, IL) |
| MRSN20176 | O11 | Yes—deletion | Afghanistan; contains the blaVIM-II gene |
| AZPAE14865 | O11 | No | Respiratory tract infection, India (Chennai) |
| AZPAE14900 | O11 | Yes—deletion | Intraabdominal infection, India (Chennai) |
| RW109 | O10/O19 | No | Unilever culture collection |
| WH-SGI-V-07698 | O10/O19 | Yes—insertion of a transposase gene in orf8 (IS3 family) and orf12 (IS30 family) | Hospital isolate |
| AR439 | O6 | Yes—insertion of a transposase gene in wbpR (IS30 family) | NA |
| AZPAE14827 | O6 | No | Intra-abdominal infection, USA (Detroit, MI) |
| AS012587 | O6 | No | Pneumonia, USA |
| WH-SGI-V-07237 | O2/O5/O18/O20 | Yes—insertion of a transposase gene in wbpA (IS5 family) | Hospital isolate |
| VRFPA09 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Septicemia, India; multidrug resistant |
| CMC_VB_PA_B22862 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, blood, India (Vellore); extremely drug resistant |
| B14130 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, blood, India |
| SP4527 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, sputum, India |
| SP4371 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, sputum, India |
| AR_0443 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | FDA/CDC Antimicrobial Resistant Isolate Bank |
| PA-81 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Tracheal secretions, Pakistan; contains New Delhi metallo-β-lactamase, carbapenemase |
| MRSN12914 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Urine; Multidrug Resistant Organism Repository and Surveillance Network |
| B17932 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, blood, India |
| SP4528 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Respiratory tract infection, sputum, India |
| GIMC5016:PA1840 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Burn wound, Russia (Moscow); ST2592b |
| NCTC13719 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Blood, UK (London); ST654b; blaVIM metallocarbapenemase positive |
| 514747 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | NA |
| PA1 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Pneumonia, bronchial aspirate, France (Paris) |
| PA5 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Pneumonia, bronchial aspirate, France (Paris) |
| BA7823 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) and frameshifts in other genes | Bacteremia, blood, India |
| SP2230 | O11 | Yes—insertion of a transposase gene in wzy (IS3 family) | Bacteremia, sputum, India |
a
Only isolates containing the himD-wbpM locus on a single contig were analyzed.
b
Sequence type.
We reasoned that it was also possible that the O15 and O17 clusters were acquired multiple times, which led us to analyze their location in the genomes of the sequenced isolates. In the O17 isolates, we found that the chromosomal locus of the cluster was consistently positioned between emrB and rmlB (Fig. 5). Another gene, hipA, was also identified in this island, which codes for the toxin component of the HipA-HipB toxin-antitoxin module and has been implicated in bacterial persistence (55). Interestingly, downstream of emrB is the coding region for tRNAThr. tRNA genes are often the targets of site-specific recombinases that transfer genomic islands into the chromosomes of recipient bacteria. These particular islands often contain phage genes, site-specific recombinase genes, and a duplicated tRNA (56). We identified a duplicated tRNA adjacent to rmlB and found several genes that were putative homologs of integrases and mobile element proteins on this island. These observations suggest that the O17 cluster was obtained in these bacteria through site-specific integration, and our analyses map the precise borders of this unique island.
FIG 5
In the O15 isolates, we discovered several variations in the genomic locus of the OSA biosynthesis cluster. In isolates MRSN20176 and NCTC11839 (equivalent to IATS O15/ATCC 33362), the O15 cluster was flanked by emrB and rmlB. Within this region, there was considerable similarity to the O17 regions, including the duplicated tRNA, mobile element proteins, and integrases, suggesting a common origin (Fig. 5). Interestingly, the genomic island in MRSN20176 has a relatively large size of approximately 71 kb due to the presence of a cluster of predicted phage genes. Another group of isolates (Fig. 5, represented by isolate SP4528) were chromosomally encoded but at a different locus. Notably, the clusters of these isolates were truncated after wbbL. Finally, in three other O15 strains that have been completely sequenced, AR439, RW109, and PABL048, it was intriguing that the O15 OSA clusters were not harbored on the chromosome, but rather, they were localized to large plasmids (437 kb, 555 kb, and 415 kb, respectively). To our knowledge, this is the first demonstration of the presence of a plasmid-borne OSA cluster in P. aeruginosa. For the genomes of the other O15 strains from our phylogenetic analysis that were assembled only at the contig level, we extracted the sequences of the contigs containing the O15 clusters and found that they all aligned well with either the plasmid or chromosomal modalities described above, indicating that both seem to be common in the population (see Fig. S6 in the supplemental material).
Finally, due to the diversity in the loci of these clusters, we postulated that variations within the O15 and O17 clusters might have arisen as well. Surprisingly, when we compared alignments among the clusters of the O17 and O15 isolates, there were noticeable insertions and deletions localized to orf8O17 and orf4O15. A multiple-sequence alignment of the Orf8O17 amino acid sequences revealed that the cause of these insertions and deletions was a 14-amino-acid motif located between the methyltransferase and GT1 domains that was repeated 4, 9, 10, or 17 times (see Fig. S7 in the supplemental material). Analysis of the predicted secondary structure (performed by using the PredictProtein server [57]) revealed that the repeating region of Orf8 is likely comprised of a single alpha helix. Similarly, when we analyzed Orf4O15, we found a 28-amino-acid sequence within the alpha-helical coiled-coil domain that was repeated 1 to 6 or 8 times (see Fig. S8 in the supplemental material). These results demonstrate that diversity within specific domains of the chain length-determining proteins has evolved in the O17 and O15 clusters.
DISCUSSION
Prior to this study, the gene clusters responsible for the biosynthesis of P. aeruginosa O17 and O15 OSAs were unknown. Here, we used a bioinformatics approach to identify genetic loci containing homologs of LPS biosynthesis genes and designed experiments to provide evidence that they are involved in the synthesis of O17 and O15 OSAs. These OSA clusters appear to be relatively self-contained and possess most of the genes that would be required for the synthesis and transport of the polysaccharide (Tables 1 and 2). However, we cannot rule out that genes outside these clusters may also be necessary for the synthesis of OSA in these serotypes. For instance, a knockout mutation of wbpM in IATS O17 generated in our laboratory significantly reduced OSA production (58), whereas a study by Dean and Goldberg found that a similar knockout abrogated OSA biosynthesis (37). Although the discrepancy in these two results has not been resolved, both studies suggested that WbpM played a role in the synthesis of O17 OSA, but this role is difficult to discern. Biochemical evidence reported by our group has shown that WbpM catalyzes the conversion of UDP-d-GlcNAc to UDP-4-keto-d-QuiNAc, a precursor of UDP-d-FucNAc and UDP-d-QuiNAc; however, all of these N-acetylated sugar structures are absent from the O17 OSA repeat structure (59).
This study has revealed that both the ABC transporter-dependent and Wzx/Wzy-dependent pathways are used for the production of OSA in P. aeruginosa. In the O17 Δwzm Δwzt knockout, OSA was barely detected by silver staining but was easily visualized by Western blotting. Furthermore, OSA was not detected on the cell surface of this mutant when assayed by immunofluorescence. These results confirmed that Wzm and Wzt are involved in the synthesis of OSA in O17 and are consistent with the hypothesis that they transport the Und-P-linked polymer across the inner membrane. Attempts to make similar mutations in O15 proved to be difficult, suggesting an inability of this strain to deal with the stress of blocked O antigen synthesis. Since peptidoglycan is also synthesized on Und-P, sequestering of this lipid in an unusable state can be lethal to bacteria and exerts strong selective pressure for mutations that alleviate the buildup of these intermediates (60). Consistent with our observation was a report by Cuthbertson et al. that E. coli carrying a knockout mutation of wzm and wzt rapidly accumulated compensatory mutations in other genes (61). For P. aeruginosa serotype O5, our group reported that during the construction of wzx knockout strains, some mutants acquired secondary-site mutations in wbpL (62). Furthermore, in serotype O11, knockout mutations made in wbpM resulted in some strains that also exhibited defects in the core oligosaccharide region of LPS (63). Similar to these previous studies, we isolated O15 strains where secondary mutations had occurred; a clear truncation of the core oligosaccharide and no detectable OSA were observed when the LPS of these strains was analyzed by silver staining, and neither LPS species was restored by supplying the respective gene in trans (see Fig. S5 in the supplemental material). The underlying mechanisms that allow mutations to be made in the O17 OSA cluster but not that of O15 could be numerous but require further study. Overall, the apparent lethality of mutating wzm and wzt in O15, the presence of other predicted LPS biosynthesis genes in this cluster, and the sequence similarity of wzmO15 and wztO15 to other O antigen transporters imply a role in OSA translocation. Providing direct evidence for their function in OSA biosynthesis will require more complex strategies.
In the literature, E. coli and K. pneumoniae are the prototype species for the ABC transporter-dependent assembly of O antigen, and significant advances in our understanding of this process have come from studies of these organisms. However, variations of these schemes inevitably exist in other bacteria, as we have demonstrated with Orf8O17 and Orf4O15. Based on our results, Orf8O17 can be conceptually described as a hybrid of the previously characterized WbdD and WbbB proteins; Orf8O17 contains a putative methyltransferase domain (similar to WbdD) separated from two chain-extending glycosyltransferase domains (similar to WbbB). A similar domain architecture was observed in Franconibacter pulveris G3872 for a putative WbbB-like protein, which has a predicted methyltransferase domain separated from two GT2 domains by a coiled coil (47). Another protein, WsaE of Geobacillus stearothermophilus, polymerizes/terminates the S-layer glycan and possesses two rhamnosyltransferase domains and a methyltransferase domain separated by a coiled coil (64). However, a distinct feature of Orf8O17 is the presence of a long alpha helix between its domains that lacks any appreciable coiled coils. This helix and the coiled-coil domain of Orf4 are likely the “rulers” that separate the terminating domain from the membrane, allowing the proteins to measure the polysaccharide. This is supported by the observation that overexpressing recombinant proteins lacking these domains perturbs the normal chain length modality (Fig. 3). Previous reports indicated that there is a positive correlation between the length of the ruler and the length of the O antigen chain (21, 22). Therefore, our observation that Orf4 and Orf8 have variable numbers of amino acid repeats in their ruler domain indicates that these proteins may drive OSA diversity by producing different lengths of the polysaccharide in different isolates. We speculate that this may be an evolutionary adaptation to increase bacterial fitness under certain environmental conditions. Finally, we note that confirming the activities of these proteins will require in vitro biochemical assays that were outside the scope of this study.
We also observed that the expression levels of orf8 affected the chain length of the O17 OSA (Fig. 3A). Basal expression of orf8 in the Δorf8 mutant background resulted in an increased production of shorter chains, as determined by both silver staining and Western blotting. Therefore, the production and export of shorter chains might depend on the relative ratio of Orf8 to other proteins in the OSA assembly system. Indeed, the determination of chain length in some ABC transporter-dependent systems can depend on complex relationships between multiple proteins. For instance, the chain length profile of the E. coli O9a O antigen is mostly determined by the size of the WbdD coiled-coil domain but is also a factor of the relative amounts of WbdA (chain-extending glycosyltransferase) (21, 24, 65–67). Further characterization of O17 OSA biosynthesis is required to deduce the precise reason for this phenotype.
With the sequences of the O15 and O17 clusters in hand, in silico serotyping from whole-genome sequencing data will be more accurate and subsequently provide a better estimate of P. aeruginosa strains containing these OSA structures. This is particularly important because some O15 and O17 serotype strains are associated with clinically relevant genotypes. For instance, O15 strains have been found to contain the metallocarbapenemase VIM-10 and the metallo-β-lactamase VEB-1 (68, 69), while studies by Ouellet et al. (53) and Vincent et al. (54) revealed that environmental O17 isolates had the characteristics of strains adapted to a chronic infection lifestyle, such as inactivating mutations in lasR and gacS and increased biofilm production. Our finding that the identity of the OSA cluster at the himD-wbpM locus is highly varied between strains carrying the O15 cluster indicates that inferring an O15 serotype from this locus alone may result in inaccurate predictions and necessitates the inclusion of the clusters identified in this study in future analyses. Variation at the himD-wbpM locus inevitably exists between O17 strains as well but was not observed here, which was likely a factor of the low number of sequenced strains available.
The acquisition of OSA clusters by horizontal gene transfer (HGT) and recombination at the himD-wbpM locus has been described previously (39). Here, we have provided evidence that OSA clusters have also integrated into the tRNASer gene upstream of rmlB, resulting in a novel genomic island. The presence of the same predicted phage integrase within this genomic island in both MRSN20176/NCTC11839 (O15) and PPF-1 (O17) suggests a common bacteriophage origin. In MRSN20176, the genomic island contains a 50-kb region encoding predicted phage proteins, which further supports this hypothesis. The finding that the O15 cluster is localized to a plasmid in some strains was another surprising insight into the HGT of OSA clusters since this has not been previously demonstrated in P. aeruginosa. Plasmids have been found to carry genes for the full or partial biosynthesis of O antigens in other bacteria (70–73). This finding explains why the O15 cluster was found in strains occupying diverse branches of our constructed phylogenetic tree. This also raises questions regarding the possible dissemination of this plasmid/cluster to other bacterial species; megaplasmids in the same family as that from strain AR439 were shown to transfer between Pseudomonas species (74). The movement of O15 and O17 by HGT and the accumulation of insertions and deletions at the himD-wbpM locus (Table 3) point to a constant remodeling of O antigen in P. aeruginosa. Overall, these observations expand our understanding of the evolution of P. aeruginosa OSA diversity.
In summary, we have identified the elusive OSA biosynthesis clusters of P. aeruginosa serotypes O15 and O17, which will allow better tracking of serotypes in the global population. The initial characterization of the genes in these clusters reveals variations in the prototypic ABC transporter pathways of E. coli and K. pneumoniae. Further genetic and biochemical characterization is warranted to provide a comprehensive understanding of this important polysaccharide assembly system.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
Bacteria were cultured at 37°C in lysogeny broth (LB). When necessary, cultures were supplemented with the appropriate antibiotics at the following concentrations: 100 μg/ml ampicillin and 10 μg/ml gentamicin for E. coli or 300 μg/ml carbenicillin and 300 μg/ml gentamicin for P. aeruginosa. P. aeruginosa IATS O15 (ATCC 33362) and O17 (ATCC 33364) were obtained from our laboratory’s culture collection.
DNA manipulations.
PCR was performed using either Phusion Hot Start DNA polymerase or DreamTaq green polymerase (Thermo). PCR and plasmid purification were performed using GeneJet purification kits (Thermo). Restriction enzymes and T4 DNA ligase were purchased from NEB. All enzymes and kits were used according to the manufacturers’ instructions. Chromosomal deletion mutants were constructed using the two-step allelic exchange method described previously by Hmelo et al. (75). Deletion constructs were engineered to contain approximately 500 to 1,000 bp of 5′- and 3′-end homology to the target site. The constructs were engineered by gene splicing by overlap extension (gene SOEing) and cloned into pEX18Gm (76, 77). Briefly, regions upstream and downstream of the region to be deleted were amplified in independent PCRs. The resulting PCR products were designed to contain 10 to 15 bp of overlap to facilitate their annealing in a second PCR to splice them into a single gene deletion amplicon. The following restriction sites in pEX18Gm were used to clone the deletion constructs: XbaI and HindIII (ΔwzmO17 ΔwztO17), EcoRI and HindIII (Δorf8O17), EcoRI and XbaI (ΔwzmO15 ΔwztO15), and XbaI and PstI (Δorf4O15). All complementing constructs were cloned into pHERD20T (78). The following restriction sites were used: XbaI and HindIII [orf8O17, orf8O17(MT), and orf8O17(GT1-2)] and XbaI and HindIII (orf4O15). Plasmid DNA was transferred into P. aeruginosa by either the 10-min electroporation method for pHERD20T-based plasmids (79) or conjugal transfer from E. coli SM10 for pEX18Gm-based plasmids (75). The primers used in this study are described in Table S1 in the supplemental material.
Bioinformatics analyses.
Secondary structure was analyzed using the NPS@ (49) and PredictProtein (57) servers. We routinely used the RAST server to browse and annotate the genomes of various P. aeruginosa strains (80–82). The BLAST server and CDD were used to predict the function of proteins or identify their homologs (83, 84). The maximum likelihood phylogenetic tree was constructed with Parsnp using the PAO1 genome as the reference sequence (85) and visualized with the Interactive Tree of Life online server (86). Clustal Omega was used to generate multiple-sequence alignments of protein sequences, while BLAST, the BLAST ring image generator (BRIG), and the Artemis comparison tool were used to compare the alignments (87–89). ESPript 3.0 (90) was used to generate graphics of multiple-sequence alignments, and sequence logos were generated using WebLogo (91). The scaffold containing the IATS O17 cluster (orf1 to orf19) can be found under GenBank accession number LJZK01000094.1 and is located between nucleotides 126956 and 148476. The scaffold containing the IATS O15 cluster (galE to orf12) can be found under GenBank accession number NZ_LJZI01000136.1 and is located between nucleotides 2366 and 18041. The accession numbers of the genome sequences used in this study are listed in Table S2.
Production of anti-O17 polyclonal antibodies.
The O17 antigen was prepared from whole-cell bacterial extracts. First, P. aeruginosa IATS O17 was grown overnight in 10 ml of LB, and bacteria were pelleted at 5,000 × g for 10 min the next morning. The pellet was suspended in 10 ml of 0.9% NaCl, boiled for 30 min, and then stored at −20°C in 1-ml aliquots. Prior to use, aliquots were thawed and mixed 1:1 with Freund’s incomplete adjuvant. A single New Zealand White rabbit was injected subcutaneously with 1 ml of antigen. Two weeks after the first injection, a booster injection (1 ml) was administered intramuscularly, followed by a second booster 4 weeks later. Four days later, the rabbits were exsanguinated, and the collected blood was allowed to clot at room temperature. Serum was separated from the clot by centrifugation at 2,000 × g and used for Western blotting. The generation of antibodies was performed in compliance with guidelines of the Canadian Council on Animal Care, the Animals for Research Act (94), and the University of Guelph Animal Care Policy and Procedures (AUP number 3569) and adhered to international guiding principles for biomedical research involving animals.
LPS and protein analysis.
LPS was extracted from cultures grown overnight according to the method described previously by Hitchcock and Brown (92). Five microliters of extracted LPS was electrophoresed on 10% SDS-PAGE gels and visualized by either the silver staining method described previously by Fomsgaard et al. (93) or Western immunoblotting. All gels and blots are representative of results from 2 or 3 independent experiments. For Western immunoblots, LPS was transferred to a nitrocellulose membrane for 45 min at 180 mA. The membranes were washed twice with phosphate-buffered saline (PBS) and blocked for 1 h with 5% (wt/vol) skim milk in PBS. Membranes were then washed for 10 min three times in PBS before incubation overnight (room temperature) with anti-O17 rabbit antibody (1:100 dilution in PBS). The membranes were washed three more times for 10 min in PBS and then incubated for 1 h with goat anti-mouse antibody conjugated to alkaline phosphatase (diluted 1:2,000 in PBS). Finally, membranes were washed twice in PBS and once in buffer A. The blots were developed in buffer A containing 660 μl of 5% nitroblue tetrazolium chloride (NBT) and 66 μl of 5% 5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt (BCIP). For protein blots, 20 μl of whole-cell lysates was electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose at 200 mA for 30 min. The membranes were washed twice with Tris-buffered saline (TBS) (pH 7.4) containing 0.1% Tween 20 and 0.05% Triton X-100 (TBSTT) and then once in TBS. The blots were blocked overnight in blocking buffer (TBS containing 3% bovine serum albumin [BSA]). The next day, blots were washed twice in TBSTT and once in TBS and then incubated for 2 h with anti-FLAG mouse monoclonal antibody conjugated to horseradish peroxidase (HRP) diluted 1:3,000 in 10 ml of blocking buffer. Finally, membranes were washed three times with TBSTT, patted dry, and then incubated for 4 min with 2 ml of developing solution (100 mM Tris [pH 8.8], 1.25 mM luminol, 2 mM 4-iodophenylboronic acid, and 5.3 mM H2O2). Bands were visualized by chemiluminescence on a Bio-Rad GelDoc imager with ImageQuant software.
Immunofluorescence.
Three hundred microliters of a liquid culture grown overnight was centrifuged for 2 min at 8,000 × g, suspended in 1 ml of 4% paraformaldehyde (PFA), and incubated at room temperature for 20 min. The PFA-treated cells were then centrifuged for 5 min at 12,000 × g and washed three times with 1 ml of PBS. Ten microliters of this washed suspension was then spotted onto poly-l-lysine-coated multiwell microscope slides and incubated at room temperature for 15 min. The slides were then washed three times with 1 ml PBS and blocked for 30 min with 10 μl of 2% BSA in PBS per well. After three more washes with 1 ml of PBS, 10 μl of primary antibody (anti-O17 polyclonal antibody, adsorbed to O17 Δorf8 cells to remove anti-lipid A-core antibodies) diluted 1/100 in PBS with 2% BSA was added to each well, and the mixture was incubated for 1 h. Following three more washes with PBS, each well was incubated with secondary antibody (goat anti-rabbit antibody conjugated to fluorescein isothiocyanate [FITC]) at a dilution of 1/200 in 2% BSA in PBS. The slides were washed three more times and then mounted with SlowFade diamond antifade (Thermo Fisher). Microscope slides were stored at 4°C in the dark until imaging. Images were captured on a Zeiss Axiovert 200 M fluorescence microscope at a ×1,000 magnification.
ACKNOWLEDGMENTS
This work was supported by operating grants from the CIHR awarded to C.M.K. (PJT 156111) and J.S.L. (MOP-14687). S.M.H. is the recipient of an Ontario graduate scholarship.
We thank Chris Whitfield for critical review of the work and colleagues Herbert Schweizer and Tung Hoang for providing many Pseudomonas-specific plasmid vectors as tools for genetic manipulations, particularly the pEX series of plasmids for creating knockout and deletion mutants of P. aeruginosa genes.
Supplemental Material
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Information & Contributors
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Published In
Journal of Bacteriology
Volume 202 • Number 19 • 8 September 2020
eLocator: 10.1128/jb.00347-20
Editor: Thomas J. Silhavy, Princeton University
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© 2020 American Society for Microbiology. All Rights Reserved.
History
Received: 16 June 2020
Accepted: 13 July 2020
Published online: 8 September 2020
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Thomas J. Silhavy
Editor
Princeton University
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2020.
Identification of the Pseudomonas aeruginosa O17 and O15 O-Specific Antigen Biosynthesis Loci Reveals an ABC Transporter-Dependent Synthesis Pathway and Mechanisms of Genetic Diversity. J Bacteriol
202:10.1128/jb.00347-20.
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