**2. Subtractive hybridization to identify Capsular Polysaccharide 1 (CPS I)**

To identify the genetic determinants that confer enhanced virulence in *B. pseudomallei*, a method combining subtractive hybridization, insertional mutagenesis, and animal virulence studies was developed [42]. Subtractive hybridization was carried out between the virulent *B. pseudomallei* and the weakly virulent *B. thailandensis* in order to isolate DNA sequences encoding for virulence determinants unique to *B. pseudomallei*. The genomic DNA sample from *B. pseudomallei* containing the sequences of interest was known as the tester DNA, and genomic DNA from *B. thailandensis*, the reference sample, was called the driver DNA. Tester and driver DNAs were digested and subjected to two rounds of hybridization. The remaining unhybridized sequences were considered tester-specific sequences. To enrich for tester-specific sequences, excess driver DNA was added in the hybridizations. The testerspecific sequences were then amplified by PCR and cloned into the plasmids pPCR or pZErO-2.1. Screening of the subtraction library revealed a number of DNA sequences unique to *B. pseudomallei*. Fifteen distinct plasmid inserts from the library were sequenced. The DNA inserts ranged from 100 to 800 bp in length and were found to contain an average G+C content of approximately 44 to 52%, which is considerably lower than the 68% G+C content of the *B. pseudomallei* chromosome. The DNA sequences were analyzed using the NCBI BLASTX program. One of the plasmid inserts, pDD1015, was found to share limited homology with WbpX, a glycosyltransferase, from *Pseudomonas aeruginosa* [43].

130 The Complex World of Polysaccharides

inherently resistant to a number of antibiotics, and even with aggressive antibiotic therapy, the

At the time our studies were initiated some cell-associated antigens had been identified and characterized in *B. pseudomallei*. Cell-associated antigens include exopolysaccharide (EPS) and lipopolysaccharide (LPS) [28, 29, 30]. The EPS produced by *B. pseudomallei* was determined to be an unbranched polymer of repeating tetrasaccharide units with the structure -3)-2-*O*-acetyl-β-D-Gal*p*-(1-4)-α-D-Gal*p*-(1-3)-β-D-Gal*p*-(1-5)-β-D-KDO*p*-(2- [31, 32]. The role of EPS in virulence was not known, but sera from patients with melioidosis had been shown to contain antibodies against EPS [30]. Two other EPS structures were also identified; a branched 1,4-linked glucan polymer ((CP-1a) and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galactose, glucose, and glucuronic acid (CP-2) [33]. The genes involved in the synthesis of these capsules, and the role of these capsules in virulence had not been identified. The LPS of *B. pseudomallei* was structurally characterized and reported to contain two types of O-polysaccharide moieties termed type I O-PS and type II O-PS [34, 35]. Type II O-PS was found to be an unbranched heteropolymer with repeating D-glucose and L-talose residues with the structure -3)-β-Dglucopyranose-(1-3)-6-deoxy-α-L-talopyranose-(1-. Type II O-PS had been shown to be involved in serum resistance [36]. Type II O-PS mutants also demonstrated reduced virulence in three animal models of *B. pseudomallei* infection [36]. Type I O-PS was determined to be an unbranched homopolymer with the structure -3)-2-*O*-acetyl-6-deoxy-β-D-manno-heptopyranose-(1-, however, the role for this polysaccharide in infection had not

mortality rate remains high, and the incidence of relapse is common [26, 27].

been defined, nor the genes responsible for its biosynthesis been identified.

clinical isolates are not able to utilize L-arabinose [41].

*B. thailandensis* is a nonpathogenic soil organism originally isolated in Thailand [37]. Based on biochemical, immunological, and genetic data, *B. pseudomallei* and *B. thailandensis* are closely related species. However, these two organisms differ in a number of ways and have been classified into two different species [38]. The rRNA sequence of *B. thailandensis* differs from that of *B. pseudomallei* by 15 nucleotides, and there are significant differences in genomic macrorestriction patterns between these organisms [39]. The biochemical profiles of these two species differ in that *B. thailandensis* can utilize L-arabinose whereas *B. pseudomallei*  does not [38, 40]. The most distinct difference between these two species, however, is their relative virulence. The 50% lethal dose (LD50) for *B. pseudomallei* in the Syrian hamster model of acute melioidosis is <10 organisms, whereas the LD50 for *B. thailandensis* is approximately 106 organisms [38]. It has also been shown that the two species can be differentiated based on their propensity to cause disease in humans. Environmental strains isolated in Thailand that are able to assimilate L-arabinose are not associated with human infection, whereas

**2. Subtractive hybridization to identify Capsular Polysaccharide 1 (CPS I)** 

To identify the genetic determinants that confer enhanced virulence in *B. pseudomallei*, a method combining subtractive hybridization, insertional mutagenesis, and animal virulence studies was developed [42]. Subtractive hybridization was carried out between the virulent *B. pseudomallei* and the weakly virulent *B. thailandensis* in order to isolate DNA sequences

#### **2.1. Demonstration that CPS I is important for the virulence of** *B. pseudomallei*

The 373-bp DNA insert from pDD1015 was cloned into a mobilizable suicide vector, pSKM11 [44]. The resulting plasmid, pSR1015, was mobilized into wildtype *B. pseudomallei*  1026b to create the mutant strain SR1015. Since the insert from pDD1015 was found to demonstrate homology to a glycosyltransferase from *P. aeruginosa*, it was postulated that it might encode a protein involved in carbohydrate synthesis. Since three carbohydrate structures had been previously purified and characterized, antibodies to each of these polysaccharides were available. To define the phenotype of SR1015, an ELISA was performed with the EPS-specific monoclonal antibody 3015, and *B. pseudomallei* 1026b and SR1015 were both found to contain EPS [45]. SR1015 was also shown to contain type II O-PS and to be serum resistant. Immunogold electron microscopy studies using rabbit polyclonal sera specific for a type I O-PS–flagellin conjugate was performed on the parent strain, 1026b, and SR1015 (Figure 1). *B. pseudomallei* 1026b reacted with antibodies to both flagellin and type I O-PS, as was evident by the distribution of gold particles around the bacterial surface and extending out along the flagella (Figure 1A). Unlike *B. pseudomallei* 1026b, SR1015 reacted only with the antibodies to flagellin, as the gold particles were found associated only with the flagella (Figure 1B). *B. thailandensis*, the negative control, did not react with the antibodies either to flagellin or to type I O-PS (Figure 1C). *B. stabilis* LMG7000 was also shown to react to the antibodies to type I O-PS, indicating this organism may produce a similar capsule (Figure 1D). Western blot analysis of proteinase K-digested whole cells from *B. pseudomallei* 1026b, *B. thailandensis* E264, and *B. pseudomallei* SR1015 using rabbit polyclonal sera raised to O-PS–flagellin protein conjugate confirmed the lack of type I O-PS in SR1015. Type I and type II O-PS were stained in *B. pseudomallei* 1026b, while only type II O-PS was stained in the lanes corresponding to *B. pseudomallei* SR1015 and *B. thailandensis*. These results indicated that we had identified and insertionally inactivated a gene involved in the synthesis of the type I O-PS of *B. pseudomallei* [42]. SR1015 was tested for virulence in the Syrian hamster model of acute septicemic melioidosis. The LD50 for SR1015 after 48 h was 3.5 x 105 CFU, while the LD50 of the parent strain, 1026b, was <10 CFU. The LD50 for SR1015 was similar to that for

the weakly virulent *B. thailandensis* (6.8 x 105 CFU) [42]. This demonstrated that SR1015 is severely attenuated for virulence in this animal model of melioidosis and that type I O-PS is a major virulence determinant of *B. pseudomallei*. We later determined that the type I O-PS was a capsular polysaccharide (CPS I), not an O-PS moiety, which will be discussed below.

**Figure 1.** Immunogold electron microscopy of *B. pseudomallei* 1026b (A) and SR1015 (B), *B. thailandensis*  E264 (C), and *B. stabilis* LMG7000 (D). Bacteria were reacted with polyclonal rabbit antiserum directed against an O-PS–flagellin protein conjugate absorbed with *B. thailandensis* E264 to remove the antibodies directed against type II O-PS, washed, and reacted with a goat anti-rabbit IgG-gold (5 nm) conjugate. Original magnification x330,000.

## **2.2. Cloning and sequencing of the genetic loci required for CPS I production and export**

132 The Complex World of Polysaccharides

Original magnification x330,000.

the weakly virulent *B. thailandensis* (6.8 x 105 CFU) [42]. This demonstrated that SR1015 is severely attenuated for virulence in this animal model of melioidosis and that type I O-PS is a major virulence determinant of *B. pseudomallei*. We later determined that the type I O-PS was a

**Figure 1.** Immunogold electron microscopy of *B. pseudomallei* 1026b (A) and SR1015 (B), *B. thailandensis*  E264 (C), and *B. stabilis* LMG7000 (D). Bacteria were reacted with polyclonal rabbit antiserum directed against an O-PS–flagellin protein conjugate absorbed with *B. thailandensis* E264 to remove the antibodies directed against type II O-PS, washed, and reacted with a goat anti-rabbit IgG-gold (5 nm) conjugate.

capsular polysaccharide (CPS I), not an O-PS moiety, which will be discussed below.

Two methods were used to clone the genes involved in the production and export of type I O-PS. The DNA flanking the insertion of pSR1015 was cloned from SR1015 and sequenced. We also used transposon mutagenesis to clone the genes involved in production of the polysaccharide; this was done to obtain any unlinked genes that may be involved in polysaccharide production. Approximately 1,300 transposon mutants were screened for loss of type I O-PS by ELISA. Six mutants were identified, and the DNA flanking the transposon insertion was cloned and sequenced. The Tn*5*-OT182 mutants SLR5, SLR8, SLR13, SLR18, and SLR19 mapped to the same region of the chromosome. Sequence analysis of the cloned fragments revealed the presence of 26 potential open reading frames involved in the synthesis and export of type I O-PS [42]. The open reading frames that predicted proteins involved in polysaccharide biosynthesis were found to demonstrate homology to proteins involved in the synthesis of a polysaccharide structure composed primarily of mannose. The other reading frames in the locus predicted proteins involved in the transport of capsular polysaccharides in a variety of bacteria, particularly those that produce group 2 and group 3 capsular polysaccharides [8]. The genes responsible for the production of type I O-PS were found to be similar to other loci encoding for capsular polysaccharides in that they are divergently transcribed [4]. The gene cluster involved in the production of this polysaccharide is also similar to group 3 capsule gene clusters in that there are no genes encoding KpsF and KpsU, which are present in group 2 capsule gene clusters [8]. However, the organization of the *B. pseudomallei* type I O-PS gene cluster differs in that it does not contain two export regions flanking a single biosynthetic region as seen in other group 3 capsule polysaccharide clusters [46]. The biosynthetic genes identified are not organized into one continuous transcriptional unit; instead, *wcbB*, *manC*, and *wcbP* are separated from the rest of the biosynthetic genes. The overall G+C content of this region is about 58%, lower than the G+C content of the rest of the chromosome (68%). The low G+C content in these clusters suggests that polysaccharide genes have a common origin and may have been transferred horizontally between species [9]. The genes involved in the production of this polysaccharide were named according to the bacterial polysaccharide gene nomenclature scheme [47]. The gene products associated with this cluster are shown in Figure 4. Mutations constructed in a number of these genes confirmed their role in the production of this polysaccharide [42].

#### **2.3. CPS I was originally identified as a Type I O-polysaccharride moiety**

The polysaccharide with the structure -3)-2-*O*-acetyl-6-deoxy-β-D-manno-heptopyranose-(1 was originally isolated and characterized as an O-PS component of LPS in *B. pseudomallei* and was designated type I O-PS [35]. However, our results suggested that this polysaccharide was a capsule rather than an O-PS moiety. The genes involved in the production of this capsule demonstrated strong homology to the genes involved in the production of capsular polysaccharides in many organisms, including *N. meningitidis*, *H. influenzae*, and *E. coli*. In addition, the export genes associated with this cluster are not associated with the previously characterized O-PS gene cluster [36]. Western blot analysis of proteinase K cell extracts and silver staining demonstrated that this polysaccharide has a high molecular mass (200 kDa) and lacks the banding pattern seen with O-PS moieties. Studies by our laboratory indicated that mutants in the production of the core oligosaccharide of the LPS are still capable of producing this polysaccharide [48]. Based on the above criteria and the genetic similarity to group 3 capsules, we proposed that this polysaccharide is a group 3 capsule and designated this capsule CPS I. This conclusion was further supported by Isshiki *et al* who separated this polysaccharide from a smooth lipopolysaccharide preparation of *B. pseudomallei* [49].
