**4. Other capsules produced by** *B.pseudomallei*

Sequence analysis of the completed genome of *B. pseudomallei* revealed four operons with the predicted function of capsular polysaccharide biosynthesis and export [54]. One of these operons, with the gene identifiers BPSL2786-2810, corresponds to the previously characterized mannoheptose capsule designated CPS I [42, 50]. Three other operons were identified. These three capsule operons in the genome of *B. pseudomallei* were further analyzed using the BLAST program and Artemis. The operons are illustrated in Figure 4. The operon consisting of the genes BPSS0417-0429, was designated CPS II (Figure 4B). Another operon, BPSS1825-1835, was designated CPS III and the predicted homologues were investigated further (Figure 4C). A fourth operon, CPS IV, was found to contain genes that may be involved in the synthesis of a capsule with the gene identifiers BPSL2769-2785 (Figure 4D) [54, 55].

**Figure 4.** Organization of the chromosomal regions containing the genes comprising the *B. pseudomallei* capsule operons. The direction of transcription is represented by arrows, and the gene names demonstrating the highest degree of homology to the *B. pseudomallei* open reading frames are indicated. The relative sizes of each locus are indicated. (A) *B. pseudomallei* capsule cluster I (CPS I). (B) *B. pseudomallei* capsule cluster II (CPS II). (C) *B. pseudomallei* capsule cluster III (CPS III). (D) *B. pseudomallei* capsule cluster IV (CPS IV).

#### **4.1. Distribution of capsule loci between three** *Burkholderia* **species**

Comparative analysis of the genomes of three *Burkholderia* species, *B. pseudomallei*, *B. mallei* and *B. thailandensis*, was performed to determine whether all of the predicted *B. pseudomallei* capsule operons were present in *B. mallei* and *B. thailandensis* as well. CPS II, III, and IV were found to be present in *B. pseudomallei* and *B. thailandensis,* but not *B. mallei*. This is in contrast to CPS I, which is present in *B. pseudomallei* and *B. mallei*, but not *B. thailandensis* [42, 50, 54, 55, 56, 57]. CPS II was found to be identical between *B. pseudomallei* and *B. thailandensis*, but *B. thailandensis* was found to contain two flanking hypothetical genes not present in *B. pseudomallei*. The CPS II genes were found to be deleted entirely from *B. mallei*. A large chromosomal region ranging from open reading frames BPSS0404 to BPSS0491 including CPS II was shown to be deleted in *B. mallei* compared to *B. pseudomallei* and replaced with a large chromosomal region containing open reading frames BMAA0555 (IS407A *orfB*) to BMAA1784, a unique hypothetical protein not found in *B. pseudomallei*. The two genomes align with the presence of the alkyl hydroperoxidase reductase genes *ahpC* and *ahpF*, but these are organized in the opposite orientation in *B. mallei* compared to *B. pseudomallei*. The entire CPS III operon and flanking genes were shown to be the same in both *B. pseudomallei* and *B. thailandensis*. In contrast, the majority of the CPS III cluster was deleted from *B. mallei* with the exception of the *wcaJ* and *manC* genes, as well as the two flanking hypothetical genes on one side, and another hypothetical gene on the other side of the deleted region. The deletion of the CPS III genes in *B. mallei* was found to be replaced with the IS407A *orfA* and *orfB* genes. The entire CPS IV region was found to be replaced in *B. mallei* and flanked by two IS407A elements. The open reading frame BPSL2785, which encodes a hypothetical protein, is present in *B. mallei* (BMA2284.1) ATCC 23344 as well as a number of other *B. mallei* strains, but is organized in the opposite orientation. The genomes of *B. pseudomallei*, *B. thailandensis*, and *B. mallei* all diverge upstream of the CPS IV region, but all three organisms were found to align at the location of the *ompA* and hypothetical genes [55].

#### **4.2. CPS III does not contribute to the virulence of** *B. pseudomallei*

140 The Complex World of Polysaccharides

(Figure 4D) [54, 55].

capsule cluster IV (CPS IV).

were investigated further (Figure 4C). A fourth operon, CPS IV, was found to contain genes that may be involved in the synthesis of a capsule with the gene identifiers BPSL2769-2785

**Figure 4.** Organization of the chromosomal regions containing the genes comprising the *B. pseudomallei*

demonstrating the highest degree of homology to the *B. pseudomallei* open reading frames are indicated.

Comparative analysis of the genomes of three *Burkholderia* species, *B. pseudomallei*, *B. mallei* and *B. thailandensis*, was performed to determine whether all of the predicted *B. pseudomallei* capsule operons were present in *B. mallei* and *B. thailandensis* as well. CPS II, III, and IV were found to be present in *B. pseudomallei* and *B. thailandensis,* but not *B. mallei*. This is in contrast to CPS I, which is present in *B. pseudomallei* and *B. mallei*, but not *B. thailandensis* [42, 50, 54, 55, 56, 57]. CPS II was found to be identical between *B. pseudomallei* and *B. thailandensis*, but *B. thailandensis* was found to contain two flanking hypothetical genes not present in *B. pseudomallei*. The CPS II genes were found to be deleted entirely from *B. mallei*. A large

capsule operons. The direction of transcription is represented by arrows, and the gene names

The relative sizes of each locus are indicated. (A) *B. pseudomallei* capsule cluster I (CPS I). (B) *B. pseudomallei* capsule cluster II (CPS II). (C) *B. pseudomallei* capsule cluster III (CPS III). (D) *B. pseudomallei*

**4.1. Distribution of capsule loci between three** *Burkholderia* **species** 

In order to assess the role of CPS III in virulence a mutant in the CPS III operon was tested for virulence compared to wild type *B. pseudomallei* in the Syrian hamster model of melioidosis. Syrian golden hamsters were inoculated intraperitoneally with 101 to 103 cells of either wild-type *B. pseudomallei* 1026b or the capsule mutant SZ1829. After 48 h, the LD50 values were determined. SZ1829 had LD50 values of <10 CFU, identical to that of wild type *B. pseudomallei*, indicating that this capsule is not required for virulence. In addition, the bacterial load in the blood of the infected hamsters was similar to that of wild-type and significantly higher than that of the non-pathogenic *B. thailandensis* E264 and the CPS I mutant, *B. pseudomallei* SR1015, both of which are incapable of establishing bacteremia [37, 50]. This indicates that CPS III does not contribute to persistence in the blood. Similar results were obtained for CPS II and CPS IV mutants, but we went on to further characterize CPS III.

#### **4.3. Expression of CPS III in host and environmental conditions**

A *lux* reporter strain was constructed in the CPS III operon by cloning an internal fragment of one of the genes into pGSV3-*lux*, a suicide vector containing a promoterless *lux* operon from *Photorhabdus luminescens* [53]. Regulation of this capsule in an environment similar to that encountered in the host was determined by growing the *lux* reporter strain, SZ1829, in the presence of M9 plus 1% glucose versus M9 plus 1% glucose plus 30% normal human serum (NHS). Absorbance (OD540) and luminescence (in relative light units) measurements were taken every hour. The expression of CPS III (SZ1829) was higher in M9 plus 1% glucose alone compared to M9 plus 1% glucose plus 30% NHS. The expression of SZ1829 was 3-4 fold lower in 30% NHS. This was in contrast to the expression of CPS I (SZ211) (see section 3.5), which was significantly more highly expressed in 30% NHS at a level of 3-4 fold

compared to growth in M9 plus 1% glucose alone [55]. Although CPS III demonstrated higher expression initially in 30% NHS, this may have been due to the fact that the addition of NHS caused precipitation in the media which affected the optical density of the cultures.

The expression of the *lux* operon in reporter strains SZ211 (CPS I- ) and SZ1829 (CPS III- ) was also measured in water to determine whether CPS III was induced in this environment. Overnight cultures of SZ211 and SZ1829 were inoculated into sterile water and incubated at 37oC without shaking. Capsule expression was determined as described above, but the luminescence/absorbance calculations for water were compared to the values for these strains when grown in LB. CPS III was found to be induced in water compared to LB. The expression of SZ1829 was found to be significant with an increase of 2-3 fold over the course of the experiment. The expression of CPS I was found to be greater than 4 fold higher in LB compared to water [55].

Microarray analysis of capsule expression was performed using a low-density DNA microarray. RNA was isolated from the livers and lungs of hamsters infected with *B. pseudomallei* and from *B. pseudomallei* grown in LB. The results of the microarray experiment are shown in Table 1 [55]. The level of gene expression, or fold change, is represented as the ratio of gene expression in the hamster compared to growth in LB. As shown in Table 1, CPS III genes were not found to be significantly expressed *in vivo* since most of the fold changes were determined to be less than 2-fold. Many of the genes had negative fold change values, indicating that these genes are suppressed in the host environment. The highest fold change result was 2.337866 for BPSS1827, a predicted glucose-6-phosphate isomerase, which is still much lower than the fold changes observed for CPS I genes, which were significantly higher [58].


\*Note: Gene expression representative of the liver and lungs of infected hamsters.

**Table 1.** Microarray analysis of *B. pseudomallei* CPS III expression following intraperitoneal inoculation in the hamster model of melioidosis.

#### **4.4. Carbohydrate composition of CPS III**

Glycosyl composition analysis was performed on the purified capsule by combined gas chromatography/mass spectrometry (GC/MS). GC/MS results indicated that CPS III is composed of galactose, glucose, mannose, xylose, and rhamnose residues, with the highest proportion of carbohydrate being galactose and glucose. Glycosyl linkage analysis was also performed [55]. The predominant glycosyl residue detected was a terminally-linked heptopyranosyl (t-Hep) at a percentage of 23.2. Other residues detected were a terminallylinked and a 4-linked glucopyranosyl (t-Glcp) (4-Glcp) at percentages of 14.6 and 10.8, respectively [55].

## **5. Conclusion**

142 The Complex World of Polysaccharides

compared to water [55].

[58].

compared to growth in M9 plus 1% glucose alone [55]. Although CPS III demonstrated higher expression initially in 30% NHS, this may have been due to the fact that the addition of NHS caused precipitation in the media which affected the optical density of the cultures.

also measured in water to determine whether CPS III was induced in this environment. Overnight cultures of SZ211 and SZ1829 were inoculated into sterile water and incubated at 37oC without shaking. Capsule expression was determined as described above, but the luminescence/absorbance calculations for water were compared to the values for these strains when grown in LB. CPS III was found to be induced in water compared to LB. The expression of SZ1829 was found to be significant with an increase of 2-3 fold over the course of the experiment. The expression of CPS I was found to be greater than 4 fold higher in LB

Microarray analysis of capsule expression was performed using a low-density DNA microarray. RNA was isolated from the livers and lungs of hamsters infected with *B. pseudomallei* and from *B. pseudomallei* grown in LB. The results of the microarray experiment are shown in Table 1 [55]. The level of gene expression, or fold change, is represented as the ratio of gene expression in the hamster compared to growth in LB. As shown in Table 1, CPS III genes were not found to be significantly expressed *in vivo* since most of the fold changes were determined to be less than 2-fold. Many of the genes had negative fold change values, indicating that these genes are suppressed in the host environment. The highest fold change result was 2.337866 for BPSS1827, a predicted glucose-6-phosphate isomerase, which is still much lower than the fold changes observed for CPS I genes, which were significantly higher

**Gene ID Predicted function Fold Change** 

BPSS1825 Glycosyltransferase 1.757155 BPSS1826 Glycosyltransferase 0.093565 BPSS1827 Glucose-6-phosphate isomerase 2.337866 BPSS1828 Glycosyltransferase -0.66607 BPSS1829 Glycosyltransferase -0.18061 BPSS1830 Capsule export, tyrosine-protein kinase -0.30655 BPSS 1831 Capsule export, outer membrane protein 0.725849 BPSS1832 Transport, tyrosine-protein phosphatase 1.52665 BPSS1833 UDP-glucose-6-dehydrogenase -0.71411 BPSS1834 Sugar transferase -0.07963 BPSS 1835 Mannose-1-phosphate guanyltransferase -0.75075

\*Note: Gene expression representative of the liver and lungs of infected hamsters.

in the hamster model of melioidosis.

**Table 1.** Microarray analysis of *B. pseudomallei* CPS III expression following intraperitoneal inoculation

) and SZ1829 (CPS III-

**(***in vivo* **vs.** *in vitro***)** 

) was

The expression of the *lux* operon in reporter strains SZ211 (CPS I-

Although significant advances have been made in the field, melioidosis continues to be a public health concern in many regions of the world [59]. Completion of the sequencing of the *B. pseudomallei* genome has revealed potential virulence determinants and comparative genomics between the genomes of *B. pseudomallei*, *B. thailandensis*, and *B. mallei* species has contributed to a better understanding of the organism. Further studies are ongoing to define the pathogenesis of *B. pseudomallei* and to identify effective vaccine candidates and diagnostic targets [54, 59].

To obtain virulence determinants unique to *B. pseudomallei*, we used subtractive hybridization between this organism and a related nonpathogenic organism, *B. thailandensis*. Analysis of the subtractive hybridization library revealed that *B. pseudomallei* contains a number of DNA sequences that are not found in *B. thailandensis*. One of the subtraction clones, pDD1015, demonstrated weak homology to a glycosyltransferase, WbpX, from *P. aeruginosa* [43]. The insert from pDD1015 was cloned into a mobilizable suicide vector for insertional inactivation of the glycosyltransferase gene in wild-type *B. pseudomallei*. The resulting strain, SR1015, was markedly less virulent than the parent strain in an animal model. We determined that SR1015 harbored a mutation in a glycosyltransferase gene involved in the production of a capsular polysaccharide which we subsequently designated as CPS I. We then identified the operon involved in the biosynthesis and transport of this capsular polysaccharide (CPS I) [42]. The genes identified encode for proteins that are similar to proteins involved in the biosynthesis and export of capsular polysaccharides, particularly those involved in the production of group 3 capsular polysaccharides. Group 3 capsules include the *E. coli* K10 capsule and may also include the *H. influenzae* group b capsule and the capsule produced by *N. meningitidis* serogroup B [8]. Group 3 capsules are always coexpressed with O serogroups, are not thermoregulated, are transported by an ABC-2 exporter system, and do not contain the *kpsU* and *kpsF* genes, and usually the gene clusters map near the *serA* locus [8]. Thus far, no *serA* locus that is associated with the type I O-PS cluster was identified, but this polysaccharide is coexpressed with O antigen and lacks the *kpsU* and *kpsF* genes, and genes encoding for a putative ABC-2 transporter have been identified. The genes involved in the production of group 3 capsules are organized into regions and are divergently transcribed. Regions 1 and 3 are generally conserved and contain genes involved in export of the polysaccharide. These regions flank region 2, which contains the biosynthetic genes and is not conserved between serotypes [4]. The genetic organization of the CPS I is also similar to that of other capsule gene clusters in that the genes are organized into more than one transcriptional unit and appear to be divergently transcribed [42].

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 is 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 showed that this polysaccharide has a high molecular mass (200 kDa) and lacks the banding pattern seen with O-PS moieties. This conclusion was further supported by another group of researchers that demonstrated this polysaccharide is a capsule rather than an O-PS component of LPS because it lacks a lipid A moiety and was not capable of macrophage activation [49]. Studies by our laboratory have 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 capsule.

Virulence genes of a number of pathogenic bacteria are located on pathogenicity islands (PAIs), regions on the bacterial chromosome that are present in the genome of pathogenic strains but rarely present in those of nonpathogenic strains. The PAIs may range in size from about 30 kb to 200 kb and often differ in G+C content from the remaining bacterial genome; the PAIs are often associated with the carriage of many virulence genes. These genetic units are often flanked by direct repeats and may be associated with tRNA genes or insertion sequence (IS) elements at their boundaries. They may also be associated with the presence of mobility genes, such as IS elements, integrases, transposases, and origins of plasmid replication. These DNA regions are considered to be unstable in that they may be subject to deletion with high frequency or undergo duplications and amplifications [7]. A number of PAIs have been described for both gram-positive and gram-negative bacteria, and the application of subtraction hybridization has been used to successfully identify such genetic elements [7]. The subtractive hybridization that was carried out between *B. pseudomallei* and *B. thailandensis* led to the identification of a number of sequences that were found to be A-T rich compared to the rest of the *B. pseudomallei* chromosome. This, combined with the fact that insertional mutagenesis of the glycosyltransferase gene identified by this method resulted in an avirulent strain, suggests that we may have identified DNA sequences from a putative PAI and that the capsular polysaccharide gene cluster may be located on this island. It is possible that *B. pseudomallei*, *B. mallei*, and *B. stabilis* acquired DNA encoding for capsule as well as other potential, yet unidentified virulence factors by horizontal transfer recently in evolution. *B. pseudomallei* and *B. mallei* are known to contain IS elements that are present in *B. cepacia* but not in *B. thailandensis* [56, 60].

Capsule production has been correlated with virulence in many bacteria, particularly those causing serious invasive infections of humans [61]. Our studies demonstrated that CPS I is critical for the virulence of *B. pseudomallei* [42, 50]. A number of functions have been suggested for polysaccharide capsules: prevention of desiccation for transmission and survival, adherence for colonization, resistance to complement-mediated phagocytosis and complement-mediated killing, and resistance to specific host immunity due to a poor antibody response to the capsule [4].

144 The Complex World of Polysaccharides

genes involved in export of the polysaccharide. These regions flank region 2, which contains the biosynthetic genes and is not conserved between serotypes [4]. The genetic organization of the CPS I is also similar to that of other capsule gene clusters in that the genes are organized

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 is 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 showed that this polysaccharide has a high molecular mass (200 kDa) and lacks the banding pattern seen with O-PS moieties. This conclusion was further supported by another group of researchers that demonstrated this polysaccharide is a capsule rather than an O-PS component of LPS because it lacks a lipid A moiety and was not capable of macrophage activation [49]. Studies by our laboratory have 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

into more than one transcriptional unit and appear to be divergently transcribed [42].

similarity to group 3 capsules, we proposed that this polysaccharide is a capsule.

Virulence genes of a number of pathogenic bacteria are located on pathogenicity islands (PAIs), regions on the bacterial chromosome that are present in the genome of pathogenic strains but rarely present in those of nonpathogenic strains. The PAIs may range in size from about 30 kb to 200 kb and often differ in G+C content from the remaining bacterial genome; the PAIs are often associated with the carriage of many virulence genes. These genetic units are often flanked by direct repeats and may be associated with tRNA genes or insertion sequence (IS) elements at their boundaries. They may also be associated with the presence of mobility genes, such as IS elements, integrases, transposases, and origins of plasmid replication. These DNA regions are considered to be unstable in that they may be subject to deletion with high frequency or undergo duplications and amplifications [7]. A number of PAIs have been described for both gram-positive and gram-negative bacteria, and the application of subtraction hybridization has been used to successfully identify such genetic elements [7]. The subtractive hybridization that was carried out between *B. pseudomallei* and *B. thailandensis* led to the identification of a number of sequences that were found to be A-T rich compared to the rest of the *B. pseudomallei* chromosome. This, combined with the fact that insertional mutagenesis of the glycosyltransferase gene identified by this method resulted in an avirulent strain, suggests that we may have identified DNA sequences from a putative PAI and that the capsular polysaccharide gene cluster may be located on this island. It is possible that *B. pseudomallei*, *B. mallei*, and *B. stabilis* acquired DNA encoding for capsule as well as other potential, yet unidentified virulence factors by horizontal transfer recently in evolution. *B. pseudomallei* and *B. mallei* are known to contain IS elements that are present in *B. cepacia* but not in *B. thailandensis* [56, 60].

To establish a correlation between capsule production and clinical infection a number of *B. pseudomallei* strains isolated from clinical specimens were tested for CPS I production. All 55 strains tested were found to produce CPS I by western blot analysis [51]. In addition 10 strains of *B. thailandensis* were tested and found negative for CPS I production, confirming the importance of CPS I in virulence as well as clinical infection.

CPS I production by *B. pseudomallei* was shown to contribute to the persistence of the organism in the blood of the host. All CPS I mutants tested in the animal model could not be isolated from the blood following infection. The addition of purified capsule was shown to increase the virulence of the CPS I mutant strains SR1015 and SZ210 in the animal model. Differences in tissue distribution between wild type *B. pseudomallei* and SR1015 in infected hamsters indicated that SR1015 was cleared from the blood because the numbers of SR1015 in the blood of infected hamsters was 10,000-fold lower than that of wild type 1026b and lower than the initial inoculum of 100 cfu/ml [50].

CPS I production was shown to be responsible for persistence in the blood by evasion of the complement cascade and the mechanism for this was determined to be through the reduction of C3b deposition and opsonophagocytosis. The addition of purified CPS I to serum bactericidal assays showed that the capsule contributes to increased resistance of serum sensitive strains lacking the O-polysaccharide moiety (O-PS) of LPS to the bactericidal effects of normal human serum. However, CPS I mutants themselves were not found to be serum sensitive because they still produced O-PS, which was previously shown to be responsible for serum resistance, because it prevents lysis by the MAC complex [36]. This led us to postulate that CPS I was affecting the complement cascade through some other mechanism and it was found that this mechanism was through the reduction of C3b deposition and opsonization [50]. Both Western blot analysis and immunofluorescence microscopy experiments using a mouse monoclonal antibody to human C3b demonstrated the inhibition of C3b deposition by CPS I. In both experiments C3b deposition was more pronounced on the surface of the CPS I mutant compared to wild type. Also evident was that some C3b deposition occurred in the wild type, but this was expected since bacterial capsules are known to allow the diffusion of some C3b to the bacterial surface and *B. pseudomallei* is capable of activating the alternative pathway of complement culminating in the formation of the MAC complex [36, 62]. The accumulation of C3b affects the amplification step of the complement cascade and therefore, the less C3b deposited the less C5a is generated for phagocyte recruitment [63]. This explains the increased clearance of CPS I mutants from the blood. This conclusion was supported by the fact that *B. thailandensis,* the non-pathogenic organism which lacks CPS I, has been shown to be serum resistant, but is not capable of establishing a bacteremia in the Syrian hamster model of acute melioidosis [36, 37, 42]. Effective opsonization of invading bacteria results in enhanced phagocytosis and clearance of organisms form the blood of an infected host [52]. Quantitative radiolabelled phagocytic assays were also performed to establish a correlation between opsonization of the bacteria and phagocytosis by polymorphonuclear leukocytes. In the presence of serum, the CPS I mutant was more readily phagocytosed than wild type [50].

The expression of CPS I in the presence of normal human serum was found to be significantly elevated, also confirming that this capsule contributes to survival in the host. The presence of CPS I enables *B. pseudomallei* to survive in the blood through the inhibition of complement factor C3b deposition and phagocytosis [50]. The presence of this capsule facilitates survival as well as spreading to other organs, which can explain the overwhelming septicemia that is common in culture-positive melioidosis patients [64]. Therefore CPS I production is critical to the virulence of *B. pseudomallei* and further research will enhance the development of preventative strategies for melioidosis since this polysaccharide is one of the components of a *B. pseudomallei* subunit vaccine [28, 65].

Sequence analysis of the genome of *B. pseudomallei* revealed the presence of four operons possibly involved in polysaccharide capsule biosynthesis. One of these operons, (CPS I), corresponded to the previously identified and characterized mannoheptose capsule that was shown to be responsible for virulence and comprises one of the currently proposed melioidosis and glanders subunit conjugate vaccine [28, 66, 42, 50, 67]. The CPS I capsule cluster is present in the genome of *B. mallei* as well, but the complete cluster is not found in the genome of *B*. *thailandensis* [56, 57, 68]. This correlates with previous studies that have shown that this capsule is produced by *B. mallei*, but not by *B. thailandensis* [36, 37, 42, 56].

Three other putative capsule operons were identified by sequence analysis and all of these operons were found to be present in *B. pseudomallei* and *B. thailandensis*, but not *B. mallei*. Since these capsules are found in *B. thailandensis* and *B. pseudomallei*, they may be required for either survival in the host or in the environment; however, further studies are required to determine the roles of CPS II and CPS IV.

CPS III, located on chromosome 2, was found to contain 11 genes involved in the biosynthesis of a polysaccharide and was shown to be present in the genomes of *B. pseudomallei* and *B. thailandensis*, but not *B. mallei*. A mutation in the CPS III cluster did not affect production of CPS I and so it can be concluded that this operon encodes for gene products responsible for the biosynthesis of a separate capsule. CPS III was not found to contribute to the pathogenesis of *B. pseudomallei*. This capsule was not shown to be highly expressed *in vivo* by microarray analysis and was not required for virulence in the animal model. The CPS III mutant, SZ1829, which contains a mutation in the BPSS1829 gene as a result of insertional inactivation, was found to be as virulent in the animal model as wild type *B. pseudomallei*. The expression of this capsule was shown to be elevated when incubated in water, but suppressed in the presence of normal human serum [55]. The presence of the CPS III cluster in *B. pseudomallei* and *B. thailandensis*, both of which can survive for long periods in the environment compared to *B. mallei*, the increased expression of this capsule in water, and the low level of expression of this capsule *in vivo*, suggests that this capsule may contribute to the survival of *B. pseudomallei* in the environment [69].

146 The Complex World of Polysaccharides

[50].

resistant, but is not capable of establishing a bacteremia in the Syrian hamster model of acute melioidosis [36, 37, 42]. Effective opsonization of invading bacteria results in enhanced phagocytosis and clearance of organisms form the blood of an infected host [52]. Quantitative radiolabelled phagocytic assays were also performed to establish a correlation between opsonization of the bacteria and phagocytosis by polymorphonuclear leukocytes. In the presence of serum, the CPS I mutant was more readily phagocytosed than wild type

The expression of CPS I in the presence of normal human serum was found to be significantly elevated, also confirming that this capsule contributes to survival in the host. The presence of CPS I enables *B. pseudomallei* to survive in the blood through the inhibition of complement factor C3b deposition and phagocytosis [50]. The presence of this capsule facilitates survival as well as spreading to other organs, which can explain the overwhelming septicemia that is common in culture-positive melioidosis patients [64]. Therefore CPS I production is critical to the virulence of *B. pseudomallei* and further research will enhance the development of preventative strategies for melioidosis since this

polysaccharide is one of the components of a *B. pseudomallei* subunit vaccine [28, 65].

Sequence analysis of the genome of *B. pseudomallei* revealed the presence of four operons possibly involved in polysaccharide capsule biosynthesis. One of these operons, (CPS I), corresponded to the previously identified and characterized mannoheptose capsule that was shown to be responsible for virulence and comprises one of the currently proposed melioidosis and glanders subunit conjugate vaccine [28, 66, 42, 50, 67]. The CPS I capsule cluster is present in the genome of *B. mallei* as well, but the complete cluster is not found in the genome of *B*. *thailandensis* [56, 57, 68]. This correlates with previous studies that have shown that this capsule is produced by *B. mallei*, but not by *B. thailandensis* [36, 37, 42, 56].

Three other putative capsule operons were identified by sequence analysis and all of these operons were found to be present in *B. pseudomallei* and *B. thailandensis*, but not *B. mallei*. Since these capsules are found in *B. thailandensis* and *B. pseudomallei*, they may be required for either survival in the host or in the environment; however, further studies are required

CPS III, located on chromosome 2, was found to contain 11 genes involved in the biosynthesis of a polysaccharide and was shown to be present in the genomes of *B. pseudomallei* and *B. thailandensis*, but not *B. mallei*. A mutation in the CPS III cluster did not affect production of CPS I and so it can be concluded that this operon encodes for gene products responsible for the biosynthesis of a separate capsule. CPS III was not found to contribute to the pathogenesis of *B. pseudomallei*. This capsule was not shown to be highly expressed *in vivo* by microarray analysis and was not required for virulence in the animal model. The CPS III mutant, SZ1829, which contains a mutation in the BPSS1829 gene as a result of insertional inactivation, was found to be as virulent in the animal model as wild type *B. pseudomallei*. The expression of this capsule was shown to be elevated when incubated in water, but suppressed in the presence of normal human serum [55]. The presence of the CPS III cluster in *B. pseudomallei* and *B. thailandensis*, both of which can

to determine the roles of CPS II and CPS IV.

Previous studies have demonstrated that *B. pseudomallei* produces three other capsular polysaccharides in addition to CPS I and these have been structurally characterized. One is an acidic polysaccharide 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-, which is recognized by patient sera [32]. The other two are: a branched 1,4-linked glucan polymer ((CP-1a) and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galatose, glucose, and glucoronic acid (CP-2) [49]. Combined GC/MS analysis of CPS III revealed that the composition of this capsule demonstrates some similarity to the composition of the previously described capsule CP-2 composed of rhamnose, mannose, galactose, glucose, and glucoronic acid; however, the proportions of carbohydrate residues were not similar, and the CPS III capsule was also found to contain xylose and not glucoronic acid. In addition, CPS III was determined to be composed primarily of heptose [55]. Therefore it is evident that the capsule identified in this study is not one of the previously described capsule structures. Some of the previously characterized capsules produced by *B. pseudomallei* have been shown to be produced under unique conditions [32, 49]. Strain variation, differences in expression of the capsules, and discrepancies between purification strategies may also explain why a number of capsules have been shown to be produced by this organism. Nevertheless, the genes BPSS 1825-1835 appear to be involved in the biosynthesis of a capsule with this composition. Further analysis by 2D NMR would be required to definitively establish a connection between CPS III and one of the other published structures.

Studies by another laboratory have also focused on the presence of these capsule clusters in *B. pseudomallei*. Sarkar-Tyson *et al*. identified two polysaccharide clusters, one of which corresponds to the CPS III presented in this paper, but the authors identified this cluster as type IV O-PS (2007). The type IV O-PS was found to be involved in virulence in a mouse model [70]. However, a mutant in this polysaccharide did not demonstrate any difference in hydrophobicity compared to wild-type, indicating that this polysaccharide does not contribute to making the cell surface more hydrophobic, which is an advantageous characteristic for some pathogenic bacteria. The differences in virulence compared to the current work can be attributed to the use of different animal models; however, all other data seem to indicate that this capsule is not required for virulence.

A study was recently published which outlines the identification of another capsule produced by *B. pseudomallei* [71]. This capsule was determined to be composed of 1,3-linked α-D-mannose residues. This capsular polysaccharide was also found to be produced by *B. mallei*. The genes involved in the synthesis of this polysaccharide have not yet been identified and work is also underway to determine the role for this novel capsule in the pathogenesis of melioidosis and glanders.

*B. pseudomallei* is an environmental saprophyte often found in soil and stagnant water and incidence of the disease is high in rice farmers in Southeast Asia [22, 69]. This organism harbors a large genome which explains its ability to survive for long periods of time in the environment as well exist as a significant pathogen in both humans and animals. The presence of multiple polysaccharide clusters in the genome and the production of multiple capsule structures under differing conditions may contribute to the ability of this organism to adapt to a variety of conditions. As demonstrated in this study, capsule expression is dependent on the particular environment, which indicates that *B. pseudomallei* produces these capsules to promote a survival advantage either in the host or in the environment. Further studies aimed at characterizing the capsules of *B. pseudomallei* will be beneficial to understand the pathogenesis of this organism and to advance further vaccine development.
