**3. Role of the** *B. pseudomallei* **capsule in virulence**

The role of CPS I in the pathogenicity of *B. pseudomallei* was investigated by performing further animal studies, serum bactericidal assays, complement protein C3b deposition assays, and radio-labelled phagocytic assays [50]. These experiments were facilitated by constructing a deletion strain harbouring a mutation in one of the CPS I genes and by complementation of this strain. An in-frame deletion was constructed in *wcbB*, a gene which encodes a glycosyltransferase, resulting in the capsule-minus strain SZ210. To confirm the role of *wcbB* in the biosynthesis of capsule, SZ210 was complemented by the introduction of a wild-type copy of the *wcbB* gene cloned into the mobilizable broad-host-range plasmid pBHR1 (MoBiTec). Western blot analysis of proteinase K-digested whole cells was performed using mouse monoclonal antibody directed to *B. pseudomallei* capsule to assess capsule production by these strains. Similar to the capsule minus strain SR1015 and *B. thailandensis* E264, which is known to lack this capsule, SZ210 was found to be negative for CPS I production, as indicated by the absence of a 200 kDa band that is present for wild-type 1026b. Complementation of SZ210 by providing the wild type *wcbB* gene in *trans* restored capsule production. Whole-cell extracts from the complemented strain SZ210(pSZ219) reacted to the capsule antibody producing the 200-kDa band corresponding to the *B. pseudomallei* capsule.

## **3.1. Production of CPS I correlates with clinical infection**

To establish a correlation between CPS I production and clinical infection a number of strains of *B. pseudomallei* isolated from a variety of clinical specimens were tested for capsule production by western blot analysis with polyclonal rabbit antisera to *B. pseudomallei* CPS I. Out of the 55 clinical strains tested for capsule production, 52 were found to produce this capsule. Three strains, 420a, 415c, and 375a were found to be negative for capsule production, similar to *B. thailandensis* E264. However, one of the capsule genes, *wzt2*, was successfully amplified from these three strains and following inoculation in the animal model, all three of these strains were found to produce capsule by western blot analysis. This indicated that CPS I production may be regulated in some strains and its expression may be induced *in vivo*. Therefore all of the 55 clinical strains of *B. pseudomallei* tested were found to produce capsule, establishing a 100% correlation between CPS I production and clinical infection [51].

#### **3.2. CPS I promotes survival of** *B. pseudomallei in vivo*

134 The Complex World of Polysaccharides

*pseudomallei* capsule.

clinical infection [51].

lipopolysaccharide preparation of *B. pseudomallei* [49].

**3. Role of the** *B. pseudomallei* **capsule in virulence** 

**3.1. Production of CPS I correlates with clinical infection** 

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

The role of CPS I in the pathogenicity of *B. pseudomallei* was investigated by performing further animal studies, serum bactericidal assays, complement protein C3b deposition assays, and radio-labelled phagocytic assays [50]. These experiments were facilitated by constructing a deletion strain harbouring a mutation in one of the CPS I genes and by complementation of this strain. An in-frame deletion was constructed in *wcbB*, a gene which encodes a glycosyltransferase, resulting in the capsule-minus strain SZ210. To confirm the role of *wcbB* in the biosynthesis of capsule, SZ210 was complemented by the introduction of a wild-type copy of the *wcbB* gene cloned into the mobilizable broad-host-range plasmid pBHR1 (MoBiTec). Western blot analysis of proteinase K-digested whole cells was performed using mouse monoclonal antibody directed to *B. pseudomallei* capsule to assess capsule production by these strains. Similar to the capsule minus strain SR1015 and *B. thailandensis* E264, which is known to lack this capsule, SZ210 was found to be negative for CPS I production, as indicated by the absence of a 200 kDa band that is present for wild-type 1026b. Complementation of SZ210 by providing the wild type *wcbB* gene in *trans* restored capsule production. Whole-cell extracts from the complemented strain SZ210(pSZ219) reacted to the capsule antibody producing the 200-kDa band corresponding to the *B.* 

To establish a correlation between CPS I production and clinical infection a number of strains of *B. pseudomallei* isolated from a variety of clinical specimens were tested for capsule production by western blot analysis with polyclonal rabbit antisera to *B. pseudomallei* CPS I. Out of the 55 clinical strains tested for capsule production, 52 were found to produce this capsule. Three strains, 420a, 415c, and 375a were found to be negative for capsule production, similar to *B. thailandensis* E264. However, one of the capsule genes, *wzt2*, was successfully amplified from these three strains and following inoculation in the animal model, all three of these strains were found to produce capsule by western blot analysis. This indicated that CPS I production may be regulated in some strains and its expression may be induced *in vivo*. Therefore all of the 55 clinical strains of *B. pseudomallei* tested were found to produce capsule, establishing a 100% correlation between CPS I production and Syrian golden hamsters were inoculated intraperitoneally with 101 to 105 cells of either wild type *B. pseudomallei* 1026b, capsule mutants SR1015 and SZ210, or the complemented strain SZ210(pSZ219). One group of animals inoculated with SR1015 also received 100 µg of purified *B. pseudomallei* capsule. After 48 h, the LD50 values were calculated, and the blood of the infected animals was diluted and plated for bacterial quantitation. The addition of purified capsule significantly increased the virulence of the capsule mutant strain SR1015. The LD50 value was calculated to be 34 CFU, similar to the LD50 value of wild-type *B. pseudomallei* 1026b (<10 CFU). In contrast, the LD50 value for SR1015 without the addition of purified capsule was calculated to be 3.5 x 105 CFU, 10,000- fold higher than when capsule was added to the inoculum. In addition, purified capsule enhanced the survival of SR1015 in the blood. Bacteria could not be detected in the blood of hamsters inoculated with SR1015 alone. However, the number of SR1015 CFU recovered from the blood of infected animals was 9.0 x 102 CFU/ml when capsule was added to the inoculum, an almost-1,000-fold increase. This number was comparable to the number of wild-type *B. pseudomallei* 1026b bacteria recovered from the blood. The addition of capsule was not toxic to the hamsters, as hamsters inoculated with 100 µg of purified capsule alone survived for the duration of the experiment without any ill effects. The LD50 value for the capsule mutant strain SZ210 containing an in frame deletion of the *wcbB* gene was calculated to be 9.6 x 104 CFU, and the number of bacteria in the blood was determined to be 10 CFU/ml. Complementation of this strain restored virulence in the animal model, resulting in an LD50 value of 12 CFU, comparable to that of wild type *B. pseudomallei* 1026b. Furthermore, the number of bacteria in the blood of animals infected with the complemented strain, SZ210(pSZ219), was determined to be 4.9 x 105 CFU/ml, similar to the number of bacteria recovered from animals infected with 1026b [50].

To further demonstrate the role of the capsule in infection by *B. pseudomallei*, an experiment was designed to investigate differences in tissue distribution between the capsule mutant strain and the wild type in infected hamsters. Animals were inoculated with 102 CFU of either wild-type *B. pseudomallei* 1026b or the capsule mutant SR1015. At different time points, the animals were sacrificed, and the numbers of bacteria in the blood, liver, lungs, and spleen of each animal were determined. As seen in Figure 2, the numbers of *B. pseudomallei* 1026b and SR1015 bacteria were nearly undetectable at 12 h (Figure 2A). By 24 h, the numbers of 1026b bacteria recovered from the blood, lung, liver, and spleen increased, while SR1015 was detected only in the spleen (Figure 2B). By 48 h, very high numbers of 1026b bacteria were recovered from all of the organs taken, representing a dramatic increase compared to the inoculum (Figure 2C). In contrast, all of the organs taken from hamsters infected with SR1015 contained fewer bacteria (Figure 2C). Of particular interest was the fact that the number of SR1015 bacteria recovered from the blood at 48 h was lower than in the inoculum, suggesting that the capsule mutant was cleared from the blood more effectively than the wild type. The number of SR1015 bacteria recovered from the spleen was higher than the number of SR1015 bacteria in the blood, suggesting that SR1015 was being cleared from the blood and sequestered in the spleen. The difference in virulence between the two strains can be attributed to capsule production, since the CPS I mutant strain was found to have a growth rate similar to that of the wild-type strain 1026b [50].

**Figure 2.** Differences in tissue distribution between *B*. *pseudomallei* strains 1026b and SR1015 in the Syrian hamster model of acute melioidosis. Female Syrian hamsters (three per group) were inoculated intraperitoneally with 102 CFU of either strain, and at 12, 24, and 48 h, two groups of animals were sacrificed and bacterial quantitation of the tissues was determined. The data represent the average number of bacteria found in each tissue and the standard deviation for a given time point.

## **3.3. CPS I production by** *B. pseudomallei* **is responsible for persistence in the blood by inhibiting complement**

136 The Complex World of Polysaccharides

strains can be attributed to capsule production, since the CPS I mutant strain was found to

**Figure 2.** Differences in tissue distribution between *B*. *pseudomallei* strains 1026b and SR1015 in the Syrian hamster model of acute melioidosis. Female Syrian hamsters (three per group) were inoculated intraperitoneally with 102 CFU of either strain, and at 12, 24, and 48 h, two groups of animals were sacrificed and bacterial quantitation of the tissues was determined. The data represent the average

number of bacteria found in each tissue and the standard deviation for a given time point.

have a growth rate similar to that of the wild-type strain 1026b [50].

To define the role of the capsule for persistence in the blood, serum bactericidal assays were performed with the addition of purified capsule to determine if capsule had an effect on the survival of serum-sensitive strains of *B. pseudomallei*. For these experiments, we utilized a double mutant that we constructed in the laboratory, SLR5, which lacks both capsule and Opolysaccharide, since the capsule mutant SR1015 was previously found to be serum resistant [42]. The survival of SLR5 was extremely poor when incubated in the presence of 30% normal human serum (NHS). However, the addition of purified capsule increased the survival of SLR5 in NHS. The addition of 50 µg of capsule to the reaction increased the numbers of SLR5 to 5.9 x 101 CFU/ml, and the addition of 100 µg of capsule increased the survival of SLR5 by nearly 1,000-fold to 1.9 x 103 CFU/ml. Furthermore, pre-incubation of 30% NHS with 100 µg of capsule (PI-CPS) before the addition of bacteria increased the survival of SLR5 100,000-fold to 4.4 x 106 CFU/ml. This was similar to the survival of SLR5 when incubated with serum that was heat-inactivated (HI-NHS). These effects were found to be specific to capsule, since the addition of 50 or 100 µg of purified *B. pseudomallei* O-PS or preincubation of the serum with O-PS did not increase the survival of serum-sensitive SLR5 [50].

Since capsule mutants of *B. pseudomallei* are serum resistant in that they are not susceptible to lysis by the membrane attack complex (MAC) because they still produce O-PS, we postulated that the ability of the capsule to enhance survival in the blood could be due to its ability to inhibit C3b deposition and opsonization. To investigate the effect of capsule on C3b deposition, the amount of C3b deposited on the surfaces of wild-type *B. pseudomallei*  1026b and the capsule mutant, SR1015, in the presence of serum was determined by Western blot analysis using a mouse monoclonal antibody specific to human complement factor C3b. The deposition of C3b was found to be more pronounced in the capsule mutant SR1015 than in the wild type in both 10 and 30% NHS. Similar results were observed with the capsule mutant SZ210, a strain containing an in-frame deletion of the *wcbB* gene. More C3b was detected when SZ210 was incubated in both 10 and 30% NHS than with 1026b. Optical densitometry measurements were performed in order to quantitate the difference in C3b deposition between the strains. The average amount of C3b deposited on the surfaces of SR1015 and SZ210 bacteria was 3.5-fold higher than for 1026b in 10% NHS and 2.5-fold higher in 30% NHS. In addition, there was a shift in the molecular mass of C3b, which normally runs at 185 kDa, indicating a covalent attachment of the molecule to the bacterial surface. The nature of this attachment was not investigated; however, C3b is thought to covalently attach to the bacterial surface through an ester or amide linkage [50, 52].

Immunofluorescence microscopy analysis was also performed to demonstrate the difference in C3b deposition between the capsule mutant and the wild type. The same experiment described above was performed, and samples were reacted with the mouse monoclonal antibody to human complement factor C3b, except that the samples were reacted with a secondary antibody conjugated to Cy3 and stained with DAPI for visualization of bacterial cells. As shown in Figure 3, the *B. pseudomallei* capsule mutant SR1015 demonstrated more reactivity to the antibody to human C3b in the presence of serum than the wild-type 1026b. This is evident from the red fluorescence that corresponds to the C3b bound to the bacterial surface surrounding the blue DAPI-stained cells seen when the capsule mutant was incubated in the presence of 10% NHS (Figure 3D to F). In contrast, the amount of red fluorescence surrounding the DAPI-stained wild-type cells was minimal in the presence of 10% NHS (Figure 3A to C). There was a dramatic difference in the amount of C3b deposited on the surface of the capsule mutant compared to the wild type, which was detectable after only 15 min of incubation of the bacteria with human serum (Figure 3B and E). By 60 min, there was some C3b deposition on wild-type *B. pseudomallei*; however, there was still more C3b deposited on the surface of the capsule mutant (Figure 3C and F) [50]. This experiment was not performed with 30% NHS due to excessive clumping of the samples during the fixation process, which resulted in inconsistent and poor staining of the cells. Western blot analysis was also performed to determine the amount of complement factor C3b deposition on the surface of *B. thailandensis* E264, a related nonpathogenic organism. The amount of C3b deposition in *B. thailandensis* E264 was more pronounced than with *B*. *pseudomallei*  1026b and was similar to the amount of C3b deposited on the surface of the capsule mutant, *B. pseudomallei* SR1015, in the presence of human serum. The amount of C3b deposition that occurred on the surface of *B. thailandensis* was expected, since the organism is known to lack this capsule [42, 50].

**Figure 3.** Immunofluorescence microscopy analysis of decreased complement factor C3b deposition in 10% normal human serum by *B. pseudomallei* capsule. *B. pseudomallei* 1026b and SR1015 were incubated in 10% normal human serum (NHS), reacted with a mouse monoclonal antibody to human complement factor C3b, reacted with a rabbit anti-mouse IgG conjugated to Cy3 (Jackson Laboratories), and stained

with DAPI for visualization of whole bacterial cells (Sigma). (A) *B. pseudomallei* 1026b incubated in PBS; (B) 1026b incubated in 10% NHS for 15 min; (C) 1026b incubated in 10% NHS for 60 min; (D) *B. pseudomallei* SR1015 incubated in PBS; (E) SR1015 incubated in 10% NHS for 15 min; (F) SR1015 incubated in 10% NHS for 60 min. The blue fluorescence indicates the DAPI stained bacteria, and the red fluorescence indicates the binding of complement factor C3b to the bacterial surface.
