**3.4. The** *B. pseudomallei* **capsule CPS I reduces phagocytosis**

138 The Complex World of Polysaccharides

this capsule [42, 50].

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

**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

The capsule mutant SR1015 was phagocytosed more significantly by PMNL than the wildtype strain. The proportion of wild-type *B*. *pseudomallei* 1026b phagocytosed in the presence of 10% NHS was 35.9%, while the proportion of the capsule mutant SR1015 phagocytosed was 51.7% (*P* < 0.001). When each strain was incubated in the presence of 30% NHS, 59.3% of the wild-type strain 1026b was phagocytosed by the PMNL after 30 min compared to 82.3% for the capsule mutant (*P* < 0.001) [50].
