Essential Role for Cellular Phosphoglucomutase in Virulence of Type 3 Streptococcus pneumoniae

Abstract

Synthesis of the Streptococcus pneumoniae type 3 capsule requires the pathway glucose-6-phosphate (Glc-6-P) 3 Glc-1-P 3 UDP-Glc 3 UDP-glucuronic acid (UDP-GlcUA) 3 (GlcUA-Glc) n . The UDP-Glc dehydrogenase and synthase necessary for the latter two steps, and essential for capsule production, are encoded by genes (cps3D and cps3S, respectively) located in the type 3 capsule locus. The phosphoglucomutase (PGM) and Glc-1-P uridylyltransferase activities necessary for the first two steps are derived largely through the actions of cellular enzymes. Homologues of these enzymes, encoded by cps3M and cps3U in the type 3 locus, are not required for capsule production. Here, we show that cps3M and cps3U also are not required for mouse virulence. In contrast, nonencapsulated isolates containing defined mutations in cps3D and cps3S were avirulent, as were reduced-capsule isolates containing mutations in pgm. Insertion mutants that lacked PGM activity were avirulent in both immunologically normal (BALB/cByJ) and immunodeficient (CBA/N) mice. In contrast, a mutant (JY1060) with reduced PGM activity was avirulent in the former but had only modestly reduced virulence in the latter. The high virulence in CBA/N mice was not due to the lack of antibodies to phosphocholine but reflected a growth environment distinct from that found in BALB/cByJ mice. The reduced PGM activity of JY1060 resulted in enhanced binding of complement and antibodies to surface antigens. However, decomplementation of BALB/cByJ mice did not enhance the virulence of this mutant. Suppressor mutations, only some of which resulted in increased capsule production, increased the virulence of JY1060 in BALB/cByJ mice. The results suggest that PGM plays a critical role in pneumococcal virulence by affecting multiple cellular pathways.

Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

“…Since all of the mutants isolated in the second screen were in the acapsular background and the capsule is required for colonization (53), we backcrossed all of the mutations into a wild-type encapsulated strain before testing them for colonization. Although the capsule, S. pneumoniae's quintessential virulence factor, has been studied extensively during invasive disease (2,36), little is known about its role during colonization. Recent work by Nelson and colleagues (63) suggests that one of its functions is to help S. pneumoniae evade entrapment by the mucus covering the mucosa, allowing bacteria to reach the underlying epithelial layer, where it is thought stable colonization can take place.…”

Genetic Alteration of Capsule Type but Not PspA Type Affects Accessibility of Surface-Bound Complement and Surface Antigens ofStreptococcus pneumoniae

“…The anti-type 19 polyclonal antiserum reacts with JD611 at antiserum dilutions of Ͼ2 ϫ 10 5 (18). The amount of type 3 capsule on the cell surface does not affect binding of the bacteria to a microtiter plate (18). Likewise, strains expressing type 2 and type 3 capsules bound equivalently to microtiter plates.…”

“…For detection of other surface antigens, a polyclonal antibody specific for serotype 19 (typing serum obtained from Statens Seruminstitut, Copenhagen, Denmark; diluted 1:5,000) was used. Because whole cells are used as immunogens for this antiserum and because of the poor immunogenicity of the type 19 capsule, the majority of the antibodies are reactive with noncapsular antigens (18). The antiserum can thus be used to detect general exposure of surface antigens to antibody.…”

“…The results of the antibody binding assays were normalized to the binding observed with JD611, a nonencapsulated derivative of the type 3 strain WU2. The anti-type 19 polyclonal antiserum reacts with JD611 at antiserum dilutions of Ͼ2 ϫ 10 5 (18). The amount of type 3 capsule on the cell surface does not affect binding of the bacteria to a microtiter plate (18).…”

“…Indirect ELISAs to detect binding of antibodies to surface antigens were performed essentially as described previously (18). S. pneumoniae cultures were grown to mid-exponential phase (ϳ3 ϫ 10 8 CFU/ml) in THY, heat killed by incubation at 65°C for 20 min, and centrifuged (14,000 ϫ g, 20 min).…”