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) --> Glc-1-P --> UDP-Glc --> UDP-glucuronic acid (UDP-GlcUA) --> (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.

FIG. 1

Virulence of type 3 point and insertion mutations in BALB/cByJ mice. The type 3 locus is shown at the top (11). The arrow indicates the direction and length of the only known transcript. Parentheses indicate naturally truncated sequences (cps3M at the 3′ end, plpA at the 5′ end, and tnpA at both the 5′ and 3′ ends). plpA also contains a point mutation (·) that causes a frameshift in the remaining partial open reading frame. The partial open reading frame for TnpA is opposite (i.e., right to left) that of all others in the locus. Maps show the structure of each mutant locus, with boxes indicating the region that was used to target insertion-duplication mutations. Arrows within each locus indicate the site of the plasmid insertion, with the orientation indicating the direction of a cat reporter sequence contained in the plasmid. Strain names are indicated above or below the appropriate arrows. Where more than one strain name is given, independent isolates were tested, with approximately equal numbers of mice used for each isolate. The maps are not drawn to scale, but the target fragments are sized proportionately. The amino acid positions of the insertions and effects of the mutations are shown to the left of each map. −, expected loss of function; dn, downstream insertion that results in an intact copy of all sequences. The D⁻, dn D, and S⁻ mutants are nonencapsulated; all others produce parental amounts of capsule. For virulence studies, BALB/cByJ mice were infected i.v. with 10⁷ bacteria (similar results were obtained with lower doses; the WU2 LD₅₀ is approximately 3 × 10⁵ CFU). The number of mice infected (n) is combined for independent isolates containing the same mutation, none of which differed from each other. The downstream D insertion is polar on S (16); thus, it and the S⁻ mutant are considered together. P values were determined using Fisher's exact test to compare survival (alive versus dead) and the Mann-Whitney two-sample rank test to compare median times to death (MTD; in hours). A time to death of >504 h indicates survival. Bacteria isolated from dead mice retained the expected insertions, as determined by resistance to the antibiotic marker (erythromycin) carried on the plasmid insertion.

FIG. 2

Virulence of type 3 deletion mutants in BALB/cByJ mice. The limits of each deletion are shown below the map. All mutants produced parental levels of capsule. Mice were infected i.v. with 10⁷ bacteria. The number of mice infected (n) is combined for independent isolates of the same mutation (indicated by strain names at the left). None of the independent isolates differed from each other. Numbers in parentheses represent a set of experiments that was performed at a later time and that resulted in a shorter median time to death for the parent strain, WU2. The mutants are therefore compared to the WU2 control mice from the appropriate set of experiments. None of the mutants were statistically different from the parent with regard to survival (compared using Fisher's exact test) or median times to death (MTD; in hours; compared using the Mann-Whitney two-sample rank test).

FIG. 3

Virulence of the PGM mutant JY1060 in BALB/cByJ mice. Mice were infected i.p. (A) or i.v. (B) with either the type 3 parent (WU2, □), the PGM mutant (JY1060, ▪), or the repaired PGM mutant (GH5088, ▨). The i.p. and i.v. LD₅₀s for WU2 are approximately 50 and 3 × 10⁵ CFU, respectively. The total number of mice infected at each dose is indicated above the bar. Results for GH5088 were not significantly different from those for WU2 by either route of infection but were different from those for JY1060 (P = 0.0008 [i.p.] and 0.0007 [i.v.]; compared using Fisher's exact test). JY1060 was significantly different from WU2 at all doses (i.p., P values were <0.005 [10²], <0.0001 [10³], and <0.0001 [6 × 10⁴, compared to WU2 at 10³]; i.v., P values were 0.007 [5 × 10⁵] and <0.0001 [6 × 10⁶]). At the i.p. dose of 6 × 10⁴ CFU (*), 7 of 11 mice infected with JY1060 died. However, the bacteria recovered from these mice did not exhibit the small colony morphology characteristic of JY1060, and none of the mice were considered to have died from JY1060. (C) Blood clearance following i.v. infection with 6 × 10⁶ bacteria. Mice were bled at 1 min (time zero), 1 h, 4 h, and 20 h postinfection. Results are the means ± SEM. P values (in parentheses) were determined using Student's t test by comparison to WU2 data at each time point. □, WU2 (n = 5); ▪, JY1060 (n = 3); ●, the nonencapsulated strains JD611 and JD908 combined (n = 6). The nonencapsulated strains were undetectable at 4 and 24 h.

FIG. 4

Virulence of JY1060 suppressor mutants. BALB/cByJ mice were infected i.p. with WU2 (▪) or the suppressor mutants GH5000 (♦), GH5001 (●), GH5087 (▴), and GH5089 (▾). A time to death of >504 h indicates survival. Overall survival, compared using Fisher's exact test, was significantly different (P = 0.0064) for WU2 and the suppressors at the dose of 10³ CFU. Median times to death (indicated by the bars and compared using the Mann-Whitney two-sample rank test) were significantly different at the doses of 1 × 10³ and 6 × 10³ CFU (P = 0.0002 for each, compared to the 10³-CFU dose of WU2).

FIG. 5

Virulence of JY1060 in XID mice. CBA/N mice were infected i.v. and i.p. at the indicated doses with either WU2 (▵) or JY1060 (▴). A time to death of >504 h indicates survival. Numbers in parentheses are P values for comparison of median time to death with WU2 at the lower dose, as determined using the Mann-Whitney two-sample rank test. There were no significant differences in overall survival.

FIG. 6

Effect of decomplementation on virulence. BALB/cByJ mice were infected i.v. with WU2 (□) or JY1060 (▪), 5 to 8 h after injection with cobra venom factor (CVF). The total number of mice infected at each dose is indicated above the bar. Statistical significance was calculated using Fisher's exact test. P values at the 10⁵-CFU dose were 0.0014 (WU2 with CVF versus WU2 without CVF) and 0.007 (WU2 versus JY1060, with CVF). At the 10⁷-CFU dose, P values were 0.007 (JY1060 with CVF versus JY1060 without CVF) and 0.015 (WU2 versus JY1060, without CVF). The LD₅₀ of WU2 was reduced to <10³ CFU in CVF-treated mice (data not shown).

FIG. 7

Binding of antibodies to surface components. Reactivity of antibodies with whole cells was tested using indirect ELISAs. Binding to WU2 and JY1060 is expressed relative to the nonencapsulated JD611. The anti-type 19 polyclonal antiserum contains a high titer of antibody to noncapsule components and reacts with JD611 at antiserum dilutions of (>2 × 10⁵)-fold. It was used as a source of polyclonal antibody to S. pneumoniae surface antigens. Results are expressed as the means ± SEM (n = 3). Reactivity of WU2 and JY1060 with the anti-type 19 polyclonal antiserum was significantly different (P = 0.001, Student's t test). Binding of the other antibodies or antiserum to WU2 and JY1060 was not significantly different. TA, teichoic acid (C polysaccharide).