Surface association of Pht proteins of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a major human pathogen responsible for massive global morbidity and mortality. The pneumococcus attaches a variety of proteins to its cell surface, many of which contribute to virulence; one such family are the polyhistidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE. In this study, we have examined the mechanism of Pht surface attachment using PhtD as a model. Analysis of deletion and point mutants identified a three-amino-acid region of PhtD (Q27-H28-R29) that is critical for the process. The analogous region in PhtE was also necessary for its attachment to the cell surface. Furthermore, we show that a large proportion of the total amount of each Pht protein is released into bacterial culture supernatants. Other surface proteins were also released, albeit to lesser extents, and this was not due to pneumococcal autolysis. The extent of release of surface proteins was strain dependent and was not affected by the capsule. Lastly, we compared the fitness of wild-type and ΔphtABDE pneumococci in vivo in a mouse coinfection model. Release of Pht proteins by the wild type did not complement the mutant strain, consistent with surface-attached rather than soluble forms of the Pht proteins playing the major role in virulence. The significant degree of release of Pht proteins from intact bacteria may have implications for the use of these proteins in novel vaccines.

Fig 1

Surface attachment of PhtD. (A and B) Flow cytometric measurement of the presence of PhtD at the bacterial surface. A representative histogram of fluorescence profiles for each strain is shown. (C) Western blots assessing the amounts of PhtD in samples of the bacterial lysate and culture supernatant. WT, wild type. Approximate molecular sizes are indicated on the left; these were estimated based on the mobilities of standard markers (not shown).

Fig 2

Surface attachment of PhtD after targeted mutagenesis. (A) Alignment of the putative attachment motif in PhtD with homologous regions in PhtA, PhtB, and PhtE. The Pht proteins of S. pneumoniae D39 were aligned using ClustalW2 (available online at http://www.ebi.ac.uk/Tools/msa/clustalw2/). Only the six amino acids corresponding to RHQAGQ from PhtD are shown. Colors indicate physiochemical properties of classes of amino acids (red, small/hydrophobic; blue, acidic; black, basic; green, hydroxyl/sulfhydryl/amine). (B) Flow cytometric measurement of the presence of PhtD at the bacterial surface. A representative histogram of fluorescence profiles for each strain is shown. (C) Western blot assessing the amounts of PhtD in samples of the bacterial lysate and culture supernatant.

Fig 3

Surface attachment of PhtE after targeted mutagenesis. (A) Flow cytometric measurement of the presence of PhtE at the bacterial surface. A representative histogram of fluorescence profiles for each strain is shown. (B) Western blot assessing the amounts of PhtE in samples of the bacterial lysate and culture supernatant.

Fig 4

Proportions of pneumococcal surface protein in culture supernatants of wild-type D39, WCH43, and WU2, calculated after quantitation from Western blots. Results are presented as mean and standard error from samples grown in biological triplicate. Measurement of PhtD only was made for the D39 ΔlytA mutant strain.

Fig 5

The capsule does not affect secretion or release of PhtD. (A) Percentage of PhtD in supernatants of D39 wild-type and Δcps cultures. Results are representative of three independent experiments. (B) Percentage of PhtD in the supernatants of cultures of five serotype 3 clinical isolate strains.

Fig 6

Release of PhtD over time. (A) Western blots for PhtD and AdcR in bacterial lysates and culture supernatants of bacteria grown in the presence or absence of chloramphenicol. (B) Percentages of PhtD and AdcR in culture supernatants based on quantitations of band intensities. The results are representative of two independent experiments.

Fig 7

Flow cytometric measurement of PhtD at the bacterial surface of the indicated strain. ΔphtABDE bacteria were incubated with supernatant from wild-type or N full strains or purified PhtD as shown. Geometric mean fluorescence intensities from one experiment are shown; results are representative of two independent experiments.

Fig 8

In vivo competition between wild-type and ΔphtABDE bacteria. Mice were infected intranasally with a mixed culture of the two strains and samples collected after 24 and 48 h. Geometric mean competitive indices and 95% confidence intervals are shown. A competitive index of 1 (dashed line) would indicate no difference in fitness between the two strains. Values for all groups were significantly different from the theoretical mean of 1 (P < 0.0001 [one-sample t tests, two tailed]).