David A. Watson

Antibody to capsular polysaccharide of Streptococcus pneumoniae at the time of hospital admission for pneumococcal pneumonia

IgG to capsular polysaccharide (CPS) of Streptococcus pneumoniae is thought to provide the greatest degree of protection against pneumococcal disease. Serum obtained at hospital admission from 14 (27%) of 51 patients with bacteremic pneumococcal pneumonia and 11 (37%) of 30 with nonbacteremic pneumococcal pneumonia contained IgG to CPS of the infecting serotype; these percentages are similar to the prevalence of IgG to CPS in a control population. However, when compared with antibody from healthy adults, this IgG had far less capacity to opsonize the infecting pneumococcal serotype for phagocytosis in vitro by normal human polymorphonuclear leukocytes or to protect mice against experimental challenge. Failure to opsonize correlated closely with failure to protect mice, and each of these parameters correlated well with poor avidity for CPS. Future vaccine studies may need to examine the functional capacity of antibodies as a surrogate for infection, in addition to measuring their concentration in serum.

IgG Responses to Protein-Conjugated Pneumococcal Capsular Polysaccharides in Persons Who Are Genetically Incapable of Responding to Unconjugated Polysaccharides

We have previously shown that the capacity to make IgG to pneumococcal capsular polysaccharides (PCPs) is inherited as an autosomal, mixed codominant trait. The purpose of this study was to determine whether this genetically determined unresponsiveness could be overcome by injection of protein-conjugated pneumococcal vaccines. Seven healthy adults who had failed to produce IgG to five or more of 10 representative PCPs after receiving pneumococcal vaccine and whose parents, siblings, and/or offspring had a similar lack of responsiveness received a series of protein-conjugated polysaccharide vaccines. Excellent IgG responses to most of the PCPs tested were eventually observed in five of the seven subjects after they received octavalent diphtheria toxoid-conjugated vaccine. Administration of certain protein-conjugated PCPs leads to IgG responses in some persons who lack the capacity to respond to unconjugated PCPs.

Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

ABSTRACT We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

Streptococcus pneumoniae PstS Production Is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity

ABSTRACT Differential display-PCR (DDPCR) was used to identify aStreptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation ofpstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Global transcription profiling and virulence potential of Streptococcus pneumoniae after serial passage

Streptococcus pneumoniae, a facultative human pathogen associated with wide variety of diseases, such as pneumonia, meningitis, sepsis and otitis media. Factors involved in the initial colonization, survival and etiology of this commensal bacteria in the human host from the ex vivo environment is still not clearly understood. Here, we report alterations in global transcriptional profiles of S. pneumoniae 6304 serotype 4 after 50 passages (50P) and 100 passages (100P) on laboratory media to better understand gene expression strategies employed by the bacterium during progression from the nasopharynx to the blood. The results show that six-fold more genes were differentially expressed after 100P as compared to 50P. After 100P on blood agar plates, 726 genes (33%) of 2192 genes in the S. pneumoniae genome were differentially expressed. Moreover, the majority of these genes (68%) were expressed at higher levels with increasing passage number and from different functional groups. Significantly, all the genes present in Region of Diversity 10 (RD10) are required for virulence during blood stream infection showed enhanced expression after passage. However, there was no significant decrease in the LD(50) of serial passage strains compare to single passage strain in a mouse challenge model. Overall, our data suggest that bacteria adapt to extended laboratory passage by substantially altering gene expression. Furthermore, extended passage on blood agar plates reduces the expression of genes associated with initial colonization and adherence but enhances the expression of genes needed for systemic infection.