Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin

Abstract

Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

FIG. 1.

Incidence of serotype 1 IPD in Scotland from 2000 to 2004. MLST data for 2001 to 2004 allow the sequence type distribution within serotype 1 isolates to be recorded. Black bars represent ST306, white bars represent ST227, and diagonally striped bars represent all other sequence types. The gray bar represents the total serotype 1 IPD in the year 2000.

FIG. 2.

Alignments of the amino acid sequence for ST227 and ST306 pneumolysin compared with the D39 pneumolysin sequence. The mutations are highlighted in gray. ST227 pneumolysin differs from D39 pneumolysin by 1 amino acid substitution (D380N). ST306 pneumolysin differs from D39 pneumolysin by 4 amino acid substitutions (Y150H, T172I, K224R, and A265S) and 2 amino acid deletions (V270K271).

FIG. 3.

Transmission electron microscopy of negatively stained erythrocyte membrane treated with filtered cell extract from an ST227 isolate (a) or cell extract from an ST306 isolate (b). Original magnification, ×25,000. Bar, 200 nm. The black arrow indicates a pore, white arrows show arcs that are inserted into the membrane, and gray arrows highlight arcs of toxin that have not inserted into the membrane.