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Pneumolysin, a potent 53-kDa thiol-activated cytolysin produced by virtually all clinical isolates, is a proven virulence factor of Streptococcus pneumoniae (12, 13). Its cytolytic activity involves interaction with cholesterol in target cell membranes and insertion into the lipid bilayer, followed by oligomerization to form transmembrane pores, which bring about cell lysis. Erythrocytes are particularly susceptible to pneumolysin-induced lysis, but theoretically, the toxin can interact with any cell that has cholesterol in its plasma membrane. Indeed, in vitro studies with purified toxin have demonstrated detrimental effects on a wide range of cells and tissues, even at sublytic concentrations (12, 13). However, pneumolysin is a bifunctional toxin, and in addition to its cytotoxic properties, it is capable of directly activating the classical complement pathway in the absence of specific antibody, with a concomitant reduction in serum opsonic activity (14). This is mediated by its capacity to bind directly to the Fc region of human immunoglobulin G (IgG) (10).

Structure-function analysis of pneumolysin has demonstrated that a domain towards the C terminus of the toxin which includes the unique cysteine residue (amino acids [aa] 427 to 437) is critical for cytotoxicity. This cysteine motif is highly conserved among other members of the thiol-activated cytolysin family. Several single amino acid substitutions within this region reduce the cytotoxicity of pneumolysin by up to 99.9% (8, 17). A separate region, which has a degree of amino acid homology with human complement (C)-reactive protein, is responsible for IgG binding and complement activation, and a mutation in this domain (Asp385Asn) inhibits IgG binding and abolishes complement activation (10).

Inactivation of the pneumolysin gene (ply) by insertion-duplication mutagenesis in either a type 2 or a type 3 strain reduced virulence of S. pneumoniae for mice challenged by either the intranasal (i.n.) or intraperitoneal (i.p.) route (6, 7). Subsequent studies demonstrated that i.n. or intratracheal (i.t.) challenge with ply-negative pneumococci resulted in a less-severe inflammatory response, a reduced rate of multiplication within the lung, a reduced capacity to injure the alveolar-capillary barrier, and a delayed onset of bacteremia compared with those of the wild-type strain (9, 16). Intravenously (i.v.) administered ply-negative pneumococci also exhibited a reduced rate of multiplication in the blood relative to that of the wild-type strain (7). Benton et al. (3) subsequently reported that i.v. challenge of mice with the ply-negative mutant resulted in a chronic becteremia, with numbers of pneumococci in the blood remaining at, or below, 10⁷ CFU per ml for a week in some mice. i.v. administration of an identical dose of the wild-type pneumococcus, however, resulted in fulminant infection; mice invariably died within 28 h, at which time there were approximately 10⁹ to 10¹⁰ CFU per ml of blood. Benton et al. concluded that during the first few hours of bacteremia pneumolysin plays a critical role by preventing the generation of inflammation-based immunity, thereby permitting continued exponential net growth of pneumococci (3).

We have also constructed a series of derivatives of the type 2 S. pneumoniae strain D39 in which the wild-type ply gene has been replaced by mutated genes encoding toxins with defined point mutations affecting either or both of the cytotoxic and complement activation properties (5). In an i.p. challenge model, pneumococci carrying two ply point mutations (Cys428Gly and Trp433Phe) resulting in production of a toxin with only 0.001% residual cytolytic activity was much less virulent than wild-type D39. A D39 derivative producing pneumolysin with just the Trp433Phe mutation (with 0.1% residual cytolytic activity) was only marginally (albeit statistically significantly) less virulent than the wild-type strain. Interestingly, in this model system, pneumococci carrying the Asp385Asn ply mutation, which abolishes complement activation, was as virulent as the wild-type strain. However, by using the same pneumococcal mutants, distinct roles have been demonstrated for both toxin properties in the pathogenesis of bronchopneumonia and lobar pneumonia with mouse i.n. and i.t. challenge models. In both models, strains producing pneumolysin lacking either property were less virulent than the wild type (1, 15). In the i.t. challenge model, cytotoxic activity was required for damage to the alveolar-capillary barrier and for optimal bacterial multiplication in the alveoli and lung tissue during the first 6 h of infection. However, the complement activation property was associated with increased numbers of pneumococci in lung tissue and the blood 24 h after infection (15). In the i.n. challenge model, the complement activation property was important for growth of pneumococci in both the lungs and the blood 6 to 24 h after challenge, while the cytotoxic property was associated with increased numbers of pneumococci in the lungs after 24 h (1).

An S. pneumoniae D39 derivative carrying three ply point mutations (Asp385Asn, Cys428Gly and Trp433Phe) abrogating both complement activation and cytolytic properties of the toxin has been shown to be less virulent than strains carrying either Asp385Asn or Cys428Gly and Trp433Phe substitutions in both i.n. and i.v. challenge models (1, 4). However, in both cases, the triple point mutant was significantly more virulent than a D39 derivative in which ply was disrupted by insertion-duplication mutagenesis, leading to the suggestion that pneumolysin has an additional property which contributes to pathogenesis.

The above observations could, of course, be explained by polar effects caused by integration of the insertion-duplication mutagenesis vector pVA891 into ply, although there are no open reading frames immediately downstream of ply in the S. pneumoniae genome. Moreover, construction of the various D39 ply point mutants also involved insertion of pVA891 immediately downstream of the intact ply open reading frame; these constructs included a control strain in which insertion of pVA891 reconstituted a wild-type ply gene, and this strain was fully virulent (1, 5). An alternative explanation for the lower virulence of the ply insertion-duplication mutant could be the production of a truncated gene product, or a ply-vector encoded fusion protein, which is toxic for the pneumococcus. The insertion-duplication mutant was constructed with a derivative of pVA891 containing a 695-bp Sau3A fragment from the middle of ply cloned into the BamHI site of the vector. This was transformed into S. pneumoniae and integrated into the chromosome by a single recombination event (7). Translation of the interrupted gene would be expected to result in a fusion protein containing the first 322 aa of pneumolysin plus an indeterminate number of vector-encoded amino acids. Theoretically, fusions containing vector-encoded amino acids plus the C-terminal 380 aa of pneumolysin could also result from readthrough from vector sequences into the 3' portion of ply. Furthermore, smaller truncated pneumolysin C-terminal fragments could result from initiation at internal ATG codons in the distal portion of ply.

In the present study we have eliminated these complications by constructing a D39 derivative with an in-frame ply deletion, and we compared its virulence to that of the wild-type strain and derivatives carrying insertion-duplication and point mutations in ply.

Bacterial strains. The virulent type 2 S. pneumoniae strain D39 (NCTC 7466) has been described previously (2). D39 derivatives in which ply has been disrupted by insertion-duplication mutagenesis (designated PLN-A) or in which the wild-type ply has been replaced by a mutated gene encoding pneumolysin with three amino acid substitutions (Asp385Asn, Cys428Gly and Trp433Phe) abrogating both complement activation and cytolytic properties of the toxin (designated PdT) have also been described previously (5, 7).

Virulence studies. In order to compare the virulence of the various S. pneumoniae strains, D39, PLN-A, PdT, and Ply were grown overnight on blood agar (supplemented with 0.2 µg of erythromycin/ml in the case of PLN-A and PdT), inoculated into serum broth (meat extract broth plus 10% horse serum), and incubated at 37°C for 3 h. Production of type 2 capsule was confirmed by quellung reaction by using antisera obtained from Statens Seruminstitut, Copenhagen, Denmark. In vitro growth rates of the three strains were also identical (result not shown). Cultures were then diluted to a density of 5 × 10⁴ CFU/ml, and 0.1-ml volumes were injected i.p. into groups of 12 to 13 BALB/c mice. The survival time of each mouse was recorded (Fig. 1), and differences in median survival time between groups were analyzed using the Mann-Whitney U test (two tailed). Eleven of the 12 mice challenged with the wild-type S. pneumoniae D39 succumbed to the challenge, with a median survival time of 0.9 days. Although all of the mice challenged with PdT succumbed, the median survival time (2.0 days) was significantly greater than that for the D39 group (P < 0.001). The PLN-A and Ply groups had median survival times of 2.8 and 13.8 days, respectively, both of which were significantly greater than that for either the D39 group (P < 0.001 in both cases) or the PdT group P < 0.05 and P < 0.002, respectively). Interestingly, the difference in median survival time between the PLN-A and Ply groups also reached statistical significance (P < 0.05). However, when the challenge experiment was repeated at a slightly lower dose (5 × 10² CFU) no significant difference in survival times between the PLN-A and Ply groups was observed. In this experiment, the median survival times were 8.9 and 7.0 days, respectively; overall survival rates were 6 of 12 and 5 of 12, respectively (result not presented).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Survival time of mice after i.p. challenge. Groups of 12 to 13 BALB/c mice were injected i.p. with approximately 5 × 103 CFU of the indicated strains. The survival time of each mouse is indicated. The broken lines denote the median survival time for each group.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Numbers of S. pneumoniae in tissues after i.p. challenge. Groups of 16 BALB/c mice were challenged i.p. with 5 × 102 CFU of D39, PLN-A, PdT, or Ply. Four mice chosen at random from each group were sacrificed 1, 2, 3, or 4 days after challenge (where there were sufficient survivors). Numbers of each strain in blood (expressed as log10 CFU/ml) or in homogenates of lungs, liver, spleen, or brain (expressed as log10 CFU/g of tissue) were determined as described in the text. Data are the means for each group, with the standard errors indicated by the bars.

Conclusions. In the present study we have examined the comparative virulence of the type 2 S. pneumoniae strain D39 and derivatives containing insertion-duplication, deletion, or point mutations in the ply gene. Virulence was assessed on the basis of survival time after i.p. challenge and the numbers of pneumococci in blood, lungs, brain, liver, or spleen at various times after challenge. PdT, which produces pneumolysin with three amino acid substitutions (Asp385Asn, Cys428Gly, and Trp433Phe) abrogating both complement activation and cytolytic properties of the toxin, was less virulent than the wild-type D39 but was more virulent than either the ply insertion-duplication mutant PLN-A or the ply deletion mutant Ply. A difference between the virulence of PdT and PLN-A has been observed previously with i.n. or i.v. challenge models (1, 4), but this could have been due to polar effects of the pVA891-mediated insertion-duplication event or perhaps to toxicity of truncated pneumolysin polypeptides or fusion proteins resulting from insertion of plasmid sequences into ply. Both these possibilities have been eliminated by the present study as in the i.p. challenge model the virulence of Ply is similar to that of PLN-A.