Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3.

Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.     

Comparative efficacy of pneumococcal neuraminidase and pneumolysin as immunogens protective against Streptococcus pneumoniae

Neuraminidase and pneumolysin were purified from cultures of Streptococcus pneumoniae and used, either singly or in combination, to immunize juvenile mice which were subsequently challenged intranasally with virulent S. pneumoniae. In each of two independent trials, a small but significant (P less than 0.05) increase in survival time (compared with that of non-immunized mice) was observed in groups which had been immunized with neuraminidase, but only if the enzyme had been pre-treated with 3.4% (v/v) formaldehyde. The median extension in survival time was significantly less (P less than 0.01) than that of mice which had been immunized with pneumolysin alone. The median survival time for mice which had received both formaldehyde-treated neuraminidase and pneumolysin was not significantly different from that of mice which had received pneumolysin alone. While these findings provide direct evidence that neuraminidase contributes to the pathogenicity of the pneumococcus in mice, they suggest that this protein may be of less value than pneumolysin as a vaccine component in the present experimental model.     

Effect of defined point mutations in the pneumolysin gene on the virulence of Streptococcus pneumoniae.

The thiol-activated toxin pneumolysin is a known pneumococcal virulence factor, with both cytotoxic (hemolytic) and complement activation properties. Copies of the pneumolysin gene carrying defined point mutations affecting either or both of these properties were introduced into the chromosome of Streptococcus pneumoniae D39 by insertion-duplication mutagenesis. The virulences of these otherwise isogenic strains were then compared. There was no significant difference in either the median survival time or overall survival rate between mice challenged with D39 derivatives producing the wild-type toxin and those expressing a pneumolysin gene with an Asp-385-->Asn mutation, which abolishes the complement activation property. However, mice challenged with strains carrying either His-367-->Arg or Trp-433-->Phe plus Cys-428-->Gly mutations, which reduce hemolytic activity to approximately 0.02 and 0.0001% of the wild-type level, respectively, had significantly greater median survival times and overall survival rates than mice challenged with D39 derivatives expressing a wild-type pneumolysin gene. No additional reduction in virulence was observed when mice were challenged with a D39 derivative carrying Trp-433-->Phe, Cys-428-->Gly, and Asp-385-->Asn, rather than Trp-433-->Phe and Cys-428-->Gly, mutations in the pneumolysin gene. Thus, it appears that in the intraperitoneal challenge model, the contribution of pneumolysin to virulence is largely attributable to its hemolytic (cytotoxic) properties rather than to its capacity to activate complement. Interestingly, however, the amount of pneumolysin required for full virulence may be very small, as D39 derivatives carrying the Trp-433-->Phe mutation (which reduces hemolytic activity to 0.1% of the wild-type level) had intermediate virulence.     

Development of a vaccine against invasive pneumococcal disease based on combinations of virulence proteins of Streptococcus pneumoniae

Current global efforts are focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. One such strategy involves the use of one or more pneumococcal protein antigens common to all serotypes, to provide cheap, non-serotype-dependent protection. In this study, we evaluated the protective efficacy of immunization of mice with PdB (a pneumolysin toxoid), PspA, PspC (CbpA), PhtB, and PhtE in an invasive-disease model. The antigens were administered in alum adjuvant, either alone or in various combinations. Protection against intraperitoneal challenge with virulent type 2 and 6A strains was assessed in two murine strains. Our findings show that in some situations, different individual proteins gave the best (and worst) protection. However, in many cases, a synergistic/additive effect was seen by using multiple proteins even where the individual proteins showed little value by themselves. For instance, the median survival times for mice immunized with combinations of PdB and PspA, PdB and PspC, or PspA and PspC were significantly longer than those for mice immunized with any of the single antigens. To date, the combination of PdB, PspA, and PspC offers the best protection.     

Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization-intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.     

Detection of pneumolysin and autolysin genes among antibiotic resistant Streptococcus pneumoniae in invasive infections

Purpose: To detect the presence of autolysin and pneumolysin genes among Streptococcus pneumoniae strains isolated from different disease entities among Indian patients. The study also attempted to determine antimicrobial susceptibility of the isolates. Materials and Methods: A total of 24 S. pneumoniae isolates were checked for the presence of lytA gene coding for autolysin and ply gene coding for pneumolysin using polymerase chain reaction (PCR). All the isolates were subjected to susceptibility testing by disc diffusion method for 10 different therapeutically relevant antibiotics. Minimum inhibition concentration (MIC) was determined using broth dilution method for ampicillin, penicillin and ciprofloxacin. Results: Eleven isolates from ocular infections and 13 isolates from different invasive diseases showed susceptibility to most of the antibiotics tested except chloramphenicol and ciprofloxacin. Fifty percentage of the isolates showed resistance to chloramphenicol and ciprofloxacin. A moderate level of resistance of 18% was noted for cefepime and ceftriaxone. Only 6% of resistance was observed for amoxicillin and ceftazidime. MIC levels ranged from 0.015 to 1 μg/mL for ampicillin and only one isolate had an MIC of 1 μg/mL. The MIC levels for penicillin ranged from 0.062 to 4 μg/mL, wherein nine isolates showed high levels of MICs ranging from 2 to 4 μg/mL. Six isolates had a very high resistance levels for ciprofloxacin with MIC ranging from 32-128 μg/mL. The presence of lytA was observed in 23 out of 24 isolates tested whereas only 17 isolates were positive for pneumolysin. Four ocular isolates and one isolate from ear infection were negative for pneumolysin. Conclusion: Emerging resistance observed for cefepime and ceftriaxone might be due their increased and frequent usage nowadays. Presence of pneumolysin appears to be more critical for pathogenesis of invasive infections than the ocular infections. However, presence of lytA gene in all the isolates signifies that irrespective of site of isolation, kind of infection caused, autolysin is an obligate necessity for this organism.     

A C-terminal truncated mutation of licC attenuates the virulence of Streptococcus pneumoniae

LicC has been identified as a virulence factor of Streptococcus pneumoniae. However, its role in virulence is still not fully understood because deletion of licC is lethal for the bacterium. In this study, a mutant with 78-bp truncation at the C-terminus of licC was obtained from a signature-tagged mutagenesis (STM) library. The mutant was viable with a large reduction in enzymatic activity as CTP:phosphocholine cytidylyltransferase detected in vitro using a firefly luciferase assay. The mutation attenuated the adhesion and invasion of S. pneumoniae ST556 (serotype 19F) to epithelial cells by 72% and 80%, respectively, and increased the phagocytosis by macrophages for 16.5%, compared to the parental strain. When the mutation was introduced into the encapsulated D39 strain (serotype 2), it led to attenuated virulence in mouse models either by intranasal colonization or by intraperitoneal infection. In addition, the phosphocholine (PCho) on cell surface was decreased, and the choline binding proteins (CBPs) were impaired, which may explain the attenuated virulence of the mutant. These observations indicate that C-terminus of licC is accounted for the main activity of LicC in PCho metabolism and is essential for the virulence of S. pneumoniae, which provides a novel target for drug design against pneumococcal infection.     

Production and purification of Streptococcus pneumoniae hemolysin (pneumolysin).

Pneumolysin was found to be produced by 112 of 113 clinical isolates of Streptococcus pneumoniae and to be an intracellular hemolysin. A 10-liter-scale fermentor production and purification procedure was developed for this hemolysin. The culture was concentrated by filtration 10 times before centrifugation. The cellular content was purified by ion-exchange chromatography, covalent thiopropyl gel chromatography, and gel filtration. One batch operation resulted in 6 mg of highly purified pneumolysin, with a yield of 66% and a specific activity of 1,400,000 hemolytic units per mg. The pneumolysin had a molecular weight of 53,000 and an isoelectric point of 5.2. The purification method developed will be of value in future studies on this hemolysin.     

Role of novel choline binding proteins in virulence of Streptococcus pneumoniae

The choline binding proteins (CBPs) are a family of surface proteins noncovalently bound to the phosphorylcholine moiety of the cell wall of Streptococcus pneumoniae by a conserved choline binding domain. Six new members of this family were identified, and these six plus two recently described cell wall hydrolases, LytB and LytC, were characterized for their roles in virulence. CBP-deficient mutants were constructed and tested for adherence to eukaryotic cells, colonization of the rat nasopharynx, and ability to cause sepsis. Five CBP mutants, CbpD, CbpE, CbpG, LytB, and LytC, showed significantly reduced colonization of the nasopharynx. For CbpE and -G this was attributable to a decreased ability to adhere to human cells. CbpG, a putative serine protease, also played a role in sepsis, the first observation of a pneumococcal virulence determinant strongly operative both on the mucosal surface and in the bloodstream.     

临床分离肺炎链球菌的毒力基因及血清型分析

目的 了解肺炎链球菌的5种常见毒力基因(psaA、pspA、nanA、ply、lytA)在临床分离株中的分布情况.方法 以2006-2008年从临床分离133株肺炎链球菌为研究对象,采用PCR法对5种常见的毒力基因进行检测.结果 lytA、psaA、nanA、pspA、ply这5种基因在133株菌中的检测阳性率分别为94.7%、85.0%、84.2%、60.2%、82.7%;5种毒力基因在87株常见血清型菌中的阳性率分别为100%、87.4%、89.7%、67.8%、86.2%.结论 5种毒力基因在本地区肺炎链球菌中的阳性检测率均较高,在常见血清型菌株中的阳性检测率较其他血清型高.这5种基因是肺炎链球菌致病的主要毒力基因,可能作为新刑肺炎蛋白疫苗的候选基因.     

Pneumolysin is a key factor in misidentification of macrolide-resistant Streptococcus pneumoniae and is a putative virulence factor of S. mitis and other streptococci

We evaluated the applicability of ply PCR for confirmation of the identification of Streptococcus pneumoniae. lytA PCR, 16S rRNA sequencing, and amplified-fragment length polymorphism were used as reference methods. In contrast to the lytA gene, the ply gene proved to be not specific for S. pneumoniae. The presence of the ply gene in other streptococci, in particular Streptococcus mitis, suggests that pneumolysin plays a pathogenic role.     

Distribution and clonal relationship of cell surface virulence genes among Streptococcus pneumoniae isolates in Japan

Streptococcus pneumoniae resides on mucosal surfaces in the nasopharynx, where selection for horizontal transfer of antimicrobial resistance genes and virulence factors may provide a survival advantage. We investigated the distribution of genes for pneumococcal cell surface proteins and their correlations with multilocus sequence typing (MLST), Pneumococcal Molecular Epidemiology Network (PMEN) clones and antimicrobial resistance, to identify pneumococcal virulence factors predicting prevalent clones from 156 pneumococcal isolates recovered from adult patients with community-acquired pneumonia in Japan. Pneumococcal eno, pavA, piuA, cbpA and cbpG were present in all isolates, and hyl and piaA were distributed among the clinical isolates. In contrast, pneumococcal rlrA, pclA, psrP, nanC and pspA family 1-type genes were variably distributed and significantly associated with MLST (Wallace coefficients (W) were over 84%). Serotype was a weaker predictor of sequence type (W, 0.75) than vice versa (W, 0.97). A multiple logistic regression analysis adjusted to the presence of virulence genes, pspA family 1 genes and carriage serotypes revealed that pclA and rlrA correlated with PMEN clones and antimicrobial resistance, and are likely to contribute to the selection of prevalent clones.     

Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae.

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.     

Molecular characterization of genes encoding the quinolone resistance determining regions of Malaysian Streptococcus pneumoniae strains

Genes encoding the quinolones resistance determining regions (QRDRs) in Streptococcus pneumoniae were detected by PCR and the sequence analysis was carried out to identify point mutations within these regions. The study was carried out to observe mutation patterns among S. pneumoniae strains in Malaysia. Antimicrobial susceptibility testing of 100 isolates was determined against various antibiotics, out of which 56 strains were categorised to have reduced susceptibility to ciprofloxacin (>or=2 microg/mL). These strains were subjected to PCR amplification for presence of the gyrA, parC , gyrB and parE genes. Eight representative strains with various susceptibilities to fluoroquinolones were sequenced. Two out of the eight isolates that were sequenced were shown to have a point mutation in the gyrA gene at position Ser81. The detection of mutation at codon Ser81 of the gyrA gene suggested the potential of developing fluoroquinolone resistance among S. pneumoniae isolates in Malaysia. However, further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia.     

肺炎链球菌核酸疫苗在恒河猴体内的免疫原性研究

目的 研究肺炎链球菌溶血素(pneumolysin,PN)核酸疫苗在恒河猴体内的免疫原性.方法 将肺炎链球菌溶血素全长基因和切去羧基端33个核苷酸的基因分别插入到质粒pVAX1载体中,构建成Ppn和Ppnd两种核酸疫苗.采用体内电转染初免结合相应蛋白质抗原加强的策略免疫恒河猴.结果 切去肺炎链球菌溶血素基因羧基端33个核苷酸.表达的重组截短溶血素蛋白保留了抗原性但去除了溶血活性,从而保证了疫苗的安全性.给恒河猴肌肉内注射500μg核酸疫苗加电转染能诱导出特异性抗体免疫应答,用相应蛋白质加强免疫后血清抗体几何平均滴度提高了约4倍.结论 采用电转染技术结合蛋白质加强免疫的策略,能增强肺炎链球菌溶血素核酸疫苗在恒河猴体内的特异性抗体水平.     

Distribution of putative virulence genes in Streptococcus mutans strains does not correlate with caries experience

Streptococcus mutans, a member of the human oral flora, is a widely recognized etiological agent of dental caries. The cariogenic potential of S. mutans is related to its ability to metabolize a wide variety of sugars, form a robust biofilm, produce copious amounts of lactic acid, and thrive in the acid environment that it generates. The remarkable genetic variability present within the species is reflected at the phenotypic level, notably in the differences in the cariogenic potential between strains. However, the genetic basis of these differences is yet to be elucidated. In this study, we surveyed by PCR and DNA hybridization the distribution of putative virulence genes, genomic islands, and insertion sequences across a collection of 33 strains isolated from either children with severe early childhood caries (S-ECC) or those who were caries free (CF). We found this genetically diverse group of isolates to be remarkably homogeneous with regard to the distribution of the putative virulence genes and genetic elements analyzed. Our findings point to the role of other factors in the pathogenesis of S-ECC, such as uncharacterized virulence genes, differences in gene expression and/or enzymatic activity, cooperation between S. mutans strains or with other members of the oral biota, and host factors.     

Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae.

psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.