Contribution of autolysin to virulence of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.     

Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.     

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.     

Identification of the psaA gene, coding for pneumococcal surface adhesin A, in viridans group streptococci other than Streptococcus pneumoniae

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.     

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans.

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.     

Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes.

The pneumococcal surface adhesin A (PsaA) is a surface-exposed protein of the gram-positive bacterium Streptococcus pneumoniae. It belongs to a group of proteins designated the lipoprotein receptor I antigen family. The gene encoding PsaA from an encapsulated strain of pneumococcal serotype 6B was cloned and sequenced. The peptide sequence was compared to that of homologs found in S. pneumoniae serotype 2, viridans streptococci, and Enterococcus faecalis. Identity values among the deduced peptides ranged from 57 to 98%. The polymorphism of psaA was examined among the 23 encapsulated vaccine serotypes by using PCR-restriction fragment length polymorphism analysis. Ten different enzymes were used to analyze 80 strains representing the 23 serotypes in a 23-valent polysaccharide vaccine. This analysis showed that restriction sites within the gene were highly conserved, with only a minor variation occurring in 10% of the strains, the result of an additional Tsp509I site. The lack of variation for the other restriction sites within the gene examined here indicates that psaA is genetically conserved, an important characteristic necessary for a candidate common protein vaccine.     

Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis

The gene encoding the pneumococcal surface adhesin A (PsaA) protein, psaA, was confirmed in all Streptococcus pneumoniae serotypes by a newly developed PCR (psaA PCR) assay. Eighty-nine of the 90 serotypes amplified produced an 838-bp fragment; the exception was a serotype 16F strain acquired from the American Type Culture Collection (ATCC). Analysis of 20 additional 16F strains from the United States and Brazil showed that the gene was amplified in all 16F strains, implying that the serotype 16F ATCC strain must be a variant. The specificity of the assay was verified by the lack of signal from analysis of heterologous bacterial species (n = 30) and genera (n = 14), including viridans group streptococci. The potential of the assay for clinical application was shown by its ability to detect pneumococci in culture-positive nasopharyngeal specimens. Demonstration of psaA in all 90 serotypes and lack of amplification of heterologous organisms suggest that this assay could be a useful tool for detection of pneumococci and diagnosis of disease.     

Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.

Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.     

Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide

PsaA of Streptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+ transporter. Mutations in psaA cause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. A psaBC deletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather, psaA mutations indirectly affect this process through the disruption of Mn2+ transport. The deficiency in Mn2+ transport also causes hypersensitivity to oxidative stress from H2O2 and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+ and Mn2+ cations after a lag of about 15 h. Addition of Mn2+ alone or together with Fe2+ allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+ alone extended the 15-h lag phase to 25 h. Thus, while Fe2+ adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2 was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.     

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] > IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.     

Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress

psaA encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex. Streptococcus pneumoniae D39 psaA mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that psaA and psaD mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in psaA and psaD mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.     

Diagnosis of pneumococcal pneumonia by psaA PCR analysis of lung aspirates from adult patients in Kenya

psaA is the gene encoding pneumococcal surface adhesin A (PsaA), a 37-kDa protein expressed on the surface of Streptococcus pneumoniae. PCR primers for psaA have been shown to amplify the target DNA sequence in all 90 serotypes of S. pneumoniae and in none of 67 heterologous pathogens and colonizing bacteria of the upper respiratory tract. Pathogenic bacteria identified in lung aspirate specimens cannot normally be dismissed as contaminants or colonizers, which limit the assay specificity of other respiratory tract specimens. psaA PCR analysis was evaluated in 171 lung aspirates from Kenyan adults with acute pneumonia. The limit of detection was one genome equivalent. Sensitivity, estimated in 35 culture-positive lung aspirates, was 0.83 (95% confidence interval, 0.70 to 0.95). psaA PCR analysis extended the number of identifications of S. pneumoniae in lung aspirates from 35 on culture to 61 by both methods. Of 26 new pneumococcal diagnoses, 19 were corroborated by results of blood culture or urine antigen detection. Sequences of the PCR products from 12 positive samples were identical to the psaA target gene fragment. Using an internal control for the PCR, inhibition of psaA PCR was demonstrated in 17% (8 of 47) of false-negative specimens. The results of a control PCR for the human gene beta-actin suggested that false-negative psaA PCR results are attributable to problems of specimen collection, processing, or DNA extraction in 30% of cases (14 of 47). psaA PCR analysis is a sensitive tool for diagnosis of pneumococcal pneumonia in adults.     

Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model

Considerable evidence has implicated Streptococcus pneumoniae neuraminidase in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, an S. pneumoniae neuraminidase-deficient mutant, DeltaNA1, has been constructed by insertion-duplication mutagenesis of the nanA gene of S. pneumoniae strain D39. The relative ability of DeltaNA1 and the D39 parent strain to colonize the nasopharynx and to induce OM subsequent to intranasal inoculation and to survive in the middle ear cleft after direct challenge of the middle ear were evaluated in the chinchilla model. Nasopharyngeal colonization data indicate a significant difference in the ability of the DeltaNA1 mutant to colonize as well as to persist in the nasopharynx. The neuraminidase-deficient mutant was eliminated from the nasopharynx 2 weeks earlier than the D39 parent strain. Both the parent and the mutant exhibited similar virulence levels and kinetics during the first week after direct inoculation of the middle ear. The DeltaNA1 neuraminidase-deficient mutant, however, was then completely eliminated from the middle ear by day 10 postchallenge, 11 days before the D39 parent strain. Data from this study indicate that products of the nanA gene have an impact on the ability of S. pneumoniae to colonize and persist in the nasopharynx as well as the middle ear.     

Infection with conditionally virulent Streptococcus pneumoniae Δpab strains induces antibody to conserved protein antigens but does not protect against systemic infection with heterologous strains

Avirulent strains of a bacterial pathogen could be useful tools for investigating immunological responses to infection and potentially effective vaccines. We have therefore constructed an auxotrophic TIGR4 Δpab strain of Streptococcus pneumoniae by deleting the pabB gene Sp_0665. The TIGR4 Δpab strain grew well in complete medium but was unable to grow in serum unless it was supplemented with para-aminobenzoic acid (PABA). The TIGR4 Δpab strain was markedly attenuated in virulence in mouse models of S. pneumoniae nasopharyngeal colonization, pneumonia, and sepsis. Supplementing mouse drinking water with PABA largely restored the virulence of TIGR4 Δpab. An additional Δpab strain constructed in the D39 capsular serotype 2 background was also avirulent in a sepsis model. Systemic inoculation of mice with TIGR4 Δpab induced antibody responses to S. pneumoniae protein antigens, including PpmA, PsaA, pneumolysin, and CbpD, but not capsular polysaccharide. Flow cytometry demonstrated that IgG in sera from TIGR4 Δpab-vaccinated mice bound to the surface of TIGR4 and D39 bacteria but not to a capsular serotype 3 strain, strain 0100993. Mice vaccinated with the TIGR4 Δpab or D39 Δpab strain by intraperitoneal inoculation were protected from developing septicemia when challenged with the homologous S. pneumoniae strain. Vaccination with the TIGR4 Δpab strain provided only weak or no protection against heterologous challenge with the D39 or 0100993 strain but did strongly protect against a TIGR4 capsular-switch strain expressing a serotype 2 capsule. The failure of cross-protection after systemic vaccination with Δpab bacteria suggests that parenteral administration of a live attenuated vaccine is not an attractive approach for preventing S. pneumoniae infection.     

Pneumococcal surface adhesin A (PsaA): a review

Pneumococcal surface adhesin A (PsaA) is a surface-exposed common 37-kilodalton multi-functional lipoprotein detected on all known serotypes of Streptococcus pneumoniae. This lipoprotein belongs to the ABC-type transport protein complex that transports Mn(2+); it is also an adhesin that plays a major role in pneumococcal attachment to the host cell and virulence. PsaA is immunogenic and natural nasopharyngeal colonization of pneumococci elicits an increase in antibody towards PsaA. Hence, PsaA is being actively evaluated as a component of a vaccine in formulations composed of pneumococcal common proteins. PsaA has been expressed as an E. coli recombinant protein, purified, and evaluated in a phase one clinical trial. This article reviews PsaA, its structure and role in pneumococcal virulence, immunogenicity, and potential to reduce nasopharyngeal colonization (a major prerequisite for pneumococcal pathogenesis) as a component of a common pneumococcal protein vaccine.     

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.     

Requirement for capsule in colonization by Streptococcus pneumoniae

Nasopharyngeal colonization is a necessary first step in the pathogenesis of Streptococcus pneumoniae. Using isolates containing defined mutations in the S. pneumoniae capsule locus, we found that expression of the capsular polysaccharide is essential for colonization by the type 2 strain D39 and the type 3 strains A66 and WU2. Nonencapsulated derivatives of each of these strains were unable to colonize BALB/cByJ mice. Similarly, type 3 mutants that produced < 6% of the parental amounts of capsule could not colonize. Capsule production equivalent to that of the parent strain was not required for efficient colonization, however, as type 3 mutants producing approximately 20% of the parental amounts of capsule colonized as effectively as the parent. This 80% reduction in capsule level had only a minimal effect on intraperitoneal virulence but caused a significant reduction in virulence via the intravenous route. In the X-linked immunodeficient CBA/N mouse, the type 3 mutant producing ~20% of the parental amount of capsule (AM188) was diminished in its ability to cause invasive disease and death following intranasal inoculation. Following intravenous or intraperitoneal challenge, however, only extended survival times were observed. Our results demonstrate an additional role for capsule in the pathogenesis of S. pneumoniae and show that isolates producing reduced levels of capsule can remain highly virulent.     

肺炎链球菌的转化缺陷导致细菌毒力减弱

的探讨肺炎链球菌的自然转化与其条件致病的关系。方法采用插入失活的方法制备肺炎链球菌的转化缺陷菌株，通过黏附实验和小鼠毒力实验观察它们毒力的变化，应用RT-PCR测定黏附因子PsaA在野生和缺陷菌株中的表达。结果实验获得了3株肺炎链球菌(1、2和22)的转化缺陷菌株(1d、2d和22d)。毒力研究发现转化缺陷菌株对ECV 304细胞的黏附能力显著弱于相应的野生菌株，1和2株肺炎链球菌腹腔感染BALB／c小鼠的能力显著强于相应的转化缺陷菌株；研究还发现PsaA基因在2和22株肺炎链球菌中的表达量显著高于其缺陷菌株。结论肺炎链球菌具有自然转化的能力有助于其毒力的表现，且可能通过黏附因子PsaA等相关毒力因子表达增加而增强其毒力。     

Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins

Although the polysaccharide capsule of Streptococcus pneumoniae has been recognized as a sine qua non of virulence, much recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The individual contributions of pneumolysin (Ply), the major neuraminidase (NanA), autolysin (LytA), hyaluronidase (Hyl), pneumococcal surface protein A (PspA), and choline-binding protein A (CbpA) have been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and comparing the impact on virulence in a mouse intraperitoneal challenge model. Mutagenesis of either the ply, lytA, or pspA gene in S. pneumoniae D39 significantly reduced virulence, relative to that of the wild-type strain, indicating that the respective gene products contribute to pathogenesis. On the other hand, mutations in nanA, hyl, or cbpA had no significant impact. The virulence of D39 derivatives carrying a ply deletion mutation as well as an insertion-duplication mutation in one of the other genes was also examined. Mutagenesis of either nanA or lytA did not result in an additional attenuation of virulence in the ply deletion background. However, significant additive attenuation in virulence was observed for the strains with ply-hyl, ply-pspA, and ply-cbpA double mutations.     

Contribution of a response regulator to the virulence of Streptococcus pneumoniae is strain dependent

Bacterial two-component signal transduction systems (TCS) enable bacteria to respond to environmental changes and regulate a range of genes accordingly. They have a crucial role in regulating many cellular responses and have excellent potential as antibacterial-drug targets. We have constructed mutations in a TCS response regulator gene for two different strains of the human pathogen Streptococcus pneumoniae. These mutants have been analyzed in our murine model of infection. Data suggest that in a D39 background the response regulator gene is essential for virulence; an isogenic mutant is avirulent via intraperitoneal, intranasal, and intravenous routes of infection. This mutant, which does not show impaired growth in vitro, is unable to grow in the lung tissue or in blood. Mutation of the response regulator in a 0100993 background results in a strain that is fully virulent intraperitoneally and intravenously but shows decreased levels of bacteremia and increased murine survival following intranasal infection. The ability to grow in the lung tissue is not impaired in this mutant, suggesting that it has an impaired ability to disseminate from the lungs to the systemic circulation. Our data highlight the importance of assessing the contribution of putative virulence factors to the infection process at different sites of infection and provide evidence that virulence determinants can behave very differently based on the genetic background of the bacterial strain. These important findings may be relevant to other bacterial pathogens.