肺炎链球菌粘附和毒力因子A研究进展

肺炎链球菌（Streptococcus pneumoniae,SP）普遍定植于呼吸道,是人类重要的侵袭性病原菌之一,是社区获得性肺炎、中耳炎、脑膜炎、菌血症、鼻窦炎的主要病原菌。肺炎链球菌粘附和毒力因子A（pneumococcal adherence and virulence factor A,PavA）是肺炎链球菌早期感染和侵袭过程中关键的毒力因子。体外试验表明,缺失PavA的肺炎链球菌的突变株其粘附和侵入上皮细胞和内皮细胞的能力明显下降。作为一种保护性抗原,其诱导的细胞和体液免疫可以有效的抵抗肺炎链球菌的感染,是肺炎链球菌新一代疫苗的候选蛋白。但是,PavA在肺炎链球菌与人肺上皮细胞交互对话中作用机制的研究尚属空白,本文就肺炎链球菌粘附和毒力因子A得最新研究进展作一综述。     

Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in Streptococcus gordonii Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.     

Drastic reduction in the virulence of Streptococcus pneumoniae expressing type 2 capsular polysaccharide but lacking choline residues in the cell wall

The role of capsular polysaccharides and several virulence-related proteins in the pathogenic potential of Streptococcus pneumoniae has been studied extensively. Much less information is available about the role of the pneumococcal cell wall in virulence. In this communication we describe an experimental system that has allowed us to test - in a global way - the role of choline, a structural component of the pneumococcal cell wall, in virulence. We constructed double mutants of S. pneumoniae which have lost the auxotrophic requirement for choline and which were also blocked from utilizing choline from the growth medium. Such a double mutant expressing type 2 capsule but completely lacking choline residues from its cell wall grew well both in vitro and also in the blood of infected mice, but showed striking reduction of virulence approaching that of a capsule-free strain in several models of pneumococcal disease including the capacity to attach and invade a human nasopharyngeal cell line; nasal colonization and intraperitoneal and intravenous inoculation in the mouse. The findings allow one to separate the choline requirement of S. pneumoniae into two sharply defined classes: the need for choline in growth and replication which can be effectively bypassed and the need for choline in pneumococcal virulence that appears to be irreplaceable. The double mutant should be a useful experimental tool to dissect the mechanism of choline requirement in various stages of pneumococcal virulence.     

Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.     

Mapping the recognition domains of pneumococcal fibronectin‐binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin‐binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non‐human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both β‐sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII¹, FnIII⁵ or FnIII¹⁵ bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.     

SP0454, a putative threonine dehydratase, is required for pneumococcal virulence in mice

Increasing pressure in antibiotic resistance and the requirement for the design of new vaccines are the objectives of clarifying the putative virulence factors in pneumococcal infection. In this study, the putative threonine dehydratase sp0454 was inactivated by erythromycin-resistance cassette replacement in Streptococcus pneumoniae CMCC 31203 strain. The sp0454 mutant was tested for cell growth, adherence, colonization, and virulence in a murine model. The Δsp0454 mutant showed decreased ability for colonization and impaired ability to adhere to A549 cells. However, the SP0454 polypeptide or its antiserum did not affect pneumococcal CMCC 31203 adhesion to A549 cells. The sp0454 deletion mutant was less virulent in a murine intranasal infection model. Real-time RT-PCR analysis revealed significant decrease of the pneumococcal surface antigen A expression in the sp0454 mutant. These results suggest that SP0454 contributes to virulence and colonization, which could be explained in part by modulating the expression of other virulence factors, such as psaA in pneumococcal infection.     

猪链球菌2型的纤连蛋白/血纤维蛋白原结合蛋白基因的序列分析

目的 为确定猪链球菌2型(SS2)江苏分离株9801是否具有纤连蛋白／血纤维蛋白原结合蛋白基因(fbps)。方法 根据已发表的SS2 fbps序列,设计并合成1对引物,以SS2江苏分离株的基因组为模板,采用聚合酶链反应(PER)方法扩增fbps,克隆于pMD-T18载体。测定阳性质粒插入序列。利用DNAstar软件,比较所测序列与不同来源SS2的fbps和FBPS与链球菌属其他种同源蛋白的同源性。结果 扩增的fbps为1 938 bp,江苏分离株与猪源致病性SS2荷兰分离株的fbps序列有5个碱基不同,与ATCCA3765株有3个碱基不同,同源性均达99．99％以上。推导的氨基酸序列比较,分别有4个和2个氨基酸不同,同源性均为99％以上。纤连蛋白结合蛋白(FBPS)无前导序列,无锚定序列,与链球菌属其他种的同源Fn结合蛋白的同源性为68．8％～76．0％。结论 FBPS是一种无锚的黏附素。     

Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene.

Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.     

The cell wall-associated serine protease PrtA: a highly conserved virulence factor of Streptococcus pneumoniae

The surface-associated subtilisin-like serine protease PrtA was identified by screening a genomic expression library from Streptococcus pneumoniae using a convalescent-phase serum. In Western blot analysis two forms of PrtA were detected in whole cell lysate and a truncated form only in culture supernatant suggesting that PrtA is produced as a precursor protein, translocated to the cell surface, truncated, and released into the surroundings. A 5' fragment of the gene was found highly conserved among 78 pneumococcal isolates of clinical relevance. Immunogenicity of PrtA, limited genetic variation, and the involvement in pneumococcal virulence demonstrated in in vivo experiments might identify PrtA as a promising candidate for a protein based vaccine.     

The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains

A biochemical approach to identify proteins with high affinity for choline-containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC ) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted M r of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was purified from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 30 degrees C. The DNA region encoding the 253 C-terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but significant, choline-independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self-alignment of the N-terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline-binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline-binding domain and the catalytic domain are located, respectively, at the N-terminal and the C-terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline-binding proteins to fuse separated catalytic and substrate-binding domains and create new and functional mature proteins.     

The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8-11 and SCR19-20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8-11 and SCR19-20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38-158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8-11 and SCR19-20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.     

SpsA, a novel pneumococcal surface protein with specific binding to secretory Immunoglobulin A and secretory component

The interaction of pathogenic bacteria with host serum and matrix proteins is a common strategy to enhance their virulence. Streptococcus pneumoniae colonizes the human upper respiratory tract in healthy individuals and is also able to cause invasive diseases. Here, we describe a novel pneumococcal surface protein, SpsA, capable of binding specifically to human secretory immunoglobulin A (SIgA). The dissociation constant of SIgA binding to SpsA was 9.3 × 10⁻⁹ M. Free secretory component (SC) also binds to S . pneumoniae , whereas serum IgA does not, suggesting that pneumococcal binding to SIgA is mediated by the SC. To our knowledge, this is the first defined interaction of SC with a prokaryotic protein. The spsA gene encodes a polypeptide of 523 amino acids with a predicted molecular mass of 59 151 Da. The SIgA- or SC-binding domain is located in the N-terminal part of SpsA and exhibits no significant homology to any other proteins. The purified SIgA-binding domain of SpsA could completely inhibit the binding of SIgA to pneumococci. SpsA was expressed by 73% of the tested S . pneumoniae isolates and was substantially conserved between different serotypes. The interaction between S . pneumoniae and SC via SpsA represents a novel biological interaction that might increase virulence by the impairment of bacterial clearance.     

clpE基因缺失对肺炎链球菌毒力的影响

【目的】 探索clpE基因缺失对肺炎链球菌毒力的影响。【方法】 用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpE基因,用PCR、测序鉴定缺失菌株,通过动物实验观察clpE基因缺失株毒力改变情况, 同时用细胞实验比较clpE基因缺失株和野生菌对宿主细胞的粘附和侵袭能力,最后用实时荧光定量PCR分析自溶素(major autolysin A,lytA)、表面黏附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、肺炎球菌表面蛋白A（pneumococcal surface protein A, pspA)和神经氨酸酶（neuraminidase, nanA）的表达。 【结果】小鼠毒力实验表明野生菌株半数致死时间54h,而缺失株半数致死时间为21d,两者比较有统计学差异（P<0 .0l）;缺失菌在对宿主细胞的粘附能力明显低于野生菌株（P<0.05）。实时荧光定量PCR显示clpE缺失株的五个毒力因子mRNA表达水平均低于野生菌,两者比较有统计学差异（P<0. 05）;【结论】ClpE通过调控肺炎链球菌多种毒力因子表达,而影响其毒力。     

Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.     

Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage

Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.     

A solute binding protein of Streptococcus pneumoniae iron transport

Streptococcus pneumoniae causes considerable morbidity and mortality throughout the world. Iron acquisition is an important virulence factor for bacterial pathogens. Two loci, piu and pia, were identified as responsible for the hemoglobin utilization of S. pneumoniae. The binding activity and surface accessibility of the solute binding protein of PiuA were studied. PiuA is a lipoprotein, binds hemin and hemoglobin, resides on the cytoplasmic membrane, and is not exposed on the surface of S. pneumoniae. The localization of PiuA has implications in its role in hemoglobin utilization and possible use as a pneumococcal vaccine.     

The use of microarray technology for the analysis of Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen associated with pneumonia, septicaemia, meningitis and otitis media. It is estimated to result in over 3 million child deaths worldwide every year and an even greater number of deaths among the elderly. Prior to the complete sequencing of the genomes of S. pneumoniae TIGR4 (serotype 4) and S. pneumoniae R6 (serotype 2), we designed a custom miniarray consisting of 497 pneumococcal genes. The overall objectives of our microarray investigations were, first, to assess the genetic diversity between different S. pneumoniae serotypes, clinical isolates and also different Streptococcus species; second, we aimed to use microarray technology to examine the mechanisms by which environmental factors influence pneumococcal gene expression, and ultimately to further the understanding of how these changes in gene expression are achieved and how they may alter the virulence of the organism.     

Characterization of selected strains of pneumococcal surface protein A.

Several proteins, in addition to the polysaccharide capsule, have recently been implicated in the full virulence of the Streptococcus pneumoniae bacterial pathogen. One of these novel virulence factors of S. pneumoniae is pneumococcal surface protein A (PspA). The N-terminal, cell surface exposed, and functional part of PspA is essential for full pneumococcal virulence, as evidenced by the fact that antibodies raised against this part of the protein are protective against pneumococcal infections. PspA has recently been implicated in anti-complementary function as it reduces complement-mediated clearance and phagocytosis of pneumococci. Several recombinant N-terminal fragments of PspA from different strains of pneumococci, Rx1, BG9739, BG6380, EF3296, and EF5668, were analyzed using circular dichroism, analytical ultracentrifugation sedimentation velocity and equilibrium methods, and sequence homology. Uniformly, all strains of PspA molecules studied have a high alpha-helical secondary structure content and they adopt predominantly a coiled-coil structure with an elongated, likely rod-like shape. No beta-sheet structures were detected for any of the PspA molecules analyzed. All PspAs were found to be monomeric in solution with the exception of the BG9739 strain which had the propensity to partially aggregate but only into a tetrameric form. These structural properties were correlated with the functional, anti-complementary properties of PspA molecules based on the polar distribution of highly charged termini of its coiled-coil domain. The recombinant Rx1 PspA is currently under consideration for pneumococcal vaccine development.     

Species-specific interaction of Streptococcus pneumoniae with human complement factor H

Streptococcus pneumoniae naturally colonizes the nasopharynx as a commensal organism and sometimes causes infections in remote tissue sites. This bacterium is highly capable of resisting host innate immunity during nasopharyngeal colonization and disseminating infections. The ability to recruit complement factor H (FH) by S. pneumoniae has been implicated as a bacterial immune evasion mechanism against complement-mediated bacterial clearance because FH is a complement alternative pathway inhibitor. S. pneumoniae recruits FH through a previously defined FH binding domain of choline-binding protein A (CbpA), a major surface protein of S. pneumoniae. In this study, we show that CbpA binds to human FH, but not to the FH proteins of mouse and other animal species tested to date. Accordingly, deleting the FH binding domain of CbpA in strain D39 did not result in obvious change in the levels of pneumococcal bacteremia or virulence in a bacteremia mouse model. Furthermore, this species-specific pneumococcal interaction with FH was shown to occur in multiple pneumococcal isolates from the blood and cerebrospinal fluid. Finally, our phagocytosis experiments with human and mouse phagocytes and complement systems provide additional evidence to support our hypothesis that CbpA acts as a bacterial determinant for pneumococcal resistance to complement-mediated host defense in humans.