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。     

Genome of the Bacterium Streptococcus pneumoniae Strain R6

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.     

Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC

The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.     

Activity of the two-component regulatory system CiaRH in Streptococcus pneumoniae R6

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a variety of processes such as competence development, autolysis, bacteriocin production, host colonization, and virulence. While the targets of the regulator CiaR are known, the role of phosphorylation in CiaR regulation has not been defined. To address this issue, the presumed phosphorylation site of CiaR, aspartic acid at position 51, was replaced by alanine. The mutant CiaRD51A protein was no longer able to activate CiaR-dependent promoters, strongly suggesting that the phosphorylated form of CiaR is active in regulation. However, depending on the growth medium, inactivation of the kinase gene ciaH resulted in a subtle increase of CiaR-dependent promoter activities or in a strong reduction. Therefore, CiaH may act as a kinase or phosphatase and CiaR is apparently able to obtain its phosphate independently of CiaH. On the other hand, promoter measurements in cells with an intact CiaRH system demonstrated a high, nearly constitutive, expression level of the CiaR regulon independent from the growth medium. Thus, in contrast to many other two-component regulatory systems, CiaRH has apparently evolved to maintain high levels of gene expression under a variety of conditions rather than responding strongly to a signal.     

Albomycin uptake via a ferric hydroxamate transport system of Streptococcus pneumoniae R6

The antibiotic albomycin is highly effective against Streptococcus pneumoniae, with an MIC of 10 ng/ml. The reason for the high efficacy was studied by measuring the uptake of albomycin into S. pneumoniae. Albomycin was transported via the system that transports the ferric hydroxamates ferrichrome and ferrioxamine B. These two ferric hydroxamates antagonized the growth inhibition by albomycin and salmycin. Cross-inhibition of the structurally different ferric hydroxamates to both antibiotics can be explained by the similar iron coordination centers of the four compounds. [(55)Fe(3+)]ferrichrome and [(55)Fe(3+)]ferrioxamine B were taken up by the same transport system into S. pneumoniae. Mutants in the adjacent fhuD, fhuB, and fhuG genes were transport inactive and resistant to the antibiotics. Albomycin, ferrichrome, ferrioxamine B, and salmycin bound to the isolated FhuD protein and prevented degradation by proteinase K. The fhu locus consisting of the fhuD, fhuB, fhuG, and fhuC genes determines a predicted ABC transporter composed of the FhuD binding lipoprotein, the FhuB and FhuG transport proteins, and the FhuC ATPase. It is concluded that active transport of albomycin mediates the high antibiotic efficacy in S. pneumoniae.     

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.     

Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria

In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.     

Streptococcus pneumoniae Serine Protease HtrA,but Not SFP or PrtA,Is a Major Virulence Factor in Pneumonia

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.     

Effects of Low PBP2b Levels on Cell Morphology and Peptidoglycan Composition in Streptococcus pneumoniae R6

Streptococcus pneumoniae produces two class B penicillin-binding proteins, PBP2x and PBP2b, both of which are essential. It is generally assumed that PBP2x is specifically involved in septum formation, while PBP2b is dedicated to peripheral cell wall synthesis. However, little experimental evidence exists to substantiate this belief. In the present study, we obtained evidence that strongly supports the view that PBP2b is essential for peripheral peptidoglycan synthesis. Depletion of PBP2b expression gave rise to long chains of cells in which individual cells were compressed in the direction of the long axis and looked lentil shaped. This morphological change is consistent with a role for pneumococcal PBP2b in the synthesis of the lateral cell wall. Depletion of PBP2x, on the other hand, resulted in lemon-shaped and some elongated cells with a thickened midcell region. Low PBP2b levels gave rise to changes in the peptidoglycan layer that made pneumococci sensitive to exogenously added LytA during logarithmic growth and refractory to chain dispersion upon addition of LytB. Interestingly, analysis of the cell wall composition of PBP2b-depleted pneumococci revealed that they had a larger proportion of branched stem peptides in their peptidoglycan than the corresponding undepleted cells. Furthermore, MurM-deficient mutants, i.e., mutants lacking the ability to synthesize branched muropeptides, were found to require much higher levels of PBP2b to sustain growth than those required by MurM-proficient strains. These findings might help to explain why increased incorporation of branched muropeptides is required for high-level beta-lactam resistance in S. pneumoniae.     

Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors

Streptococcus pneumoniae is an important human pathogen and the most common cause of community-acquired pneumonia. Both adaptive and innate immune mechanisms provide protection from infection. Innate immunity to S. pneumoniae in mice is mediated by naturally occurring anti-phosphocholine (PC) Abs and complement. The human acute-phase reactant C-reactive protein (CRP) also protects mice from lethal S. pneumoniae infection. CRP and anti-PC Ab share the ability to bind to PC on the cell wall C-polysaccharide of S. pneumoniae and to activate complement. CRP and IgG anti-PC also bind to Fc gamma R. In this study, Fc gamma R- and complement-deficient mice were used to compare the mechanisms of protection conferred by CRP and anti-PC Ab. Injection of CRP protected wild-type, FcR gamma-chain-, Fc gamma RIIb-, and Fc gamma RIII-deficient mice from infection. Complement was required for the protective effect of CRP as cobra venom factor treatment eliminated the effect of CRP in both gamma-chain-deficient and wild-type mice, and CRP failed to protect C3- or C4-deficient mice from infection. Unexpectedly, gamma-chain-deficient mice were extremely sensitive to pneumococcal infection. This sensitivity was associated with low levels of natural anti-PC Ab. Gamma-chain-deficient mice immunized with nonencapsulated S. pneumoniae produced both IgM- and IgG PC-specific Abs, were protected from infection, and were able to clear the bacteria from the bloodstream. The protection provided by immunization was eliminated by complement depletion. The results show that in this model of systemic infection with highly virulent S. pneumoniae, protection from lethality by CRP and anti-PC Abs requires complement, but not Fc gamma R.     

Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator

PcsB is a protein of unknown function(s) that influences the cell morphology of several pathogenic species of streptococcus. PcsB contains a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain found in bacterial murein hydrolases; however, direct links between steps in cell wall biosynthesis and PcsB function(s) have not been demonstrated. We show here that pcsB is essential in the human respiratory pathogen, Streptococcus pneumoniae, that depletion of PcsB is bacteriostatic and that alanine substitutions in the conserved cysteine and histidine residues of the CHAP domain appear to be lethal. We stained wild-type parent and mutant bacteria deficient in expression of PcsB with fluorescent vancomycin and DAPI to determine patterns of cell wall synthesis and nucleoid segregation respectively. The wild-type parent strain exhibited ordered, simultaneous septal and equatorial cell wall synthesis. In contrast, reduced expression of PcsB resulted in formation of long chains of cells in which peptidoglycan synthesis occurred at nearly every division septum and cell equator. Severe depletion of PcsB led to abnormal, uncontrolled cell wall synthesis at misplaced septa and around large cells. Together, these physiological properties are consistent with a role for PcsB as a murein hydrolase that balances the extent of cell wall synthesis in S. pneumoniae. Finally, we show that the defects in morphology and cell wall synthesis that result from depletion of PcsB strongly resemble those caused by depletion of the essential VicRK two component regulatory system (TCS). This result and the essentiality of pcsB support the hypothesis that the essentiality of the VicRK TCS results from its positive regulation of PcsB expression.     

Virulence of Streptococcus pneumoniae serotype 6C in experimental otitis media

Increases in colonization with serotypes of Streptococcus pneumoniae not contained within the 7-valent pneumococcal conjugate vaccine (PCV) have been reported among children following introduction. Serotype 6C has emerged as prevalent in nasopharyngeal colonization and acute otitis media (AOM), though it is uncommonly recovered from children with invasive pneumococcal disease. Vaccine serotypes within PCV7 have been replaced by nonvaccine serotypes without significant changes in the overall carriage rate. We hypothesize 1) that serotypes vary in their ability to evade host defenses and establish AOM following colonization and 2) the observed reduction in pneumococcal otitis results from a reduced disease potential by some ‘replacement serotypes’. We compared the capacity of S. pneumoniae serotypes 6C and 19A to produce experimental otitis media (EOM) in a chinchilla model. The proportion of chinchillas that developed culture positive EOM and density of middle ear infection was evaluated. EOM was found in 28/82 (34%) ears challenged with 6C compared to 13/18(72.2%) with 19A [p = 0.0003]. When disease due to 6C did occur, it was characterized by low-density infection. Our findings demonstrate that challenge with serotype 6C results in EOM less frequently than 19A. These data support the need for greater knowledge regarding differences among serotypes to produce AOM.     

Resistance mechanism of chloramphenicol in Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis.

The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacetoxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.