KR100814616B1 - ClpP VACCINE COMPRISING RECOMBINANT CLPP PROTEIN OF STREPTOCOCCUS PNEUMONIAE - Google Patents

Abstract

The present invention relates to a vaccine comprising a recombinant ClpP protein derived from pneumococcal as an antigen, which is an antigenic protein with high immunogenicity and conservatively present in all pneumococci, and thus has low immunogenicity or protection against all serotypes. It can be effectively used as an effective vaccine against pneumococcal infection without showing the disadvantages of existing pneumococcal infection prevention vaccines that have no efficacy.

Pneumococcal, Recombinant, ClpP, Protein, Antigen, Vaccine

Description

Vaccine containing recombinant pneumococcal ClpP protein {VACCINE COMPRISING RECOMBINANT CLPP PROTEIN OF STREPTOCOCCUS PNEUMONIAE}

The present invention relates to a vaccine comprising recombinant ClpP (Caseinolytic protease P) protein of Streptococcus pneumoniae as an antigen.

Streptococcus pneumoniae is a transformable Gram-positive bacterium that causes a variety of infections in humans and animals, such as bacterial pneumonia, otitis media, bacteremia, and meningitis [Willett, HP 1992. Streptococcus pneumoniae . In Zinsser Microbiology . Joklik, WK, Willet, HP, Amos, DB and Wilfert, CM, (eds). Prentice-Hall International, London, pp. 432-442]. Pneumococcal infection is difficult to treat with antibiotics due to the emergence of multidrug-resistant bacteria. In order to prevent pneumococcal infections, currently available 23-valent polysaccharide vaccines [Pneumovax 23 (Merck) and Pnu-Imune 23 (Wyeth-Lederle)] have capsular polysaccharides (APS) as effective antigens, but have low antibody production rates in infants and young children. The disadvantage is that it does not work and there is no memory response. The 7-valent conjugate vaccine [Prevnar (Wyeth-Lederle)] developed to solve the disadvantages of the 23-valent vaccine was prepared by conjugating 7 CPSs to a carrier protein. As it has a protective effect against only seven types of pneumococci, its use as a prophylactic vaccine for pneumococcal infection is very limited. Therefore, attempts have been made to develop vaccines using high antigenic proteins to prevent pneumococcal infections. Pneumolysin (Ply) toxoid is known as a virulence factor of pneumococcal that binds to the cholesterol of host cells and attempts to develop attenuated pneumolysine (PdB) as a vaccine. However, pneumolysine is highly toxic in vivo and in vitro and has no effect on PdB alone, such as pneumococcal surface protein A (PspA), choline binding protein (CbpA), pneumococcal surface adhesion A (PsaA), LytA, etc. increased the survival rate for pneumococcal infections only (Ogunniyi, AD et al., 2000, Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae .Infect. Immun. 68: 3028-3033). As such, candidate antigenic proteins of existing pneumococcal infection prevention vaccines have low immunogenicity or do not have protection against all serotypes, and thus have high immunogenicity and are conservatively present in all pneumococci. The development of a vaccine is required.

Pneumococci are present in the nasopharynx of healthy individuals, which is the main reservoir for pneumococcal infections. Pneumococci are subject to various environmental stresses in vivo. Just as pneumococci penetrate the bloodstream in the nasopharynx, changes in environmental niche in the host can cause not only changes in gene expression but also rapid morphological changes. For example, nasopharyngeal pneumococci have been shown to be predominantly transparent colony phenotypes and to tend to express small amounts of capillaries and large amounts of choline binding protein A (CbpA). On the other hand, pneumococci in the bloodstream are predominantly opaque colonies and tend to produce large amounts of capillaries and small amounts of CbpA (Kim, J. 0. et al., 1998, Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae.J . Infect.Dis. 177: 368-377). In addition, pneumococci can be found in the nasal mucosa (30-34 ° C.) (Lindemann, J. et. Al., 2002, Nasal mucosal temperature during respiration. Clin. Otolaryngol. 27: 135-139) during the pathogenesis. After infiltrating at (37 ° C), it is subjected to thermal stress. This temperature change can serve as an important reason for the rapid and temporary increase in the synthesis of a highly conserved protein set called heat shock protein (HSP) (Neidhardt, FC et. Al.,, And RA Van Bogelen. 1987. .. Heat shock response, p 1334-11345 In FC Neidhardt, JL Ingraham, KB Low, B. Magasanik, M. Schaechter, and HE Umbarger (ed.), E coli and Salmonella typhimurium:.. Cellular and molecular biology ASM Press , Washington, DC). Induction of HSP by elevated temperature, or by exposure to ethanol, oxidative stress, or heavy metals, functions to protect bacteria against these adverse effects, thereby increasing the survival rate of bacteria. Thus, a full understanding of the thermal shock response can provide useful information about the adaptation of pneumococci to the hostile environment that pneumococci face.

HSPs are classified into Hsp100, Hsp70, Hsp60, and small Hsp families by molecular weight and are present everywhere in prokaryotes and eukaryotes. A type of HSP hsp100 / Clp (casein protease solubility: c l asein ytic p rotease) family is the eukaryotic protein 104kDa present in the form of, but is present as 80 to 95kDa protein in prokaryotes. It functions as a chaperone and also participates in proteolysis to remove damaged and denatured proteins. Protein degradation by Clp requires a serine-type peptidase ClpP subunit and a regulatory ATPase subunit (Schirmer, EC et. Al., 1996, HSP100 / Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21: 289-296). Regulatory Clp subunit proteins generally comprise two classes, class I and clpM, N, X, and Y, which include clpA, B, C, and D and contain two ATP binding regions and only one It can be designated as Class II containing only ATP binding regions. Clp is classified by the size of the central spacer segment, the need for gaps in the alignment of the entire sequence, and sequence similarity in well-conserved regions and N- and C-terminal segments, with variable leader regions having very different sequences in each subfamily. (Schirmer, EC et al., 1996, supra).

Significant advances have been made in understanding the mechanism of action of the Clp family in Gram-negative bacteria, such as E. coli, but little is known about the Clp of Gram-positive bacteria. The clpP gene and clpC operon are negatively regulated by CtsR, which recognizes a directly repeated effector sequence (A / GGT CAA ANA NA / GG TCA AA), but clpX does not have these sequences and their specific mechanism of action No detail has been determined (Derre, I. Et. Al., 2000, The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37 C. Mol. Microbiol. 38: 335-347).

Since various environmental signals, including temperature and nutrient availability, can regulate the expression of virulence factors, in previous studies we examined the protein profile of the thermal shock response in pneumococci after exposing the cells to various stresses. The main proteins induced by thermal shock were 62, 72, and 84 kDa in size, which were later identified as GroEL, DnaK, and ClpL, respectively. However, certain conditions that are known to induce stress responses in E. coli and B. subtilis as a result of pulsed labeling of proteins with [ ³⁵ S] -methionine result in high molecular weight HSPs, for example It has not been shown to induce GroEL and DnaK murines. However, similar to the temperature change encountered by the pneumococcus after moving from the nasal mucosa to the lung, DnaK and GroEL were induced by changing the temperature from 30 ° C to 37 ° C in vitro (Choi, IH et al., 1999, Limited stress response in Streptococcus pneumoniae.Microbiol.Imunmun . 43: 807-812). The nucleotide sequence of ClpL of various gram positive organisms is known ( L. lactis [X62333]; S. aureus [AP003365, AP003137]; S. pyogenes [AE006538, AE004092]; Lactobacillus rhamnosus [AF323526]), for ClpL. Functional research has been limited. Pneumococcal clpP mutants have recently been found to be susceptible to high temperatures, H ₂ O ₂ and puromycin, and have significantly reduced toxicity in mice (Robertson, GT et al., 2002, Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence.J. Bacteriol. 184: 3508-3520). The specific role of other heat shock genes such as clpC, clpE, and clpX is not fully understood (Charpentier, E. et. al., 2000, Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC Mol Microbiol 37: 717-726; Chastanet, A. et al., 2001, Regulation of Streptococcus pneumoniae clp genes and their role in competence development and stress survival.J. Bacteriol. 183: 7295-7307).

Thus, the present inventors to develop a vaccine with the antigen protein, and this, to universally present in all pneumococci, first, and then study the effect of thermal shock on ClpL and ClpP synthesis, for the in vitro expression of the major pneumococcal virulence genes clpL And the effect of the clpP mutation. In addition, the effects of clpL and clpP mutations on the virulence of pneumococci were evaluated in a mouse intraperitoneal challenge model. Here, we demonstrated that the thermal shock process induces the expression of pneumolysine (Ply) in wild-type pneumococci and regulates the expression of other virulence factors. We also found that mutations in clpP increased mRNA expression at elevated temperatures but did not increase the activity of Ply. We then completed the present invention by investigating the underlying mechanism by which ClpP attenuates toxicity and assessing whether immunization with ClpP can protect mice against challenge with toxic pneumococci.

The present invention provides a vaccine comprising a recombinant ClpP protein derived from pneumococcal as an antigen.

The present invention also aims to provide a method for producing a recombinant pneumococcal ClpP protein for use as a vaccine.

Another object of the present invention is to provide a method for immunizing a human or animal against pneumococcal infection by administering a vaccine comprising pneumococcal ClpP protein to a human or animal in an immunologically effective amount.

Another object of the present invention is to provide a pneumococcal mutant as an attenuated live vaccine.

For this purpose, we studied the effect of thermal shock on ClpL and ClpP synthesis in one embodiment and investigated the effect of clpL and clpP mutations on in vitro expression of important pneumococcal virulence genes. In another embodiment, the effects of clpL and clpP mutations on pneumococcal toxicity in a mouse intraperitoneal challenge model were examined, and thermal shock in wild-type pneumococci induced expression of pneumolysine (Ply) and expression of other virulence factors. It was confirmed that the control. In another embodiment, we also found that clpP mutations increased mRNA expression at elevated temperatures but did not increase the activity of ply. In another embodiment, the inventors also investigated the underlying mechanism by which ClpP attenuates toxicity and found that immunization with ClpP can protect mice against challenge with toxic pneumococci.

In our study, ClpP mutants increased pneumolysine mRNA expression but did not increase pneumolysine levels and hemolyticity after heat shock. ClpP that if the mutants tested infect mice are significantly increased survival time and survival rate of the mice significantly compared to the wild type, ClpP bar appeared to be a weak toxicity of the mutant, which can potentially be used as a live attenuated vaccine body ClpP mutation present. In addition, the biochemical fractions of pneumococci suggest that ClpP migrates from the cytoplasm to the wall of the cell in response to thermal shock, suggesting that it can expose ClpP to host cells due to the stress that pneumococci cause when they infect the host. Suggest. In addition, experiments on the immunoprotective ability of ClpP against pneumococcal were performed by intraperitoneal injection of 10 μg of ClpP protein into Balb / c mice three times per two weeks, followed by toxic 1x10 ⁵ CFU of pneumococcal D39 strain (type 2) was challenged and the survival time was measured. As a result, the survival time was comparable to that of the experimental group administered with attenuated pneumolysine (PdB). This shows that ClpP is exposed to the outside of cells by the stress experienced in the host cell to act as an antigen, as well as a protective effect against pneumococcal infection comparable to attenuated pneumolysine. Therefore, according to the present invention, ClpP protein can be used as an effective vaccine against pneumococcal infection.

The pneumococcal ClpP protein of the present invention is a heat shock protein, a serine protease having a molecular weight of 21 kDa (Genebank AE008443). Recombinant ClpP protein in the present invention can be prepared by isolation after mass expression according to conventional genetic engineering methods in the art. In brief, the ClpP protein clones the open reading frame (ORF) of the ClpP gene (SEQ ID NOs: 5416 to 6006 in Genebank AE008443) into an expression vector, for example pET30 (a) (Novagen), thereby plasmid pET30 (a ) -ClpP (FIG. 1) can be prepared and expressed by insertion into host cells, eg, animal cells, plant cells or E. coli, followed by purification. Expression vectors, hosts, culture conditions, gene insertion methods and the like can be appropriately selected within the ordinary knowledge of those skilled in the art. One embodiment of the present invention describes the preparation of recombinant ClpP protein using E. coli. ClpP protein is involved in the regulation of mRNA and protein levels of pneumococcal virulence factors pneumolysine, PsaA, CbpA, PspA and the like.

Vaccines according to the invention can be administered by a variety of routes, including parenteral, intradermal, transdermal (by use of sustained release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration. . The vaccine is administered in an immunologically effective amount, and an "immunologically effective amount" is a dose suitable for inducing an immune response. The dose depends on factors such as the age, weight and physical condition of the animal or human subject for vaccination and the ability of the animal's immune system to synthesize antibodies and the degree of protection desired. Effective doses can be readily set by those skilled in the art through routine attempts to establish dose response curves. Immunization may be provided by a single dose of the vaccine or may require administration of several booster doses. Doses of ClpP typically range from about 1 μg to about 50 μg, or higher or lower where appropriate. Vaccines according to the invention can be prepared by adding the ClpP protein to an immunologically acceptable diluent or carrier in a conventional manner. Diluents or carriers include, but are not limited to, water, saline, dextrose or glycerol. In addition, pH stabilizers, tonicity agents, wetting agents or emulsifiers may be added. In addition, the vaccine may further comprise other pharmaceutically acceptable adjuvants such as aluminum hydroxide, alum, QS-21, monophosphoryl lipid A and 3-0-deacylated monophosphoryl lipid A (3D-MPL). have. The vaccine is typically prepared injectable in the form of an aqueous solution or suspension, or may be formulated in a solid form that is dissolved or suspended prior to use. In addition, it may be formulated into an intranasal preparation, an oral preparation, and the like by methods conventional in the art. Intranasal preparations may include excipients that do not cause irritation to the nasal mucosa or severely impair ciliary function, and may include diluents such as water, saline. Intranasal preparations may also contain preservatives such as chlorobutanol and benzalkonium chloride. Surfactants may also be included to enhance the uptake of protein antigens by the nasal mucosa. Oral liquid preparations may be, for example, in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be in dry form in tablet form or product for reconstitution with water or other suitable excipients prior to use. have. Liquids may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous excipients (which may include cooking oil), or preservatives. To prepare a vaccine, purified ClpP protein can be lyophilized and stabilized.

1 is a diagram showing the structure of a pET30 (a) -ClpP expression vector.

2 shows the structure of a pKHY004 expression vector.

3 shows the relative positions of the pneumococcal clpL locus.

4a to 4c show the experimental results for the transient induction and stability of the heat shock protein after heat shock in pneumococcal.

5A and 5B show that ClpL increases steadily after thermal shock.

6A-6C show the growth of pneumococcal D39 and its clpL and clpP mutants.

7a and 7b show the results of induction of ClpL in the clpP mutant of pneumococcal 1200.

8 is a diagram showing the induction of toxicity-related genes by thermal shock.

9 shows the relative mRNA concentrations of cbpA, cps2A, ply and psaA measured by real-time RT-PCR before and after heat shock in pneumococcal D39, clpL and clpP mutants.

Fig. 10 shows survival time of mice after intraperitoneal challenge.

11A to 11C show the results of detecting the relative mRNA stability of cps2A and ply by real-time RT-PCR.

12A to 12C show the results of evaluation of bacterial recovery from nasopharynx of CD1 mice for 4 days after intranasal challenge with pneumococcal D39 and its allogeneic clpP derivative. Figures are mean ± standard error of the mean (n = 5) for each time point.

Figure 13 shows the survival rate of pneumococcal clpP mutant in macrophage cells.

14 shows the transition of ClpP after thermal shock.

15A and 15B show the purification of total cell lysates of pneumococcal D39 (FIG. 15 a) and PdB (53 kDa), PspA fragment (43 kDa) and ClpP (21 kDa), showing the specificity of the antibody response to protein antigens. Is a diagram showing Western immunoblot analysis of the prepared product (FIG. 15B).

Figure 16 shows the survival time of mice when intraperitoneally challenged with toxic D39 strain after three immunizations with pneumococcal ClpP and known antigenic proteins.

17A and 17B show the results of immunoblot experiments showing the reactivity of pneumococcal ClpP antibodies with other organism-derived proteins (numbers on the left of the figure indicate the molecular weight of proteins, D39 and Spn1049 represent pneumococcal D39 and clinical strain 1049, respectively). Sth is Streptococcus thermophilus , A549 is human lung cancer A549 cell line, Sce is Saccharomyces cerevisiae , Bsu is Bacillus subtilis , and Pae is Pseudomonas. Pseudomonas aeruginosa , Eco for E. coli, Sty for Salmonella typhi ).

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in more detail based on an Example. However, the following Examples are for illustrating the present invention, and the scope of the present invention is not limited by these Examples. In addition, the references described herein are incorporated by reference in the present invention.

Example 1 Effect of Thermal Shock and ClpL and ClpP Mutations on the Expression of Virulence Genes of Pneumococci

In Example 1, the effects of thermal shock on ClpL and ClpP synthesis were studied, and the effects of clpL and clpP mutations on the in vitro expression of important pneumococcal virulence genes were evaluated. In addition, the effects of clpL and clpP mutations on the virulence of pneumococci were evaluated in a mouse intraperitoneal challenge model.

1. Materials and Methods

Iii) bacterial strains, growth conditions, and transformation

The bacterial strains used in this study are shown in Table 1. Pneumococcal CP1200 (C Choi, IH et al., 1999), a derivative of Rx-1 (a nonpathogenic pneumococcal without a capillary), was used in this study, and it was used at 37 ° C. Incubated in basal medium up to the middle stage of exponential growth phase: 1 L of CAT basal medium was 10 g enzyme casein hydrolysate (Difco Laboratories, USA), 5 g Tryptophan (Difco Laboratories), 1 g yeast extract (yeast) extract) (Difco Laboratories), 5 g NaCl, 5 mg choline (Sigma, USA), 0.2% glucose (Sigma, USA), 16.6 mM dipotassium phosphate (Sigma, USA) Contained. Complete transformation medium was prepared by adding 147 mg CaCl ₂ and 2 g fetal calf serum (fraction V; Sigma) per liter to CAT broth. Competence is regulated by the addition of competence specific peptides and is described in Havarstein, LS et al., 1995, An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae.Proc . Natl. Acad. USA 92: 11140-11144) was quantified as a novobiocin resistant transformant obtained after exposure of the cells to DNA in the culture medium. Capsular strain D39 (type 2) was grown in brain heart injection broth (Difco Laboratories, USA) or Todd Hewitt broth (Difco Laboratories, USA) and described in the literature (Bricker, AL et al., Transformation of a). type 4 encapsulated strain of Streptococcus pneumoniae.FEMS Microbiol. Lett. 172: 131-135). To select pneumococcal transformants, erythromycin or novobiocin were added to the growth medium at concentrations of 2.5 μg / ml or 10 μg / ml, respectively. E. coli strains (BL21 (DE3), DH5α, XL1-Blue in Table 1) were grown in Luria-Bertani (LB) broth or LB agar. Plasmids were introduced into E. coli by transformation as described in Hanahan, D. et al., 1983, Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580. To screen for E. coli transformants, ampicillin (100 μg / ml) was added to the growth medium. The plasmid vectors with the newly generated transformants in this study are listed in Table 1.

Table 1. Bacterial Strains and Plasmids Used in Example 1

ii) preparation of antisera

Generation of HSP antibodies against pneumococcal DnaK and GroEL has been described in the prior art (supra Choi et al., 1999, supra). To prepare antibodies against ClpL and ClpP, the exponential growth phase culture of pneumococcal CP1200 was incubated at 42 ° C. for 30 minutes; Cells were sonicated and proteins were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) and brightly stained with Coomassie Brilliant Blue. 84 kDa and 21 kDa protein bands were cut out and electroeluted. 100 μg of protein per ml of saline was mixed with 1 ml of Freund's incomplete adjuvant. This mixture was then injected intramuscularly and subcutaneously in rabbits. Two booster doses were administered at two week intervals, and antiserum was taken six weeks later. Preparation of serum for CbpA, pneumococcal surface antigen A (PsaA), and Ply was essentially identical to the method described in the prior art (Ogunniyi, A. D. et al., 2000, supra).

iii) protein labeling and gel electrophoresis

For protein labeling experiments, cells were grown to A ₅₅₀ (absorbance at 550 nm) = 0.2 in CAT medium and then divided into 2 ml aliquots. Cells were then collected and resuspended in fresh warmed, low content methionine labeled medium and equilibrated at 30 ° C. for 10 minutes. 10 μ Ci of [ ³⁵ S] -methionine (1000 Ci / mmol, Amersham) was added and the temperature of the culture was 42 ° C. for thermal shock. Cells were collected and 20 μl of lysis buffer (5 mM Tris [pH 8.0], 30 mM ethylenediamine tetraacetic acid [EDTA], 0.1% Triton X-100, 0.025% [w / v] phenylmethanesulfonyl fluoride [PMSF] , 1 mM dithiothreitol) and then completely dissolved by sonic crushing (on ice) as described in the prior art (choi et al., 1999, supra). SDS-PAGE (10 or 15% polyacrylamide gel) was performed as described in Laemli et al., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. The protein was visualized by Coomassie Brilliant Blue staining. The image was obtained by exposing the polyacrylamide gel to radiation sensitive imaging plates for several days. Radiographic image data was quantified using an imaging analysis system (Fujix Bio-imaging Analyzer BAS2500, Fuji Photo Film Co.).

Iii) immune blotting

Proteins isolated by 10% SDS-PAGE were electroblotted onto polyvinylidene difluoride (PVDF) membranes, and then rabbit antiserum 1: 100 dilution or pneumococci produced against the heat shock proteins of pneumococci as primary antibodies. Were reacted with a 1: 5,000 dilution of mouse antiserum produced against toxic proteins of (CbpA, PsaA, and Ply). Secondary antibodies were goat anti-rabbit or goat anti-mouse IgG 1: 2,000 dilutions conjugated to horseradish peroxidase (Sigma) or alkaline phosphatase (Bio-Rad).

Reverse transcription (RT) -PCR

Green(Molecular Probes)이 최종 농도 1:50,000 로 첨가된 마스터 혼합물로부터 설정하였다 (얼음 위에). 혼합물을 얼음 위에 각 상류 및 하류 프라이머를 함유하는 튜브에 분취하고 천천히 교반하여 완전히 혼합시켰다. 각 혼합물을 최종적으로 0.1 ml 반응 튜브 내로 분취하고 Rotor-Gene 2000 Real-Time Cycler (Corbett Research, Australia)에 넣었다. RT-PCR 사이클링 조건을 48℃에서 39 분간 1 사이클 (제 1 가닥 cDNA 합성을 위해) 94℃에서 2 분간 1 사이클 (AMV 역전사효소 불활성화 및 RNA/cDNA/프라이머 변성을 위해) 후에 변성(94℃에서 30 초), 프라이머 어닐링 (60℃에서 30 초), 및 연장 (72℃에서 39초)으로 구성된 PCR 증폭 40 사이클로 구성하였다. 증폭 데이터를 연장 단계에서 얻어서 비교 임계 역치 (Δ Δ C_T)를 이용하는 Corbett Research Software Version 4.4 로 분석하였다. 추출물간에, 표적 전사체의 수준을 내부 16S rRNA 대조에 대해 얻은 전사체 수준을 참조하여 정규화하였다. 모든 실험은 4 중으로 수행하였다.The high temperature acid phenol method is described as described in Ogunniyi, AD et al., 2002, The genes encoding virulence-associated proteins and the capsule of Streptococcus pneumoniae are upregulated and differentially expressed in vivo.Microbiol. 148: 2045-2053. Total RNA was extracted. The levels of mRNA for ply, psaA, cbpA and cps2A were quantified by one step real time reverse transcription (RT-PCR) using the Promega Access RT-PCR System (Promega Biotech, Cat. # A1250). Specific primers used in various RT-PCR assays are described in Ogunniyi et al., 2002, above, and used at a final concentration of 50 nM per reaction. Primers specific for 16S rRNA as internal control (forward, 5′-GGT GAG TAA CGC GTA GGT AA-3 ′: SEQ ID NO. 1; reverse, 5′-ACG ATC CGA AAA CCT TCT TC: SEQ ID NO. 2, Bioneer Co.) was used. Sybr a separate RT-PCR reaction (only the constituent primers are different)

Green (Molecular Probes) was set from the master mixture added at a final concentration of 1: 50,000 (on ice). The mixture was aliquoted into tubes containing each upstream and downstream primer on ice and slowly mixed thoroughly. Each mixture was finally aliquoted into 0.1 ml reaction tubes and placed in a Rotor-Gene 2000 Real-Time Cycler (Corbett Research, Australia). RT-PCR cycling conditions were denatured (48 ° C.) for 1 minute (for 1 strand cDNA synthesis) at 94 ° C. for 1 minute for 2 minutes (for AMV reverse transcriptase inactivation and RNA / cDNA / primer denaturation) (94 ° C.) 30 seconds), primer annealing (30 seconds at 60 ° C.), and extension (39 seconds at 72 ° C.) consisted of 40 cycles of PCR amplification. Amplification data were obtained in the extension step and analyzed with Corbett Research Software Version 4.4 using a comparative threshold threshold (ΔΔ C _T ). Between extracts, levels of target transcripts were normalized with reference to transcript levels obtained for internal 16S rRNA controls. All experiments were performed in quadruple.

Ⅵ) clpL  And clpP  Deletion Mutant Construction

To make a deletion-insertion mutation (Δ clpL :: ermB ) of pneumococcal clpL , a 860-bp ermB cassette (obtained from Dr. Claverys of Toulouse CNRS, France, Vasseghi, H., and JP Claverys. 1983. Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae .Gen 21: 285-292) from erythromycin resistant E. coli chromosomal DNA prs3 (5'-CCG GGC CCA AAA TTT GTT TGA T-3 ': SEQ ID No 3) and prs4 (5'-AGT CGG CAG CGA CTC ATA GAA T-3 ': SEQ ID No. 4) and used to disrupt clpL . A 410 bp fragment ( clpL- up) containing part of the 5 'end of clpL and ermB was transferred from CP1200 DNA to hlp3 (5'-CGG TAC CAT GAA CAA TAA TTT TAA C-3': SEQ ID No. 5) and hlpl (5'-ATC AAA CAA ATT TTG GGC CCG GTC AGA TGT TTC TTG AAT TTC C-3 ': SEQ ID No. 6). A 300 bp fragment ( clpL- down) containing the downstream clpL sequence and part of the 3 'end of ermB was transferred from CP1200 DNA to hlp2 (5'-ATT CTA TGA GTC GCT GCC GAC TGT TCT AGA TGA TGG TCG TTT G-3': SEQ ID NO. 7) and hlp4 (5′-GGC CGA GCT CTT AGA CTT TCT CAC GAA TAA C-3 ′: SEQ ID No. 8). The three PCR products were used as mixed templates for PCR using hlp3 and hlp4 to produce 1.6kb fragments having a 1,301 bp deletion portion of clpL substituted by the ermB gene (Genebank AE008411 SEQ ID NOs: 6374 to 7674). It was. Subsequently, three portions of the 1.6 kb fragment were introduced into the pneumococcal CP1200 or D39 strain by transformation, and recipient bacteria in which the recombinant fragment was inserted into the chromosome by homologous recombination were selected by erythromycin resistance. Transformants were screened for correct deletion by PCR and immunoblot analysis (not shown). Each of the clpL mutants HYK1 and HYK304 of CP1200 and D39 contained the correct deletion in clpL and was used for further studies. For clpP up (234-bp) hpp3 (5'-CGA ATT CAT GAT TCC TGT AGT TAT-3 ': SEQ ID No. 9) and hpp11 (5'-ATT CTA TGA GTC GCT GCC GAC TCA GAA CCA CCT GGT GTA TTG A-3 ': using SEQ ID No. 10) primer and hpp10 (5'-ATC AAA CAA ATT TTG GGC CCG GAT CGC ATC AAG TGG AGC AAA A-3' for clpP -down (319-bp) : SEQ ID No. 11) and hpp6 (5′- CGA GCT CTT AGT TCA ATG AAT TGT TG-3 ′: SEQ ID No. 12) 95 bp at CP1200 and D39 using the same method except that ClpP mutants having the deletion portion of (SEQ ID NOS: 5621 to 5715 in GenBank AE008443) were made and named HYK2 and HYK302, respectively.

Vi) overexpression of ClpL in E. coli.

To overexpress His ₆ -tagged ClpL in E. coli, clpL ORF was amplified using hlp3 and hlp4 of CP1200 DNA. It was digested with KpnI and SacI and the fragment cloned into the pET30 (a) (Novagen) KpnI and SacI sites of the plasmid was generated pKHY004 (Fig. 2). His ₆ -tag protein was expressed in E. coli and eluted with a gradient of 0.1-0.4 M NaCl by DEAE-Sepharose fast flow ™ chromatography (Amersham Pharmacia). Fractions containing ClpL were pooled and purified on a nickel-nitriloacetic acid column according to a method which slightly modified the manufacturer's instructions. Bound His ₆ -tag protein was washed with 40 mM imidazole buffer, eluted with 0.4 M imidazole buffer (pH 7.9) and dialyzed against 20 mM Tris-HCl (pH 7.8), 5 mM MgCl ₂ . The purity of the protein was> 95% as determined by SDS-PAGE and the protein was stained with Coomassie Brilliant Blue R250 (data not shown).

Viii) determination of chaperone activity.

The method described in the prior art (Kudlicki, W. et al., 1997, Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing. J. Biol. Chem. 272: 32206-32210) According to the modified method as described above, the chaperone activity of ClpL was determined. Rhodanese (Sigma, USA) (9 μM) was denatured for 1 h at 25 ° C. in 200 mM potassium phosphate buffer (pH 7.6) containing 1 mM β-mercaptoethanol and 8 M urea. 2.5 μl of denatured enzyme in 8 M urea was diluted to a final volume containing 50 mM Tris-HCl (pH 7.8), 200 mM β-mercaptoethanol, 5 mM sodium thiosulfate, 10 mM MgCl ₂ , 10 mM KCl Spontaneous ClpL assisted refolding was initiated with 250 μl of solution. The final concentration of Rhodanez in the refolding reaction was 90 nM. The refolding reaction was carried out at 25 ° C. for 30 minutes. ClpL chaperone activity was measured by refolding into the native form of Rhodanez. The enzymatic activity of Rhodanez was determined as described in Sorbo. BH et al., 1953, Crystalline rhodanese. I. Purification and physicochemical examination. Acta Chem. Scand. 7: 1129-1136.

ix) toxicity studies

Effect of clpL or clpP mutations on the toxicity of pneumococcal by performing intraperitoneal (ip) challenge with the highly toxic capsular type 2 strain (D39) and its allelic clpP and clpL mutants (HYK302 and HYK304, respectively) Was evaluated. Bacteria at 10 ° C. [vol / vol] horse serum at 37 ° C. overnight on brain heart injection agar (Difco Laboratories, USA) or Todd Hewitt agar (Difco Laboratories, USA) (supplemented with erythromycin as needed) After incubation, in a brain heart injection broth (Difco Laboratories, USA) or Todd Hewitt broth (Difco Laboratories, USA) with serum broth (10% [vol / vol] horse serum) Growth over time gave a bacterial culture of about 10 ⁸ CFU / ml (Ogunniyi, AD et al, 2000, supra). Each bacterial culture was then diluted with serum broth to approximately 10 ⁶ CFU / ml and 10 BALB / c mouse groups were infected intraperitoneally with 0.1 ml volume of D39, HYK302 or HYK304. Survival of the challenged mice was monitored 4 times a day for the first 5 days, twice a day for the next 5 days, and daily until 21 days after challenge.

x) pneumolysine assay

Hemolytic activity was determined according to a method slightly modified from the method described in the prior art (Hanhan, D., 1983, supra). Pneumococci (D39, HYK302, HYK304) grown in the THY broth to the early to mid-stage phase of the exponential growth phase (A ₆₀₀ = 0.05-0.1) were collected by 3900 × g centrifugation at 4 ° C. for 10 minutes and then in phosphate buffered saline. Resuspend. Sodium deoxycholate was added at a final concentration of 0.1% and then incubated at 37 ° C. for 10 minutes. After centrifugation of the sample, the supernatant was recovered and serially diluted. Hemolytic activity was determined by incubation with an equivalent of 1.5% washed human erythrocytes in a 96 well microtiter plate. The hemolytic titer was determined as the inverse of the estimated dilution at which 50% of the red blood cells were dissolved at 540 nm.

xi) statistical processing

Statistical analysis was performed using paired or unpaired Student's t test. Data presented are mean ± standard deviation for 2 to 4 independent experiments. The difference in mean survival time between groups was analyzed by the Mann-Whitney U test (2-tailed), and the difference in overall survival between groups was analyzed by Fisher's exact test (Fisher Exact test).

2. Results

Iii) Characterization of ClpL

84 kDa HSP was conventionally identified as ClpL by N-terminal amino acid sequencing (Cho et al., 1999, supra). Members of the Clp family contain two highly conserved ATP-binding regions (ATP-1 and ATP-2), each containing consensus sequences for adenine nucleotide binding (Gottesman, S. et al. , 1990, Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes.Proc. Natl. Acad. Sci, USA 87: 3513-3517). To confirm that putative pneumococcal ClpL is actually a member of the Clp family, oligonucleotides from the N-terminal amino acid sequence (5'-GAT GAA YAA YAA YTT YAA YAA YTT YAA-3 ': SEQ ID NO. 13) and the Clp family A second ATP binding site for a Clp member that is the best conserved amino acid sequence of (PTGVGKT) (5'-GTY TTN CCN CAN CCN GYN GG-3 ', where Y = T or C, N = A, C, G or T: SEQ ID NO. 14) was used for PCR amplification of CP1200 chromosomal DNA. PCR yielded 1.37 kb DNA fragments expected from L. lactis ClpL size. This was cloned into pGEM-T (promega) to generate plasmid pG8413. Sequencing the cloned fragments showed homology to the L. lactis clpL and bovine clp genes (data not shown). Next, a complete clpL gene was identified in the TIGR pneumococcal type 4 genome using BLAST analysis. In addition, clpL supernatant in pneumococcal R6 showed 98% identity with type 4 clpL supernatant and CP1200 clpL showed high sequence homology with R6 clpL (data not shown). The configuration of this region of the genome is shown in FIG.

A detailed analysis of the sequence of clpL revealed an ORF of 2103 bp encoding a putative polypeptide having 701 amino acids with a molecular weight of 77,699 daltons and a pi (isoelectric point) of 4.99. Analysis of the nucleotide sequence showed that pneumococcal clpL had a sigma A type promoter (TTGACC-17-bp-TATATT) 240 bp upstream of the ATG codon. clpL the upstream has a standing CtsR binding sequence GTC AAA CAA NAN RGT A (R = A or G) (SEQ ID NO. 15 ) repressor, it was determined that close to the clp genes in several organisms. Thus, clpL can be regulated by CtsR. The gene 619 bp upstream from clpL encodes the putative undecaprenyl-p-UDP-MurNAC-pentapeptide transferase and is present in the same orientation. The gene downstream of clpL encoding LuxS is in the opposite orientation (FIG. 3), suggesting that clpL is configured as a monocystron transcription unit.

BLAST analysis shows that pneumococcal ClpL has a high degree of homology to all members of the Clp family in the two conserved ATP-binding regions (p-loops) at amino acids 121-128 (GDAGVGKT) and 391-398 (GSTGVGKT). Appeared. Eight amino acids (MDDLFNQL) from positions 11 to 18 in the hydrophilic N-terminal region were absolutely conserved for bovine Clp-like protein and L. lactis ClpL. Pneumococcal ClpL ATPase exhibits the strongest homology with bovine Clp like protein (76% identity and 88% similarity) and L. lactis ClpL (59% identity and 76% similarity). It also shows high homology with other species (data not shown).

Ii) transient induction of ClpL and high stability after thermal shock

The major HSPs, ClpL, DnaK, and GroEL, with molecular weights of 84 kDa, 73 kDa, and 65 kDa, respectively, were identified by N-terminal amino acid sequencing of the corresponding pneumococcal protein after thermal shock. Since no cooperative or independent regulation of HSP expression was determined, we investigated the kinetics of HSP synthesis by pulse labeling with [ ³⁵ S] -methionine. To determine the induction kinetics of heat shock proteins, CP1200 cells (A ₅₅₀ = 0.2) at 30 ° C exponential growth were subjected to thermal shock by changing the temperature to 42 ° C, followed by pulse labeling with [ ³⁵ S] -methionine for 10 minutes. 2 ml of the culture was taken and the cells were lysed by sonication in lysis buffer, the cell lysates were analyzed by SDS-PAGE and the protein bands were visualized by autoradiography. As a result, the induction of HSP peaked 10 minutes after the change in temperature and then drastically decreased to the baseline level (FIG. 4A). After incubating the cells for 10 min at 42 ° C., the synthesis of ClpL, DnaK, and GroEL increased 11.3 ± 0.8, 5.0 ± 0.3, and 2.7 ± 0.2 fold, respectively, relative to the control. The GroEL band was very similar to the nearest protein band, but it was clearly seen that GroEL was induced as a result of magnification of autoradiography at high magnification (FIG. 4B).

In addition, to determine the stability of the heat shock protein, CP1200 cells (A ₅₅₀ = 0.2) in the mid-exponential growth phase at 30 ° C. were subjected to thermal shock at 42 ° C. for 10 minutes, followed by pulse labeling with [ ³⁵ S] -methionine, and then the cells. Cultures were returned to 30 ° C. and excess non-radioactive methionine was followed for the indicated time. After 2 ml of culture was collected and the cells were lysed by sonication, the cell lysates were analyzed by SDS-PAGE and the protein bands were visualized by autoradiography (FIG. 4b). After the first 10 minutes of exposure at 42 ° C., the level of major HSP synthesis rapidly decreased to 2.0 ± 0.2, 2.2 ± 0.3, and 1.2 ± 0.1 times, respectively, compared to the control without thermal shock treatment, indicating that the synthesis of HSP is in a new steady state. It indicates that it has reached. Similar to the results shown in FIG. 2A, 2.5 minute pulse labeling with [ ³⁵ S] -methionine from 0 to 15 minutes resulted in the initial generation of GroEL, DnaK, and ClpL, and the induction of HSP was approximately 5 minutes in temperature rise change. The peak was reached afterwards, but after 7.5 minutes, it fell to a steady state, showing a net increase of 1.5 to 2 times compared to the control (data not shown). This result indicates that these HSPs have the same induction kinetics even if they belong to different classes. In addition, the increase in the synthesis rate during thermal shock is similar to the increase in the mRNA levels of clpL and groEL in the fixed growth phase of pneumococcal pneumoniae, respectively (Saizieu, A. et al., 1998, Bacterial transcript imaging by hybridization of total RNA to olignucleotide arrays. Biotechnol. 16: 45-48). Since the ATPase subunit of the Clp member can form a complex with the ClpP serine protease, a thermal shock was applied to CP1200 cells ( A ₅₅₀ = 0.2) in the mid-exponential growth phase at 30 ° C. for 10 minutes at 42 ° C., followed by [ ³⁵ S]- After pulse labeling with methionine, the cell culture was returned to 30 ° C. and excess non-radioactive methionine was followed for the indicated time. 2 ml of culture were collected and cells were lysed by sonication. The obtained protein was analyzed by 15% SDS-PAGE, and protein bands were visualized by autoradiography (FIG. 4C). As a result, 21 kDa HSP was induced after thermal shock, and it was confirmed that the 21 kDa HSp was ClpP by N-terminal amino acid sequencing. Lane C in Figure 4c shows no thermal shock. In Figures 4a-4c the number at the top of the figure is the elapsed time (min) after returning to the non-stress condition, the dark arrow represents the major HSP and the molecular size is indicated by kDa on the left side of the chart.

HSP is immunogenic in some pathogens (Kaufmann, SHE et al., 1994, Heat shock proteins as antigens in immunity against infection and self, p. 495532. In RI Morimoto, A. Tissieres, and C. Georgopoulos (ed.) , Biology of Heat Shock Proteins and Molecular Chaperones.Cold Spring Harbor, NY (Cold Spring Harbor Laboratory Press), HSP persistence can help pathogen survival in the host. Therefore, the stability of HSP was investigated. The bacteria were thermally shocked at 42 ° C. for 10 minutes, pulse labeled, returned to 30 ° C. and then followed for various lengths of time with non-radioactive methionine. When examined for HSP after 1-60 minutes, there was no detectable decrease in the amount of radioactive ClpL, DnaK, or GroEL. That is, the HSP produced during the thermal shock survived the change in temperature for 60 minutes (FIG. 5B). Interestingly, immunoblot analysis using pneumococcal HSP showed a significant and steady increase in absolute amount of ClpL during heat shock (up to 14 times less than 60 min). 5a and 5b show that ClpL steadily increases after thermal shock after thermal shock. Whole cell lysates of pneumococcal at exponential growth phase exposed to 42 ° C were immunoblot analyzed. Pneumococcal cells grown at 30 ° C. up to A ₅₅₀ = 0.3 were subjected to thermal shock at 42 ° C. for the indicated times, after which the cultures were collected and resuspended in lysis buffer. After lysing the cells by sonication, 10 μg of protein was isolated by SDS-PAGE and reacted with antiserum against ClpL, DnaK and GroEL. For ClpP, immunoblot analysis was performed after SDS-PAGE using 30 μg of protein (FIG. 5A). Relative levels of ClpL, ClpP, DnaK and GroEL were analyzed densely after thermal shock as shown in FIG. 5A. Numbers represent standard deviations of 3 independent experiments (FIG. 5B). The amount of DnaK and GroEL increased 2.4-fold and 3.4-fold, respectively, during the measured 60 minutes (FIGS. 5A and 5B), after which the amount of all HSPs decreased (data not shown). These results indicate that ClpL is quite stable in pneumococci.

iii) clpL ^-  And clpP ^-  Phenotype of the mutant.

To construct clpL ^- and clpP ^- mutants, DNA fragments containing Δ clpL :: ermB or Δ clpP :: ermB inserts are amplified by PCR and transformed into the chromosome by transformation as described in the Materials and Methods section. Inserted. Insertion mutations were confirmed by PCR and immunoblot analysis to confirm the absence of ClpL or ClpP, respectively. D39 and its homologous genotype clpL ^- (HYK304) and clpP ^- (HYK302) were cultured mutant to the absorbance of 0.1 at 550 nm. The temperature was then changed from 37 ° C. to 43 ° C. and the cultures incubated at the indicated times and the results are shown in FIG. 6. The growth rate of the D39 derivative HYK304 with Δ clpL :: ermB was similar to that of the parent strain at 30 ° C., but at 37 ° C. the doubling time was slower to 55 minutes compared to about 40 minutes in the parent strain (FIGS. 6A to 6A). 6c). Thus, ClpL does not appear to be essential for the growth of pneumococci at 30 ° C and 37 ° C. In contrast, HYK302 with Δ clpP :: ermB mutations showed that growth at 30 ° C (multiplication time = 270 minutes) and 37 ° C (multiplication time = 100 minutes) was significantly higher than that of the parent strain (100 and 40 minutes, respectively). It was found to be reduced. The growth of D39 at 43 ° C. increased during the first two hours but then decreased. The viability of the parent strain was maintained at 42 ° C. for 45 minutes, but after 45 minutes, the viability began to decrease (data not shown). At 43 ° C., growth of clpL ⁻ and clpP ⁻ mutants (HYK304 and HYK302, respectively) was reduced. In addition, the homogeneous CP1200 derivatives HYK1 and HYK2 showed growth patterns similar to those of HYK304 and HYK302, respectively (data not shown).

iv) clpP ^-  Induction of ClpL in Mutants

It was predicted that ClpL could be regulated by ClpP from previous studies showing that ClpL and ClpP are regulated by the same CtsR, and that ClpP is involved in CtsR degradation. To investigate this possibility, we used CP1200 or its clpL or clpP negative mutants to determine the amount of ClpL and ClpP. Pneumococcal CP1200 ( A ₅₅₀ = 0.3) and its homogeneous clpL ^- and clpP ^- derivatives during exponential growth were subjected to thermal shock at 42 ° C for 30 minutes. Proteins from 3 ml of culture were subjected to immunoblot analysis using anti-ClpL or ClpP polyclonal serum. In wild type (CP1200) and clpL ⁻ mutant (HYK1), ClpP was detected at 30 ° C., but after 30 minutes of thermal shock to the cells, the amount of ClpP increased marginally as shown in FIGS. 7A and 7B. ClpL was induced, but the amount of ClpL in the non-induced culture was higher in the clpP ⁻ mutant (HYK2) than in the wild type, suggesting that ClpP inhibits the expression of ClpL (FIGS. 7A and 7B).

v) ClpL's Schaferron Function

Because HSP promotes secretion and aids in proper folding and relocation of proteins (Craig, EA et al., 1993, Heat shock proteins: Molecular chaperones of protein biogenesis.Microbiol. Rev. 57: 402-414) ClpL in pneumococci The chaperone activity of was investigated. To quantitatively measure chaperone activity, a method of refolding the denatured protein in its natural form was used.

(Mendoza, J. A. et al., 1991, Unassisted refolding of urea unfolded rhodanese. J. Biol. Chem. 266: 13587-13591). Unfolded Rhodanez refolding of Rhodanez occurs relatively slowly (Mendoza et al., 1991, supra), and as Danese activity can be determined by a simple and sensitive assay, refolding of denatured Rhodanez is protein folding It has been widely used to study Histidine tag ClpL (pKHY004) (FIG. 2) was overexpressed in E. coli, purified and used to determine refolding activity. Under the test conditions, denatured Rhodanez exhibited only 2.8-7.7% of the natural Rhodanes activity as found in the prior art (Craig et al., 1993, supra), indicating that spontaneous refolding of denatured Rhodanes was 8M urea. Diluted 100 times from solution means that it occurs inefficiently. This activity is expressed as a percentage of natural Rodanese activity performed by the same method. Incorporation of ClpL in the refolding reaction mixture in approximately 3 molar excess relative to the modified Rhodanez increased the activity and returned to natural state with almost 10% of the native Rhodanez activity. However, when a 12-fold excess of ClpL was added to the modified Rhodanez, the activity increased to 30% of the natural level. Increasing the amount of ClpL above this concentration resulted in some additional natural regression in the presence of ATP (Table 2). These results demonstrate that ClpL acts independently as a chaperone to refold denatured protein as previously known for ClpA in E. coli.

Table 2. ClpL-dependent in vitro refolding of denatured Rodanese.

Denatured Rodanese (final concentration 90 nM) was incubated alone or with ClpL in the presence of 2 mM ATP at 37 ° C. for 1 hour. The activity of the refolded enzyme is measured after 60 minutes incubation at 25 ° C. and expressed as a percentage of the activity of the same amount of natural enzyme incubated at 25 ° C. under the same conditions. Mean values and standard deviations from five independent experiments are presented.

*: Significant difference P <0.05 with control (without ClpL).

**: significant difference P <0.001 from control.

vi) regulating the expression of toxicity-related factors by thermal shock

Environmental stress, including thermal shock and starvation, can affect the expression of virulence factors (Mekalanos, JJ 1992. Environmental signals controlling expression of virulence genes determinants in bacteria.J. Bacteriol. 174: 1-7). Thus, the effect of thermal shock on the expression of virulence-related factors of the capsular strain D39 and its clpP ^- mutant (HYK302) and clpL ^- mutant (HYK304), choline binding protein (CbpA), PsaA, pneumococcal surface protein A ( PspA), Ply, and by autoblot analysis using antibodies against autolysine (LytA). Exponential growth phase capsular Streptococcus pneumoniae D39 (A ₆₀₀ = 0.1) and their homologous genotype clpP ^- was added for 20 min heat shock at 42 ℃ to (HYK304) derivative ^- (HYK302) and clpL. 0.6 ml of the culture was centrifuged and the cell pellet was resuspended in lysis buffer and then boiled for 3 minutes. Subsequently, immunoblot analysis was performed on cell lysates using a mixture of polyclonal antisera generated for CbpA, Ply, and PsaA. 8 shows the relative positions of CbpA, Ply, and PsaA. Unexpectedly, Ply was induced in wild type D39 and clpL ^- mutants after thermal shock. PsaA was also slightly induced in D39 after thermal shock, but not in clpL ⁻ mutants. In contrast, in clpP ⁻ mutants, CbpA was induced, but the expression of Ply and PsaA was reduced (FIG. 8). PspA and LytA levels did not change after thermal shock regardless of the genetic background (results not shown). To confirm the increase in Ply expression after thermal shock, the hemolytic activity of Ply in cell lysates was determined. Ply activity increased 1.8 fold after heat shock in D39 but not in clpP ⁻ mutants (Table 3).

Table 3. New Mora, who is the impact of thermal shock on hemolytic activity of ^a

Hemolytic activity of the culture is equal to A ₆₀₀ = 1. 50 μl of cell lysate was serially diluted (1: 1) in 50 μl of phosphate buffered saline. Next, 50 μl of a 1.5% suspension of human red blood cells was added to each well, and the plates were incubated at 37 ° C. for 30 minutes. Hemolysis units were calculated from wells in which 50% hemolysis occurred. These results demonstrate that thermal shock increased Ply expression, which may contribute to increased toxicity under stress load in wild-type and clpL ^- mutants, but this did not occur in clpP ^- mutants.

In Yersinia enterocolitica , ClpP protease is known to inhibit the expression of both ail transcript levels and cell surface expressing Ail proteins (Pederson, KJ, S. Carlson, and DE Pierson. 1997. The ClpP protein, a subunit of the Clp protease, modulates ail gene expression in Yersinia enterocolitica . Mol. Microbiol. 26: 99-107). The present inventors investigated the regulation of virulence gene expression at the mRNA level in S. pneumoniae . RNA was prepared from the culture and mRNA levels of ply , cbpA , psaA and the capsular synthetic gene cps2A were measured by RT-PCR and the results are shown in FIG. 9. In Figure 9 the levels of individual mRNA species between RNA extracts were corrected with reference to the levels obtained for internal 16S rRNA controls. Data points represent mean ± standard deviation of quadruple samples of each RNA extract. At 30 ℃, clpL ^- has increased in the mutants (P = 0.01) ^- Expression of cbpA in mutants was decreased as compared with the case of D39 (P = 0.001), clpP . No significant changes in ply and psaA expression were detected in wild-type and clpL ^- mutants, whereas in the clpP ^- mutant the expression of ply increased 2.5 fold ( P <0.01), whereas the expression of psaA halved ( P < 0.01). After thermal shock, cbpA mRNA levels increased by 7.48, 2.39, and 3.48 times, respectively, at 30 ° C. compared to the D39, clpL ^- and clpP ^- mutants ( P <0.001, P <0.001, P = 0.001, respectively). ). Similarly, mRNA levels of ply increased by 5.27 fold, 6.0 fold, and 3.48 fold, respectively, at 30 ° C. compared to the D39, clpL ⁻ , and clpP ⁻ mutants ( P <0.001 in all cases). After thermal shock, the expression of cps2A was markedly reduced at 30 ° C. compared to the case of D39 and clpL ⁻ mutants ( P = 0.001 in both cases). The expression of cps2A in the clpP ^- mutant increased after heat shock, but the increase was not statistically significant. In contrast, thermal shock increased the mRNA levels of psaA by 1.6 fold and 5.04 fold in D39 and clpP ^- mutants, respectively ( P <0.01, P = 0.001), but decreased in clpL ^- mutants (p <0.01; FIG. 9). These results suggest that clpL ⁻ mutations may negatively affect the expression of psaA , whereas clpP ⁻ mutations may positively affect the expression of cps2A in some unknown manner. These findings provide evidence that, in addition to thermal shock, clpL and clpP regulate a variety of toxicity-related genes to cope with new environmental challenges.

vii) toxicity clpL ^-  And clpP ^-  Influence of mutations.

To further investigate the effects of clpL ^- and clpP ^- mutations on the toxicity of D39 , survival time of mice was measured after intraperitoneal injection of pneumococcal about 10 ⁵ CFU. Groups of 10 BALB / c mice were infected with about 10 ⁵ CFU of D39 or its clpP ⁻ (HYK302) or clpL ⁻ (HYK304) derivatives. The results are shown in FIG. In FIG. 10 each data point represents one mouse and the horizontal line represents median survival time for each group. Median survival times for mice in the parental (D39) and clpL ^- mutant-infected groups were 55 and 60 hours, respectively, and this difference was not statistically significant. However, the mice infected with the clpP ^- mutant became sick 2 to 3 days after infection, but most recovered gradually after 4 to 5 days of infection. Only two of the mice challenged with the clpP ^- mutant died after 67 and 119 hours (FIG. 10). clpP ^- group were infected with mutant body D39 or clpL ^- median survival time and overall survival rate of the groups was infected body mutant exhibited a significant difference in height (in all cases P << 0.001). These results indicate that ClpP function is important for the expression of virulence factors of pneumococci.

3. Review

In this study, we have identified that ATP-dependent Clp protease AAK74513 is a clpL epidermal in pneumococcal . ClpL murines have been identified in several Gram-positive organisms ( L. lactis X62333; S. aureus AP003365, AP003137; S. pyogenes AE006538, AE004092; Lactobacillus rhamnosus AF323526), but not in Gram-negative organisms (Derre, I. Et al., 1999, ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.Mol .

Using scanning densitometry of immunoblot analysis, we found that pneumococci expressed high levels of DnaK, GroEL, and ClpP at 30 ° C. except ClpL. This level doubled when the organisms were exposed to thermal shock for 40 minutes. However, pulse labeling of proteins with [ ³⁵ S] -methionine for 10 minutes demonstrated rapid and transient induction of all HSPs, indicating that DnaK, GroEL and ClpP were constitutively expressed in large quantities at 30 ° C. In addition, the persistence of ClpL, DnaK and GroEL upon return to 30 ° C. indicates that HSP is not actively degraded upon return to normal culture conditions. Since HSP functions as a chaperone, promotes the natural return of unfolded proteins and is induced during infection in various bacterial pathogens, in vivo survival may be increased by the stabilizing effect of HSP on the bacterial macromolecular complex in hostile environments. Can be. Thus, induction of toxic proteins such as PsaA and Ply by the persistence and thermal shock of HSP upon return to normal conditions may contribute to or increase the toxicity of pneumococci. DnaK, the main HSP, is highly immunogenic in pneumococci (Hamel, J., D. Martin, and BB Brodeur. 1997. Heat shock response of Streptococcus pneumoniae : identification of immunoreactive stress proteins.Microb.Pathog. 23: 11-21 Considerable evidence indicates that HSP is an immunodominant antigen in infection by various pathogens (Kaufmann, SH E, et al., 1994, supra). Whether the pathogenic lifestyle of pneumococcal requires high levels of DnaK and ClpL and whether ClpL binds to specific substrates and forms complexes with ClpP for proteolysis are the subjects of research using recombinant proteins.

Mutations in the HSP gene are well known to affect adhesion and toxicity in many pathogens. Stress induced ClpP serine protease contributes to the toxicity of Salmonella typhimurium (Webb, C., et al., 1999, Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium. Mol Microbiol 34:.. 112-123) , Yersinia Enterococcus coli urticae (to adjust the attachment permeability locus (ail) gene expression in Yersinia enterocolitica) (Pederson, KJ et al, 1997, the ClpP protein, a subunit of. the Clp protease, modulates ail gene expression in Yersinia enterocolitica.Mol.Microbiol . 26: 99-107). In Listeria monocytogenes , ClpP is essential for cellular parasitics and toxicity (Gaillot, O. et al., 2000, The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol. Microbiol. 35: 1286-1294). These results suggest that ClpP also plays an essential role in the virulence of pneumococcus, and Robertson et al. (Robertson, GT et al., 2002, Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence.J Bacteriol. 184: 3508-3520).

In this study, we demonstrated that mRNA for toxicity related genes such as cbpA, ply, and psaA was upregulated by heat shock. If, in 30 ℃ compare the gene expression in wild-type and clp mutants, clpL ^- mutant, while showing a nearly identical expression pattern in the case of the wild-type for the cbpA, ply, psaA, and cps2A, clpP ^- the mutants of the cbpA The expression was increased but the expression of ply and psaA was decreased. Thus, clpP appears to act as a negative regulator of cbpA expression but a positive regulator of ply expression. In contrast to the findings of the present inventors, the publication of Chastanet et al. Reported that Ply production was not affected by clpP ⁻ mutations (Chastanet, A. et al., 2001, Regulation of Streptococcus pneumoniae). clp genes and their role in competence development and stress survival.J. Bacteriol. 183: 7295-7307). Chestetanet et al. Quantitatively assessed by observing hemolytic halows on blood agar plates, whereas the present inventors used a quantitative hemolytic assay, and this difference may be due to differences in measurement methods for Ply activity. Since Ply is a proven virulence factor in pneumococcal bacteremia, increased expression after heat shock may be a contributing factor in the onset. This is the first report on the regulation of virulence genes by thermal shock in respiratory pathogen pneumococci.

After thermal shock, real-time RT-PCR data showed an increase in ply expression in clpP ⁻ mutants, but did not confirm an increase in immunoblot analysis and Ply activity measurement. This discrepancy may be due to the instability of the ply mRNA of the clpP ⁻ mutant at high temperatures. ClpP may be thought to be able to directly activate the generator Ply. Our immunoblot data demonstrates that c lpP ^- mutation results in high levels of ClpL expression regardless of thermal shock, suggesting that ClpP can negatively regulate ClpL. These results confirm recent microarray studies showing high levels of clpL induction in clpP ^- mutants at 37 ° C (Robertson, GT et al., 2002, Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae). and their effects on physiology and virulence.J. Bacteriol. 184: 3508-3520). In addition, after heat shock, the expression level of cps2A , the first gene of the capsular biosynthetic locus, was reduced in wild type and clpL ^- mutants, suggesting that resistance to the host immune system may be lower. In contrast, the expression level of cps2A in the clpP ⁻ mutant was not reduced. These results suggest that clpP ^- mutants should exhibit wild-type levels of resistance to host macrophages under stress load, although overall toxicity is reduced. This can lead to the development of chronic bacteremia, in which bacteria can invade the host's immune system and survive in the host, but can cause blast disease, a phenomenon previously demonstrated for pneumolysine negative mutants of D39. none.

Taken together, virulence gene regulation can be regulated by ClpL and ClpP proteases as well as thermal shock. The thermal sensitivity of the clpL ^- mutant and the refolding activity of the modified Rhodanez by recombinant ClpL provide evidence for ClpL's chaperone function. In addition, clpP has been shown to play an essential role in the regulation of ply and cbpA expression.

Example 2 Regulation of Toxicity Factor Expression of ClpP Protease in Pneumococci and Immunity to Pneumococcal Infection

In this example, we investigated the underlying mechanism by which ClpP attenuates toxicity and assessed whether immunization with ClpP could protect mice against challenge with toxic pneumococci.

1. Materials and Methods

i) bacterial strains, growth conditions, and transformation

Bacterial strains and plasmid vectors are shown in Table 4 with the newly generated combinations in this study. Pneumococcal CP1200, a derivative of Rx-1, was grown in Casitone-Tryptone (CAT) basal medium (Cho et al., 1999, supra). Pneumococcal strain D39 (type 2) was incubated in Todd Hewitt (THY) broth. For the selection of pneumococcal transformants, erythromycin was added to the growth medium at a concentration of 0.2 μg / ml. E. coli strains (BL21 (DE3), DH5α, XL1-Blue in Table 4) were grown in Luria-Bertani (LB) broth or LB agar. Plasmids were introduced into E. coli by transformation as described by Hanahan, D., 1983, supra. For selection of E. coli transformants, kanamycin (30 μg / ml) was added to the growth medium.

Table 4. Bacterial Strains and Plasmids Used in Example 2

ii) cell culture.

Human lung epithelial carcinoma A549 (ATCC CCL-185) and murine macrophage RAW264.7 cell line (ATCC TIB-71) were purchased from the American Type Culture Collection and incubated at 37 ° C. under 5% CO ₂ . A549 cells were treated with Dulbecco's modified Eagle's medium (DMEM) containing 4.5 μg / L glucose, 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, Md.), And 100 μg / ml penicillin G, and 100 μg / ml streptomycin. ) (Gibco BRL, Gaithersburg, Md). For incubation of RAW264.7 cells, RPMI 1640 medium (Gibco BRL, Gaithersburg, Md) supplemented with 10 mM HEPES, 2 mM L-glutamine, penicillin G 100 U / ml and streptomycin 100 μg / ml, and 0.2% NaHCO ₃ Was used as the basal medium and FBS (Gibco BRL, Gaithersburg, Md) was added at a 10% concentration.

iii) Specimen Polysaccharide (CPS) Species of Pneumococcal

Pneumococci ( A ₆₀₀ = 0.5) grown overnight on blood agar plates were treated with 150 mM Tris-HCI (pH 7.0); 1 mM MgSO ₄ resuspended to prepare CPS samples of D39 and its isogenic clpP ^- mutant derivatives. This is equivalent to 5 x 10 ⁹ pneumococcal cells per ml. Its 1 ml aliquot was pelleted at 10,000 × g . The bacteria were lysed by addition of 0.1% (w / v) sodium deoxycholate (Sigma, St. Louis, MD) and incubated at 37 ° C. for 15 minutes. The following samples were incubated for 18 hours at 37 ° C. with 100 U mutanolysine (Sigma), 50 μg DNase I (Roche Applied Science, Mannheim, Germany) and 50 μg RNaseA (Roche Applied Science). The samples were then incubated with 50 μg Proteinase K (Sigma) at 56 ° C. for 4 hours and then stored at −20 ° C. The cellular material was then analyzed by immunoblot using polyclonal type 2 polysaccharide specific antiserum.

iv) antisera, gel electrophoresis, and immunoblotting.

Serums for PspA and PdB (toxoid derivatives of Ply) were constructed essentially as described in the prior art (Ogunniyi, AD et al., 2000, supra). For immunoblotting, the bacteria were incubated with A ₆₀₀ = 0.3 in THY and Kwon, HY et al., 2003, Effect of Heat Shock and Mutations in ClpL and ClpP on Virulence Gene Expression in Streptococcus pneumoniae . Immun) was produced in the same manner as described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10% or 15% polyacrylamide gel) is described in Laemmli, UK 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685). Proteins were electroblotted onto nitrocellulose membranes and then reacted with 1,5000 dilutions of polyclonal mouse serum for PspA and PdB. For buccal blotting, samples were electroblotted onto nylon membranes and then reacted with a 1: 5000 dilution of anti-serumtype 2 polyclonal mouse serum. The secondary antibody was a 1: 2000 dilution of goat anti-mouse IgG (Bio-Rad) conjugated to alkaline phosphatase.

v) pneumolysine assay

According to a method slightly modified in the method described in the prior art (Lock, RA et al., 1996, Sequence variation in the Streptococcus pneumoniae pneumolysin gene affecting haemolytic activity and electrophoretic mobility of the toxin.Microb. Pathog. 21: 71-83) Hemolytic activity was determined. Briefly, pneumococci grown in the THY broth to the early-mid log phase of the exponential growth phase ( A ₆₀₀ = 0.05-0.1) were collected by centrifugation at 3900 x g for 10 minutes at 4 ° C. And resuspended in phosphate buffered saline. Sodium deoxycholate was added at a final concentration of 0.1% and then incubated at 37 ° C. for 10 minutes. After centrifugation of the sample, the supernatant was collected and serially diluted. Hemolytic activity was determined by incubation at 37 ° C. for 30 minutes with equal volumes of 1.5% washed human erythrocytes (containing [Merck] with 0.001% 2-mercaptoethane) in a 96 well microtiter plate. Hemolytic titers were determined as the inverse of the estimated dilution at which 50% of the red blood cells were dissolved at A ₅₄₀ .

vi) RNA techniques

1.5 ml aliquots of the culture suspension were periodically taken for total RNA extraction. To measure mRNA half-life, rifampicin (100 μg / ml) was added. Total RNA was extracted using the hot acid phenol method as described in the prior art (Ogunniyi, AD et al., 2002, supra). MRNA levels for cps2A and ply were quantified by one-step real-time reverse transcription (RT-PCR) using the Promega Access RT-PCR System (Promega Biotech, Cat. # A1250). Internal control (16S rRNA) primers specific for these reactions have been described in the prior art (Ogunniyi et. Al., 2002, supra). Preparation of RT-PCR reactions, cycling conditions and analysis of data were essentially identical to those described in the prior art (Kwon et. Al., 2003, supra). All reactions were performed in a Rotor-Gene 2000 Real-Time cycler (Corbett Research, Australia). Analysis of mRNA half-life was performed by the SigmaPlot curve fitting program (non-linear least squares fitting for the sum of the index values). Two models have been proposed, one phase decomposition or two phase decomposition. In each case the model that fitted the data with the minimum deviation was retained as more valid.

vii) Cloning, Expression, and Purification of ClpP in Escherichia Coli .

clpP ORF (SEQ ID NOs: 5416 to 6006 in Genebank AE008443) was converted from CP1200 DNA to forward and reverse primers (5'-CGA ATT CAT GAT TCC TGT AGT TAT-3 ': SEQ ID NO. 9, and 5'-CGA GCT PCR amplification using CTT AGT TCA ATG AAT TGT TG-3 ′: SEQ ID NO. 12, primers are inserted at Eco RI and Sac I sites, respectively). The PCR fragment was digested with the same enzyme and cloned into the corresponding restriction site of pET30 (a) (Novagen) to make plasmid pET30 (a) -clpP (FIG. 1). This plasmid was transformed into Escherichia coli BL21, followed by expression for 3 hours using 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) in E. coli BL21 (DE3) strain. Cells were collected by centrifugation at 6,000 x g for 10 minutes, followed by lysis buffer (50 mM sodium phosphate, PH 8.0; 2 M NaCl; 40 mM already added protease inhibitor phenylmethyl-sulfonyl fluoride at a final concentration of 1 mM). Resuspended). The cells were then lysed at 12,000 1b / in ² in French pressure cells (SLM Aminco, Inc.) and centrifuged at 100,000 x g for 1 hour. The supernatant containing His ₆ -tag protein was loaded into a nickel-nitriloacetic acid column (Probond, Invitrogen) and then washed with 10 column volumes of 10 mM sodium phosphate, 20 mM imidazole, and 1 M NaCl, pH 6.0. It was. Nickel bound His ₆ -tag protein was eluted with 30 ml of 0-500 mM gradient imidazole in 10 mM sodium phosphate (pH 6.0) buffer and dialyzed against 10 mM sodium phosphate buffer (pH 7.0). Protein was> 95% pure as judged by SDS-PAGE and Coomassie Brilliant Blue R250 (data not shown).

viii) Isolation and Positioning of Subcellular Fractions

Cells of exponential growth phase are collected by centrifugation and protoplast formation induced by sucrose is described in Vijayakumar, MN et al., 1986, Localization of competence induced proteins in Streptococcus pneumoniae. J. Bacteriol. 165: 689-695 It was carried out according to the method described in). Cells were converted to protoplasts by incubating with 1 M sucrose buffer (1 M sucrose, 100 mM Tris HCl pH 7.6, 2 mM MgCl ₂ , 1 mM PMSF) at 30 ° C. for 1 hour. Cell wall fractions (supernatants) were separated from protoplasts (pellets) by centrifugation at 13,000 x g for 20 minutes. Protoplasts were diluted osmolally with 19 volumes of hypotonic buffer (100 mM Tris HCl pH 7.6, 1 mM PMSF, 1 mM EDTA). Lysates were first centrifuged at 5,000 x g for 5 minutes to remove undissolved cells, and then centrifuged at 50,000 x g for 30 minutes to obtain cytosolic fractions (supernatants) and cell membrane fractions (pellets).

ix) Determination of malate dehydrogenase activity.

The enzymatic activity of maleate dehydrogenase (MDH) was determined by absorbance ( A ₃₄₀ = 6.22 / mM / cm) of 0.2 mM NADH at 340 nm and 25 ° C. in 0.15 M potassium phosphate (pH 7.6) containing 0.5 mM oxalacetate. The rate of reduction was determined by monitoring. After adding the sample, the reaction mixture was incubated at 25 ° C. for 1 minute 40 seconds and the absorbance was measured at 34 Onm. Substrate was added to initiate the reaction. MDH activity was calculated using the initial slope of the oxidation rate of NADH from the reaction for the first 1 minute 40 seconds.

x) Adhesion and Permeability Assay.

The invasive assay of pneumococcal into human lung A549 cells was performed by modifying the antibiotic protection assay described in the prior art (Vijayakumar, MN et al., 1986, supra). A549 cells were grown confluence in 24 well tissue culture plates, washed three times with phosphate buffered saline (PBS, pH 7.2) and then 1 ml of culture medium (without antibiotics) was added per well. Exponential cultures of R-type CP1200 and its heterologous clpP ^- mutant strains ( A ₅₅₀ = 0.3, 10 ⁸ CFU / ml) were pelleted by centrifugation, washed once with PBS and resuspended in DMEM. I was. The monolayers were infected with 10 ⁷ bacteria (bacteria: cell ratio, 10: 1), and the initial contact of the cell monolayers and bacteria was promoted by centrifuging at 800 x g for 10 minutes at 4 ° C and incubating at 37 ° C for 2 hours. . Fresh medium containing 10 μg / ml penicillin and 200 μg / ml gentamicin was added to each well and confirmed that this treatment was sufficient to kill all extracellular bacteria. After an additional hour of incubation, the monolayers were washed three times with PBS, treated with 100 μl of 0.25% trypsin-0.02% EDTA to remove cells from the plate and then 400 μl of Triton X-100 (0.025% aqueous solution). ) Was added to dissolve. Appropriate dilutions were plated on blood agar to determine the number of viable bacteria. To determine the total number of adherent intracellular bacteria, infected monolayers were washed as described above, then trypsinized, lysed and plated quantitatively without antibiotic treatment. All samples were triplicate assays and each assay was repeated three or more times.

xi) Survival of RAW264.7 cells .

Cell monolayers were infected with 10 ⁷ CFU pneumococci (bacteria: cell ratio, 10: 1) in antibiotic-free RPMI1640 culture medium (Sigma). For bacterial infections, the cultures were incubated at 37 ° C. for 2 hours. After the incubation, cells were washed three times with PBS, and fresh medium containing 10 μg / ml penicillin and 200 μg / ml gentamycin was added to kill extracellular bacteria (0 hour of assay). To quantify intracellular pneumococci at various times post-infection, the supernatant was removed and the cells washed three times with PBS and then lysed with Triton X-100 as described above. Serial dilutions of lysate from each well were plated on blood agar. The number of CFUs was determined after 24 hours of human cultivation at 37 ° C. Three independent assays were performed for each bacterial strain (triple assay). Statistical analyzes were performed using paired or unpaired Student's t test.

xii) colony formation study

This study was performed essentially the same as the method described in the recent literature (Ogunniyi et al., 2003, MS in preparation, above). Prior to challenge, the bacteria were incubated overnight at 37 ° C. on Todd Hewitt agar (Difco Laboratories, USA) (supplemented with erythromycin if necessary), followed by 10% [vol / vol] horse serum. The broth was grown at 37 ° C. for about 4 hours to obtain about 4 × 10 ⁷ CFU / ml ( A ₆₀₀ = 0.1) of bacteria. Each bacterial culture was then adjusted to about 10 ⁹ CFU / ml in THY broth and 10 μl of cells (about 10 ⁷ CFU) were inoculated into the nasal pores of 5 week old CD1 mice. At 1, 2, and 4 days after infection, four mice in each group were sacrificed at random to evaluate the carrier's degree of each strain. Nasopharynx, blood and lung samples were serially diluted with appropriate sterile PBS and plated in duplicate on blood agar containing suitable antibiotics. Plates were incubated at 37 ° C. for about 16 hours in an atmosphere of 95% air / 5% CO ₂ , after which colonies were counted and averaged between the replicas.

xiii) Immunization and Serum Analysis of Mice

Mice were immunized intraperitoneally as described in the prior art (Ogunniyi, AD et al, 2000, supra). Intraperitoneal immunization with ₄ groups (12 per group) consisting of 5-6 week old CBA / N mouse females with AlPO ₄ alone, genetically modified Ply toxoid (PdB) + AlPO ₄ , PspA + AlPO ₄ , or ClpP + AlPO ₄ I was. Each mouse was administered three times with 10 μg of each protein antigen at intervals of 12 to 14 days, and serum was collected from the mice by orbital posterior bleeding one week after three immunizations. Serum was pooled by group and analyzed for Ply, PspA or ClpP specific antibodies by enzyme-linked immunosorbent assay (ELISA). The serum was also analyzed by whole cell lysates of pneumococcal D39, or Western immunoblot using purified Ply, PspA or ClpP as antigen.

xiv) challenge

Immunized mice 2 weeks after the last immunization were challenged intraperitoneally with highly toxic capsular type 2 strain (D39). Prior to challenge, the immune bacteria were grown overnight at 37 ° C. on blood agar, and then meat extract broth (brain heart injection broth, Difco Laboratories, USA) or Todd Hewitt broth (Difco Laboratories, USA). ) Was inoculated into serum broth to which 10% (v / v) horse serum was added. The bacteria were then grown statically at 37 ° C. for 3 hours to obtain about 10 ⁸ CFU / ml of bacteria and the inoculum was adjusted to 7.5 × 10 ⁵ CFU per challenge dose. Serum-type specific capsular production was confirmed by quellung reaction using antisera purchased from Statens Seruminstitut, Copenhagen, Denmark. After challenge challenge, mice were monitored every 4 hours for the first 7 days and then daily for up to 21 days and the survival time of each mouse was recorded. The difference in median survival time between each group was analyzed by Mann-Whitney U test (one-tailed).

2. Results

i) clpP ^-  Mutants do not result in persistent infection in mice .

We have demonstrated that the clpP ^- mutant showed markedly attenuated toxicity. The expression level of cps2A , the first gene of the capsular biosynthetic locus, did not decrease after heat shock in this strain, but the expression of cps2A in the parent strain was significantly reduced after heat shock, indicating that the clpP ^- mutant was stress-loaded even though overall toxicity was reduced. Suggests that wild-type levels of resistance should be exhibited to the host host macrophages. This can lead to chronic bacteremia, in which bacteria can invade the host immune system and survive in the host, but do not cause blast disease, a phenomenon previously demonstrated for pneumolysine negative mutants of D39.

To confirm this hypothesis, 10 mice were injected intraperitoneally with 10 ⁵ cfu of clpP ^- mutant and blood was collected both before and after the infection. The results showed that pneumococci were not detected in any mice at days 7, 14, and 21 post infection, indicating that clpP ⁻ mutations do not cause persistent infection (data not shown).

ii) Determination of mRNA half-life .

We recently demonstrated that in clpP − mutants, ply mRNA expression was increased after heat shock, but at the same time, Ply protein and Ply hemolytic activity were not increased. In wild type, however, both ply mRNA levels as well as Ply protein and hemolytic activity were significantly increased after thermal shock (Kwon, HY et al., 2003, supra).

This discrepancy may be due to the instability of ply mRNA at high temperatures in clpP ⁻ mutants. Therefore, the ply mRNA degradation kinetics were investigated after heat shock (HS). To compare the stability of mRNA at 30 ° C., rifampicin was added to block de novo synthesis of mRNA and its degradation was monitored by real-time RT-PCR using cps2A and ply specific primers (FIG. 11A). To determine mRNA half-life at 30 ° C., pneumococcal strains were first grown at 30 ° C. and then rifampicin was added. An aliquot for the RNA extract was removed before adding rifampicin (30 ° C.) and 10 minutes (R10m) and 20 minutes (R20m) after adding rifampicin (FIG. 11A). To determine mRNA half-life after thermal shock, pneumococcal strains were grown at 30 ° C. (30 ° C.) and then thermal shock was applied at 42 ° C. After 10 minutes at 42 ° C., rifampicin was added (HS). Aliquots for RNA extracts were removed before and after thermal shock and at 10 minutes (R-10m) and 20 minutes (R-20m) after addition of rifampicin at 42 ° C. (FIG. 11B). To determine the effect of thermal shock on mRNA half-life, pneumococcal strains were grown at 30 ° C. (30 ° C.) and treated with rifampicin for 10 minutes followed by thermal shock at 42 ° C. Aliquots of the RNA extracts were withdrawn at 30 ° C. before and after the addition of rifampicin (R-10m), then at 10 minutes (R + HS10m) and 20 minutes (R + HS10m) after thermal shock ( 11C). Levels of individual mRNA species between RNA extracts were corrected with reference to the levels obtained for internal 16S rRNA controls. Data points represent mean ± standard deviation of quadruple samples of each RNA extract. Comparison of the degradation kinetics wild type and clpP ^- the half-life of ply mRNA of mutants that appear got, which is the half-life of the mutant ply 2.1 times longer than the wild-type half-life at 30 ℃ that minutes 2.75 minutes, and 5.8, respectively (Table 5). However, at 30 ° C., degradation of cps2A mRNA in clpP ⁻ mutants was only 1.31 fold compared to degradation of the parent strain (FIG. 11A and Table 5). These results suggest that ClpP protease can degrade cps2A and ply mRNA at 30 ° C. in an unknown manner.

Table 5. Effect of thermal shock on half-life of cps2A and ply mRNA ^a

For mRNA half-life measurement, rifampicin (100 μg / ml) was added, 1.5 ml of the culture suspension was taken at 10 minute intervals, and total RNA was extracted therefrom using a high temperature phenol method. Subsequently, mRNA levels were determined by real time RT-PCR. MRNA half-life was analyzed by nonlinear least squares fit to the sum of exponential values. All experiments were performed in quadruple. To compare the stability of the transcript after thermal shock, pneumococcal cells were first grown at 30 ° C. and then the temperature was changed to 42 ° C. Total RNA was made 10 minutes and 20 minutes after addition of rifampicin at 42 ° C., as well as immediately before adding rifampicin at 42 ° C. After thermal shock, the ply mRNA level of the clpP ^- mutant increased 7.5 fold (FIG. 11B), but the level of Ply protein did not increase (data not shown), confirming our previous results. Degradation kinetics data showed that the half-lives of ply transcripts in the parent strain and clpP ^- mutants were 1.75 and 3.75 minutes, respectively, indicating that ply mRNA degraded 2.1 times slower in the clpP ^- mutant than after the heat shock. It means. In addition, the parent strain and the clpP ^- And the cps2A mRNA in mutant means a slower degradation 2.05 times compared with the parent strain (Fig. 11b) ^- a half-life of the transfer member in cps2A mutants were 2.0 minutes and 4.1 minutes, respectively, after heat shock, which clpP.

Considering that the half-life of ply and cps2A mRNA transcripts after heat shock was shorter at 30 ° C than in parental and clpP ^- mutants, mRNA species are more prone to degradation at 42 ° C than at 16S rRNA or HSP other than ClpP. It is possible to decompose more quickly by Therefore, we investigated whether the thermal shock itself could affect the half-life of ply mRNA. To determine the effect of thermal shock on ply mRNA half-life, cells cultured at 30 ° C. were treated with rifampicin, then thermal shock was applied, and the degradation kinetics of mRNA was determined by RT-PCR analysis. Under these conditions, both cps2A and ply transcripts were stable for at least 20 minutes in the parent strain and clpP ⁻ mutants (FIG. 11C), which was in contrast to the scenario observed at 30 ° C. (FIG. 11A). These results demonstrated that the transcript was actually stabilized by thermal shock at 42 ° C. (FIG. 11C).

용) Hemolytic activity of Ply is not activated by ClpP

Destabilization of ClpP can lead to low stability of the ply processing product and ply primary transcript. Although ply mRNA was stabilized by thermal shock, there was no corresponding increase in the amount of Ply protein or its level of hemolytic activity. This discrepancy may be because Ply is directly activated by the ClpP protease so that hemolytic activity may increase in the parent strain but not in clpP ⁻ mutants.

To demonstrate this hypothesis, pneumococcal cells were incubated at 30 ° C. and then thermal shock was applied at 42 ° C. for 20 minutes. The cells were lysed by incubation at 37 ° C. for 10 minutes with 0.1% sodium deoxycholate. The cell lysates were then further incubated at 37 ° C. for 20 minutes, 40 minutes and 60 minutes and the hemolytic activity of Ply was determined. Hemolytic activity in both wild-type and clpP ^- mutants decreased during this period, and at 37 ° C the hemolytic titer in clpP ^- mutant was not significantly different from the hemolytic titer in the parent strain, indicating that the ClpP protease was responsible for Ply's hemolytic activity. Suggests that it is not necessary for activation (data not shown)

Iii) ClpP movement after thermal shock

Location of hsp100 by immunogold labeling using anti-hsp100 antibodies revealed that hsp100 is located in the cytoplasm and nucleus before and after heat shock in yeast Saccharomyces cerevisiae (Fujita, K. et al., 1998, Hsp 104 responds to heat and oxidative stress with different intracellular localization in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 248: 542-547). In B. subtilis, immunogold labeling with antibodies revealed that ClpC and ClpX ATPases were detected in the envelope and inside the cell (Kruger, E. et al., 2000, The clp proteases of Bacillus). subtilis are directly involved in degradation of misfolded proteins.J. Bacteriol. 182: 3259-3265) This suggests that hsp100, ClpC, and ClpX proteins are closely related to protein aggregates during thermal shock treatment.

The present inventors first attempted to fractionate subcellular proteins using a capillary strain to determine the location of ClpP in pneumococci. However, incubation of cells in 1M sucrose buffer followed by centrifugation resulted in no cell wall separation from the protoplasts due to the presence of thick capillaries (data not shown). Therefore, strains without capillaries were used for localization experiments. Since fractionation can cause partial lysis or leakage of cellular contents during or after thermal shock, MDH was used as an internal cytoplasmic marker. MDH activity in the cell wall was less than 10% of total MDH activity at 30 ° C. (data not shown). In addition, MDH activity in the cell wall did not increase even after thermal shock (data not shown), suggesting that the thermal shock does not cause lysis or leakage of the cell membrane.

After pneumococcal CP1200 cells in exponential growth phase were exposed to heat at 42 ° C. for 20 minutes and subjected to thermal shock, cell proteins were induced to form protoplasts with sucrose, and then lysed with storage buffer (see Materials and Methods). Fractionated. Subsequently, the subcellular fractions were analyzed by immunoblot using polyclonal anti-ClpP antibody (obtained by subcutaneous injection of ClpP protein in rabbits three times every two weeks, followed by serum collection) to visualize ClpP. . ClpP was mainly detected in the cytoplasmic fraction at 30 ° C., but in smaller amounts in the cell wall fraction. However, after thermal shock, the amount of ClpP in both the cytoplasm and cell wall was increased (FIG. 14), which means that ClpP is induced at normal temperature but more induced after thermal shock. 14 W: cell wall; M: cell membrane; And C: cytoplasm. These results suggest that a significant amount of ClpP can be used as a cell wall after thermal shock, so that ClpP can play an important role in the cell wall or possibly be involved in the degradation of proteins.

Ⅴ) clpP ^-  Results on colony formation and lung permeability of mutants

Conventionally, the clpP ^- mutant has been shown to be very weak in toxicity in the murine sepsis model (Kwon, HY et al., 2003, supra). Moreover, the clpP ^- mutant did not form significant colonies in the lungs of mice after endogenous challenge and no mortality was recorded during 48 hours of infection (Robertson, GT et al., 2002, supra). However, the invasion and propagation of pneumococci seems to occur through its natural location, the nasopharynx.

Therefore, the effects of ClpP on colony formation of pneumococci after intranasal challenge were evaluated. Intranasal infection with a highly toxic capsular type 2 strain (D39) and its homologous genotype clpP ^- mutant (HYK302) showed that clpP ^- mutant did not form colonies at all in the nasal mucosa and lungs for 48 hours ( 12A-12C) This suggests a defect of adhesion.

In addition, ClpC of pneumococci was found to be associated with adhesion (Charpentier, E., R. Novak, and E. Tuomanen. 2000. Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC Mol Microbiol 37: 717-726), clpC (Rouquette, C., C. de Chastellier, S. Nair, and P. Berche. 1998. in Listeria and Yersinia ) .The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages.Mol.Microbiol. 27: 1235-1245), clpE (Nair, S., C. Frehel, L. Nguyen, V. Escuyer, and P. Berche. 1999. ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes.Mol . Microbiol. 31: 185-196), and clpP (Pederson, KJ et al., supra ). Mutants of 1997) showed reduced adhesion to host epithelial cells.

Therefore, the relationship of ClpP in adhesion to host epithelial cells was investigated. Since the presence of polysaccharide capillaries significantly weakens the adhesion of pneumococcal to the surface of the host cell, we use an R type strain for clpP ⁻ The effect of the mutation was determined. The R type clpP deletion mutant HYK2 showed no significant difference in adhesion and permeability to A549 human lung cells compared to its homologous parent strain (data not shown). Thus, these results suggest that failure to form colonies was not due to defects in adhesion or permeability.

In murine macrophages RAW264.7 cell line  clpP Reduced survival of mutants

Alveolar macrophages are a primary factor in host defense against invasive pneumococci (Knapp, S. et al., 2003, Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia.Am. J. Respir. Crit.Care Ed. 167: 171-179. In this study clpP ^- Since the mutant was observed to be defective in colony formation of the nasal mucosa compared to its parent strain, it was inferred not only because the mutant grows more slowly but also because the pneumococci are cleared quickly.

Thus, clpP ⁻ in murine macrophage RAW264.7 cells The survival rate of the mutants was determined (FIG. 13). RAW264.7 cell monolayers were infected with about 10 ⁷ CFU of pneumococcus (bacteria: cell ratio = 10: 1) in RPMI1640 medium and then taken at different time points, treated with gentamicin, and intracellular pneumococci were quantified. . Three independent assays were performed in triplicate for each bacterial strain. The D39 parent strain was able to survive in macrophage during the assay process (8 hours) and remained at 160 CFU, while recoverable clpP ⁻ The number of mutants steadily decreased to zero after 8 hours of infection (FIG. 13). Survival of clpP ⁻ mutants in macrophage cells was significantly reduced after 5 (P <0.01), 6 (P <0.05), 8 (P <0.01) hours than the parent strain (FIG. 13). Two hours after the addition of the antibiotic, the number of viable cells will be about half of the number present at the time the antibiotic is added. However, the actual number of CFUs was much less than expected, and this fact is clpP ⁻ ClpP ^- rather than mutant growth defect It is due to stress sensitive trait or susceptibility to macrophages of mutants. This suggests that ClpP is required for intracellular survival in RAW264.7 cells.

Ⅶ)  clpP The cps levels of the mutants are similar to wild type strains.

In clpP ^- mutants of Streptococcus mutans and Pseudomonas fluorescence , biofilm levels were significantly reduced (Lemos, JA, and RA Burne. 2002. Regulation and Physiological significance of ClpC and ClpP in Streptococcus mutans.J. Bacteriol. 184: 6357-6366). Therefore, the amount of cps in these mutants was considered to be lower than for the parent strain type.

To investigate whether this also applies to pneumococci, the amount of cps was determined by immunoblot analysis using polyclonal type 2 polysaccharide specific antiserum using D39 and its homologous clpP ^- mutant. Both cultures were adjusted to the same optical density to rule out the possibility that differences due to variations in the growth capacity of the strain could be observed. Parent strain (D39) and the clpP ^- In both mutants (HYK302), the same amount of cps was detected (data not shown), indicating that the toxicity weakening of the clpP ⁻ mutant was not due to lower cps levels.

Iii) Protection against plp pneumoniae challenge by mice

HSPs function as antigens in some pathogens and these proteins are known to act as defenses against infectious diseases. In addition, since ClpP moves to the cell wall after the thermal shock, the protective induction ability of the protein against pneumococcal challenge was evaluated.

Proteins were separated by SDS-PAGE followed by electroblotting on nitrocellulose. Next, they were reacted with serum obtained from a group of mice immunized with the protein. 15A and 15B show the results. The nitrocellulose membrane strips of lanes 1 to 4 in FIGS. 15A and 15B are AlPO ₄ adjuvant (lane 1), PdB + AlPO ₄ (lane 2), PspA + AlPO ₄ (lane 3), and ClpP + AlPO ₄ (lane), respectively. And the serum obtained from the mice immunized with 4). Mice immunized with ClpP induced a strong specific antibody response to the antigen; ELISA titers of pooled sera of mice immunized with purified ClpP were 8,400 ± 2250, and titers obtained from mice immunized with purified PdB (8,000 ± 600) and titers obtained from mice immunized with purified PspA (8,300 ± 2). 2,400). Mice immunized with Alum (AlPO ₄ ) adjuvant alone showed a titer of 100, the limit of detection. Western immunoblot results of sera against whole cell lysates of D39 also demonstrated the specificity of the antibody response to each antigen (FIG. 15A). Serum reacted specifically with each purified protein (FIG. 15B). However, for purified ClpP, some proteolytic products were observed.

In active immune / challenge experiments, mice were challenged with D39 of about 7 × 10 ⁵ CFU. Two groups of CBA / N mice were immunized with the indicated antigens and challenged with a capsular type 2 strain D39 of about 7.5 × 10 ⁵ CFU two weeks after three immunizations. The result was shown in FIG. In FIG. 16 each data point represents one mouse and the horizontal line represents the median survival time for each group. Median survival time for mice receiving ClpP was about 2 days (FIG. 16). This was significantly longer compared to mice receiving Alum adjuvant alone ( P <0.01). Similarly, median survival time (2 days) for mice receiving PspA was significantly longer than mice receiving alum adjuvants alone ( P <0.001). For mice receiving PdB (pneumolysine toxoid), the median survival time was about 2.5 days, which was also significantly longer compared to mice receiving alum adjuvants alone ( P <0.001). However, there was no significant difference when comparing the median survival time for mice receiving ClpP with the results obtained using PdB or PspA.

3. Review

The purpose of this study was to evaluate the role of ClpP, a heat shock protein, in the development of pneumococcal disease. We demonstrated that ply mRNA expression was increased in clpP ⁻ mutants after heat shock, but the amount of Ply or hemolytic activity was not increased at the same time. This contradiction is believed to be a contributing factor to the degradation of transcripts, as ply and cps2A mRNAs were stable at both 30 ° C. and 4 ° C. in clpP ⁻ mutants. Moreover, clpP ^- after thermal shock The half-life of both cps2A and ply mRNA in the mutants was more than two times longer than in the wild-type case, clearly indicating that after heat shock the half-life of mRNA species increases in the absence of ClpP. The hemolytic activity of Ply was not further increased by incubation of cell lysates at 37 ° C., indicating that ClpP is not a direct cause for activation of hemolytic activity of Ply. We conclude from these findings that cps2A and ply mRNA are degraded by ClpP at the post-transcriptional level, but the specific mechanism for this is still unknown.

In pneumococcal cps is an important virulence factor and provides resistance to phagocytosis (Austrian, R. 1981. Some observations on the pneumococcus and on the current status of pneumococcal disease and its prevention. Rev. Infect. Dis. 3 (Suppl .): S1S17). The formation of clpP ^- mutants in S. mutans resulted in an 80% reduction in biofilm formation (Lemos, JA, and RA Burne. 2002. Regulation and physiological significance of ClpC and ClpP in Streptococcus mutans . J. Bacteriol) . 184: 6357-6366), clpP of Streptococcus pneumoniae ^- The amount of cps in the mutant is the same as the wild type. Therefore, the survival rate of the clpP ^- mutant and the lack of colony formation in the nasal mucosa in macrophages are thought to be due to the reduced growth at both 30 ° C and 37 ° C, not due to cps levels (Kwon, HY et al. al., 2003). This may also be due to stress sensitive traits due to the accumulation of denatured proteins that are normally targeted for ClpP proteases. In some pathogens, HSP is on the surface of the cell and can mediate adhesion to host cells (Marcellaro et., 1998; 59, 60). Pneumococcal (see Charpentier, E. et al., 2000) and Listeria monocytogenes (Nair, S., E. Milohanic, and P. Berche. 2000. ClpC ATPase is required for cell adhesion and invasion of Listeria monocytogenes.Infect. Immun. 68: 7061-7068), it was found that ClpC is required for cell adhesion and expression of virulence factors; The pneumococcal clpC mutant showed an adhesion defect on human type II alveolar cells and did not express the pneumolysine or choline binding protein CbpA, CbpE, CbpF, or CbpJ, which is a pleiotropic effect of ClpC on adherence. effect) (Charpentier et. al., supra). In this study, we demonstrated that the adhesion and permeability of pneumococcal clpP ⁻ mutants to host cells were not affected (data not shown). This may be due to the adverse action in the expression of proteins such as CbpA and PsaA. After thermal shock, clpP ⁻ did not show a net decrease in adhesion. Expression of CbpA in mutants was increased, whereas expression of PsaA was significantly reduced at both mRNA and protein levels (Kwon et al., Supra).

Molecular chaperones of the hsp70 and hsp100 families have been found to bind to dislocation complexes, and they interact with dislocation precursors (Berry, AM et al., 1989, Reduced virulence of a defined pneumolysin negative mutant of Streptococcus pneumoniae. Immun 57: 2037-2042; Vijayakumar, MN et al., 1986). Recently, B. subtilis ClpC and ClpX ATPase have been detected in the cell envelope and cytoplasm (Kruger, E. et al., 2000, supra). In this study, the transfer of the Clp protein itself after thermal shock or by other stresses has not been demonstrated. However, in this study, biochemical fractionation of pneumococci showed a significant increase in the amount of ClpP in cell wall fractions after thermal shock. Thus, ClpP is the first Clp protein found to migrate to the cell wall fraction after thermal shock, which interacts by breaking down pneumococcal proteins that are expected to interact or be transported / moved to host cells.

Bacterial HSP is induced during infection and mediates adhesion and permeability in addition to acting on proper folding of intracellular proteins (Charpentier et al., 2000; Nair et al., 2000; Parsons, LM, et al., 1997, Alterations in levels of DnaK and GroEL result in diminished survival and adherence of stressed Haemophilus ducreyi.Infect.Immun . 65: 2413-2419). In this study, we immunized mice with purified pneumococcal ClpP prior to challenge with toxic D39, comparable to those obtained from well-characterized pneumococcal protein vaccine candidates PspA and Ply. It has been demonstrated that protective immunity against is induced. The fact that a strong antigen specific antibody response was generated in mice immunized prior to challenge suggests that the defense may be mediated at least in part by antibodies. In conclusion, the expression of cps2A and ply can be mediated by ClpP at post-transcriptional levels. ClpP migrated to the cell wall after thermal shock, and immunization of mice with purified proteins prior to toxic pneumococcal test infection provided protective immunity to systemic diseases.

Example 3 Reactivity of Pneumococcal Group ClpP and Human ClpP

i) strains and cultures

Non-pathogenic pneumococcal (R type) Streptococcus pneumoniae CP1200 (Cho et al., 1999) strains without polysaccharide capsules were added to CAT medium (Casitone 1%, Tryptone 0.5%, NaCl 0.5%, Yeast Extract 0.1%). , Pathogenic pneumococcal D39 (type 2; Avery Avery, OT et al., 1944. Studies on the chemical nature of the substance inducing transformation of incubated in 0.175 MK ₂ HPO ₄ , and 0.2% glucose) and containing a polysaccharide capsule. pneumococcal types.Induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III.J.Exp.Hed. 79: 137-158) and the Spn1049 strain isolated from patients (Samsung Medical Center, Optochin and Sensitive to bile acids and incomplete blood reaction in blood agar medium) were incubated in Todd-Hewitt broth medium added with 0.5% yeast extract. Saccharomyces cerevisiae (ATCC 287) is used in YNB medium containing 2% glucose (Difco Laboratories, USA), Streptococcus thermophilus (KCTC 3778) is used in MRS medium (Difco In Laboratories, USA, Bacillus subtilis Marburg strain (Boylan SA, Chun KT, Edson BA, Price CW.Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis.Mol Gen Genet. 212 (2): 271-280 (1988)), Pseudomonas aeruginosa (ATCC 15522), Salmonella typhi (ATCC 27870), E. coli DH5α (Bethesda Research Laboratory), and Nutrient medium ( In Difco Laboratories, USA, human lung cancer cell line A549 (ATCC CCL 185) was cultured in DMEM medium (Gibco BRL) containing 10% FBS and 2% penicillin streptomysin.

ii) immunoblot analysis

In order to confirm the similarity between pneumococcal ClpP and other organism proteins, immunoblot with anti-ClpP antibody was performed. The lysates of pneumococci and various organisms were electrophoresed on 10% polyacrylamide gels, transferred to nitrocellulose membranes, immunoblotted with anti-ClpP antibodies, and then enzyme-labeled secondary antibodies. Search using. In other words, nitrocellulose was treated with a solution of Tris-buffered saline (TBS, mM Tris, 150 mM NaCl [pH 7.2]) containing 2% Tween 20 to block nonspecific antigen-antibody reactions. After reacting with rabbit anti-ClpP antiserum in a TBS solution containing 0.05% Tween 20 and shaking slowly for 1 hour at room temperature. Horse radish peroxidase (HRP) -conjugated goat anti-rabbit immunoglobulin G (IgG) antibody was used as a secondary antibody and reacted by diluting 1: 1000 in a TBS solution containing 0.05% Tween 20. Hydrogen peroxide and 95% ethanol dissolved N ', N', N ', N'-4methylbenzidine were reacted with a substrate and a color developer, respectively.

iii) Amino Acid Sequence Comparison of ClpP Protein

The BLAST search provided by the National Center of Biotechnology Information (NCBI, USA) was used to identify the genes adjacent to pneumococcal clpP and to confirm similarity with other organisms' ClpP proteins. The amino acid sequence deduced from the base sequence of pneumococcal clpP showed high similarity with that of other organisms. In particular, the ClpP proteins of Streptococcus salivarius and Streptococcus agalactiae were 88% and 87% consistent with 91% and 92% similarity, respectively. In addition, L. lactis ClpP and 89%, Enterococcus faecalis cock carcass (Enterococcus faecalis) ClpP and 81%, Staphylococcus cock coarse aureus (Staphylococcus aureus) and ClpP 79%, of Bacillus sub-blocks bus (B. subtilis) and ClpP 75% similarity was shown. In particular, it showed 70% similarity with Homo sapiens. Comparing the amino acid sequence of the ClpP protein showed a high level of similarity overall, especially the serine-96, histidine-121, and aspartate-172 residues, which are assumed to be active sites of the serine protease, showed very high levels of conservativeness.

iv) Similarity of pneumococcal ClpP with other ClpP members

Pneumococcal DnaK is highly antigenic (Hamel J, Martin D, Brodeur BB.Heat shock response of Streptococcus pneumoniae: identification of immunoreactive stress proteins.Microb Pathog, 23 (1): 11-21 (1997)) Antibodies did not react with human proteins (Kim SW, Choi IH, Kim SN, Kim YH, Pyo SN, Rhee DK. Molecular cloning, expression, and characterization of dnaK in Streptococcus pneumoniae.FEMS Microbiol Lett. 161 (2): 217-224 (1998)), which has been evaluated as a vaccine candidate, this possibility has been investigated for ClpP. First, cell lysis and immunoblot of other bacteria or higher organisms were performed to confirm that the ClpP protein is similar to the ClpP family of other organisms. Unexpectedly, they reacted with Gram-positive Streptococcus thermophilus (Sth) and pneumococcal D39 (D39) and pneumococcal isolates (Spn1049), but E. coli (Eco) and Saccharomycosis Cells of Saccharomyces cerevisiae (Sce), Bacillus subtilis (Bsu), Psuedomonas aeruginosa (Pae), Salmonella typhi (Sty) and human lung cancer cell line A549 It did not react with the protein at all. This result was shown to FIGS. 17A-17B. These results suggest that the ClpP protein of pneumococci can be used as a vaccine candidate, such as DnaK.

Since the vaccine containing the recombinant ClpP protein of pneumococci according to the present invention is an antigenic protein with high immunogenicity and conservatively present in all pneumococci, it has a low immunogenicity or does not have a protective effect against all serotypes. Without showing the disadvantages of the pneumococcal prophylaxis vaccine, it can effectively exhibit an immunoprotective effect against pneumococcal infection.

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Claims (15)

 A vaccine comprising recombinant pneumococcal ClpP protein as an antigen.  The vaccine of claim 1, further comprising an adjuvant.  3. The vaccine of claim 2 wherein the adjuvant is alum.  The vaccine according to any one of claims 1 to 3, wherein the vaccine is for immunizing a human or animal against pneumococcal infection.  The vaccine of claim 4, wherein the pneumococcal infection is bacterial pneumonia, otitis media, bacteremia or meningitis. delete delete delete  i) providing an expression vector to which the DNA sequence encoding pneumococcal ClpP is operably bound; ii) introducing the expression vector of i) into a host cell; And iii) A vaccine comprising recombinant pneumococcal ClpP prepared by a method comprising the step of separating and purifying recombinant pneumococcal ClpP from a host cell. delete A vaccine comprising a ( clpP ⁻ ) mutant having an attenuated pneumococcal ClpP gene. The method of claim 11 wherein the Streptococcus pneumoniae gene deletion ClpP (clpP ^-) mutant body 95 nucleotide sequence (the 206 th to the 300 th) vaccine characterized by the deletion mutated cheim. delete The vaccine of claim 9, wherein the expression vector is pET30 (a) -clpP . The vaccine of claim 9, wherein the host cell is Escherichia coli.

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