Patent Application
20120028879
The present invention is directed to a method of detecting an oral bacterium that causes hemorrhage aggravation, a method of screening for a subject at a high risk of hemorrhage aggravation, a method of determining the risk of hemorrhage aggravation in a subject, as well as detection reagents and kits for the use in these methods.
Conditions which involve hemorrhage through vascular injuries include such as hemorrhage by a rupture of a blood vessel that caused by a traumatic injury or pressure, hemorrhage at delivery and intracerebral hemorrhage. In a case of intracerebral hemorrhage, for instance, a severe disorder may be brought about by an injury of the neuronal tissue due to compression or necrosis of the brain associated with hemorrhage, or by neurologic symptoms due to a vascular spasm in cerebrum induced by bleeding in a case of subarachnoid hemorrhage etc. In order to improve the prognosis of hemorrhage, an effective treatment of hemorrhage (hemostasis) as well as the prevention of hemorrhage aggravation is necessary, and diagnosis of the risk of hemorrhage aggravation is important.
Markers used in the diagnosis of a disease which involves hemorrhage include, for example, Apo C-III, serum amyloid A, Apo C-I, antithrombin III fragment and Apo A-I (Patent literature 1) for the diagnoses of the possibility of a stroke, cerebrospinal fluid markers of cerebral ischemia such as adenylate kinase as well as β-thromboglobulin, vascular cell adhesion molecule (VCAM) and atriuretic peptide for the diagnoses of the prognosis of a stroke and cerebral injury, and von Willebrand factor (vWF), vascular endothelial growth factor (VEGF) and matrix-metalloprotease-9 (MMP-9) for the prediction of cerebral vascular spasm which occurs later (Patent literature 2). However, these are all markers for detecting already-happening bleeding in vivo, and cannot diagnose the risk of hemorrhage aggravation.
Accordingly, there have been needs for the establishment of a method of determining or screening a risk of causing aggravation of hemorrhage or a subject with such a risk, and a method of preventing or treating.
Accordingly, an object of the invention is to identify the responsible factor that causes aggravation of hemorrhage, and to construct a system for rapidly and readily specifying a patient having a risk of hemorrhage aggravation. Another object of the invention is to prevent the aggravation of hemorrhage in an individual having such a risk.
The inventors carried out an intensive study to achieve the aforementioned objects and found that hemorrhage is aggravated in a subject who has been infected with a particular strain of S. mutans. By additional studies the inventors found that the most severe virulence is exerted by bacterial strains that do not carry a protein antigen PA (Protein Antigen, also known as PAc, SpaP, antigen I/II, antigen B, SR, IF, P1, MSL-1), i.e., a major bacterial surface protein having a molecular weight of about 190 kDa, and that carry a collagen binding protein CBP (Collagen Binding Protein, also known as Cnm) having a molecular weight of about 120 kDa, and also discovered that all these virulent bacterial strains have low cell surface charge. The influences of S. mutans on hemorrhage has never been reported so far, and the findings that particular strains of S. mutans exacerbate the prognosis of hemorrhage and that PA and CBP as well as cell surface charge are involved in such virulence were therefore surprising results. Based on these findings, the inventors further proceeded with the study, and found that CBP-positive bacterium has an ability to inhibit platelet aggregation, thereby completed the invention.
Accordingly, the present invention relates to a method of detecting a hemorrhage aggravating oral bacterium, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a sample, wherein the presence of the hemorrhage aggravating oral bacterium is determined if PA is not detected and/or CBP is detected and/or the cell surface charge is negative.
Moreover, the present invention relates to a method of screening a subject at a high risk of hemorrhage aggravation, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a biological sample obtained from the subject, wherein a high risk of hemorrhage aggravation is determined if PA is not detected and/or CBP is detected and/or the cell surface charge is negative
Alternatively, the present invention relates to a method of judging the risk of hemorrhage aggravation in a subject, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a biological sample obtained from the subject, wherein a high risk of hemorrhage aggravation in the subject is determined if PA is not detected and/or CBP is detected and/or the cell surface charge is negative.
Furthermore, the present invention relates to any one of said methods wherein the hemorrhage is hemorrhage by diabrosis.
The present invention also relates to any one of said methods wherein the oral bacterium is Streptococcus mutans.
The present invention further relates to any one of said methods wherein PA is selected from the group consisting of:
The present invention further relates to any one of said methods wherein PA comprises a polypeptide consisting of an amino acid sequence according to SEQ ID NO. 1, 17, 19, 21 or 23.
The present invention also relates to any one of said methods wherein CBP is selected from the group consisting of:
The present invention also relates to any one of said methods wherein CBP comprises a polypeptide consisting of an amino acid sequence according to SEQ ID NO. 5, 9, 27 or 31.
Also, the present invention relates to a reagent for detection of a hemorrhage-aggravating oral bacterium, the reagent comprising an oral bacterial PA-detecting agent and/or CBP-detecting agent.
Furthermore, the present invention relates to an oral bacterial PA-specific antibody for detection of a hemorrhage-aggravating oral bacterium.
Moreover, the present invention relates to a kit for detection of a hemorrhage-aggravating oral bacterium and/or for screening of a subject at a high risk of hemorrhage aggravation and/or for determination of the risk of hemorrhage aggravation in the subject, the kit comprising at least:
a PA-detecting reagent, and
a CBP-detecting reagent.
Also, the present invention relates to a hemostatic agent comprising PA protein of an oral bacterium or nucleic acid encoding the PA protein.
Also, the present invention relates to an inhibitor of platelet aggregation comprising a substance that binds to an oral bacterium PA protein or to a nucleic acid encoding the PA protein.
Also, the present invention relates to a hemorrhage aggravation inhibitor comprising a substance that binds to an oral bacterium CBP or to a nucleic acid encoding the CBP protein.
Alternatively, the present invention relates to an agent for detecting collagen-denuded site in tissue comprising CBP of an oral bacterium.
Also, the present invention relates to a carrier for delivering a substance to the collagen-denuded site comprising CBP of an oral bacterium.
The present invention also relates to a therapeutic agent for hemorrhage comprising CBP of an oral bacterium and a hemostatic agent.
Moreover, the present invention relates to said therapeutic agent for hemorrhage for a subject having low sensitivity of platelet to collagen.
Also, the present invention relates to a prophylactic agent for hemorrhage aggravation comprising an oral bacterium-removing agent.
The present invention allows rapidly and easily diagnosing the risk of causing hemorrhage aggravation in a subject. Also, the method of the present invention enables detecting responsible factors of hemorrhage aggravation using readily-available biological samples such as saliva and plaque without employing any special analyzers. As such, the present invention allows to specify a high-risk population of hemorrhage aggravation, to treat the individuals belonging to this population with a regimen such as removing virulent bacteria and advising dental hygiene, and thereby to effectively prevent a hemorrhage aggravation.
The present invention provides a method of detecting a hemorrhage-aggravating oral bacterium, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a sample, wherein the presence of the hemorrhage-aggravating oral bacterium is determined by that PA is not detected and/or that CBP is detected and/or that the cell surface charge is negative.
The present invention provides, in another embodiment, a method of screening a subject at a high risk of hemorrhage aggravation, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a biological sample obtained from a subject, wherein a high risk of hemorrhage aggravation is determined by that PA is not detected and/or that CBP is detected and/or that the cell surface charge is negative.
The present invention further provide, in another embodiment, a method of determining the risk of hemorrhage aggravation in a subject, the method comprising detecting PA and/or CBP and/or cell surface charge of oral bacteria in a biological sample obtained from a subject, wherein a high risk of hemorrhage aggravation is determined in the subject by that PA is not detected and/or that CBP is detected and/or that the cell surface charge is negative.
A mutans streptococci Streptococcus mutans, an oral bacterium that is a major pathogenic bacteria of dental caries, are known to have four serotypes (c, e, f and k). S. mutans is also known to be a pathogenic bacterium of bacteremia and infective endocarditis, and reported to be relevant to cardiovascular diseases since bacterial DNA of S. mutans was detected from the specimens of cardiac valve and aortic aneurysm (Nakano et al., 2008, Japanese Dental Science Review, 44: 29-37). However, association of S. mutans to other diseases, for example its impact on cerebrovascular diseases, have never been investigated so far.
Studies by the inventors disclosed herein revealed that the intravenous administration of some of different S. mutans strains inhibits spontaneous hemostatic action and induces aggravation of hemorrhage, when mild cerebral hemorrhage has been induced by damaging the middle cerebral artery. A MT8148 strain generally isolated from the oral cavity (serotype (Minami et al., 1990, Oral Microbiol. Immunol., 5: 189-194) does not cause such effects, thought there are strains among serotype k that evokes hemorrhage aggravation. In particular, TW295 strain and TW871 strain (Nakano et al., 2004, Journal of Clinical Microbiology, 42(1); 198-202), SA53 strain (Nakano et al., 2007, J. Clin. Microbiol., 45: 2614-2625), and LJ32 strain (Nakano, K. et al., 2008, J. Dent. Res. 87: 964-968) cause a significant hemorrhage aggravation.
The inventors found that those highly virulent S. mutans strains lack PA, a major bacterial surface protein. The inventors also found that among the PA-deficient strains, the virulence of the strains carrying CBP, another bacterial surface protein, was particularly high. The inventors further confirmed that TW295 strain-like hemorrhage aggravation is not exhibited when CBP-encoding gene of TW295 strain has been deleted by genetic engineering; and that a strain in which PA-encoding gene has been deleted from MT8148 strain exhibits hemorrhage aggravation, confirming that CBP and PA are involved in hemorrhage aggravating activity of S. mutans. The inventors further found that CBP-carrying S. mutans strains are detected in the oral cavity of human patients with hemorrhagic stroke, and further confirmed that CBP-carrying S. mutans strains isolated from such patients cause aggravation of cerebral hemorrhage in vivo. The inventors further found that the cell surface charge of a highly virulent S. mutans strain is negative. Based on these findings, the inventors demonstrated that these bacterial surface protein and cell surface charge can be utilized as useful markers for detection of a S. mutans strain that exacerbates hemorrhage, for screening of a subject at a high risk of hemorrhage aggravation, and for determination of the risk of hemorrhage aggravation of a subject.
The oral bacterium detected according to the method of the present invention may exacerbate any bleeding, though, in particular, would exacerbate a hemorrhage by diabrosis caused by the occurrence of damage on the vascular wall due to a traumatic injury, an ulcer or a ruptured aneurysm. Representative examples of hemorrhage by diabrosis include such as cerebral hemorrhage (intracerebral hemorrhage, subarachnoid hemorrhage, chronic subdural hematoma), bleeding due to traumatic injury or compression, hemorrhage after delivery, subcutaneous hemorrhage associated with diseases. Also, diseases which cause bleeding tendency include connective tissue disorders (such as allergic purpura), thrombocytopenia (such as disseminated intravascular coagulation and aplastic anemia) or platelet disorders (such as thrombasthenia), or disorders in coagulation system (such as coagulation disorders associated with liver diseases and vitamin K deficiency). Endogenous or exogenous circulating anti-coagulation substances (such as lupus anticoagulant and VIII factor anti-coagulation substance) may also cause bleeding tendency.
Hemorrhage aggravation herein means that the spontaneous hemostatic action against bleeding caused by such endogenous or exogenous factor is delayed, decreased or lost as compared to a normal subject. Also, a subject at a high risk of hemorrhage aggravation means that, in said subject, the spontaneous hemostatic action by platelets is highly likely to be delayed, decreased or lost as compared to a normal subject upon the bleeding due to an endogenous or exogenous factor.
PA (Protein Antigen) is a surface protein of approximately 190 kDa found in MT8148 strain, a S. mutans wild-type strain, and also known in various other names such as PAc (Protein Antigen c), SpaP, Antigen I/II and Antigen B, P1 and MSL-1. PA polypeptide comprises 3 alanine-rich repeat domains (A-region) at N-terminal side and 3 proline-rich repeat domains (P-region) at central part, and has cell wall/membrane-spanning domain at C-terminal. It has been reported that the A-regions are involved in the attachment of bacterial cells to teeth (Matsumoto-Nakano et al., 2008, Oral Microbiology and Immunology, 23:265-270). Also, there have been reports that PA is involved in infective endocarditis by S. mutans (Nakano et al., 2008, Japanese Dental Science Review, 44: 29-37); that an antibody against PA inhibits the attachment of bacterial cells to a hydroxyapatite substrate (Kawato et al., 2008, Oral Microbiology and Immunology, 23:14-20); and that an antiserum against PA is useful as a vaccine for dental caries (Okahashi et al., 1989, Molecular Microbiology, 3(2): 221-228). Although there is a region between A-region and P-region of PA, in which amino acid sequences are highly variable between strains (for example, in MT8148 strain, residues from 679 to 827), the repeat domain and transmembrane domain are highly conserved among strains.
Also, it is reported that strains of serotype k, which are often detected in patients with infective endocarditis, lacks PA in a high percentage, and that both the hydrophobicity of the bacterial body sensitivity to phagocytosis are low in this serotype (Nakano et al., 2008, Journal of Dental Research, 87(10): 964-968).
Known PA includes, for example, PA of serotype c MT8148 (DDBJ Accession No. X14490, amino acids: SEQ ID NO. 1, nucleic acids: SEQ ID NO. 2), PA of LJ23 strain (DDBJ Accession No. AB364261, amino acids: SEQ ID NO. 17, nucleic acids: SEQ ID NO. 18), PA of SA98 strain (DDBJ Accession No. AB364285, amino acids: SEQ ID NO. 19, nucleic acids: SEQ ID NO. 20), as well as spaP gene of antigen I/II (DDBJ Accession No. X17390, Kelly et al., 1989, FEBS Lett. 258(1), 127-132, amino acids: SEQ ID NO. 21, nucleic acids: SEQ ID NO. 22) and a meningococcus Neisseria meningitidis iron binding protein fbp gene (X53469, Berish et al., 1990, Nucleic Acid Research, 18(15): 4596-4596, amino acids: SEQ ID NO. 23, nucleic acids: SEQ ID NO. 24).
CBP, i.e., another anchor protein of S. mutans (also denoted as Cnm), is a Type I collagen binding protein of approximately 120 kDa molecular weight, and has a collagen binding domain (CBD, residues from 152 to 316), B repeat domain (residues from 328 to 455) and LPXTG motif (residues from 507 to 511) (Sato et al., 2004, Journal of Dental Research, 83(7): 534-539). CBP gene-carrying frequency of S. mutans is about 10 to 20%, and CBP-positive strain is predominantly expressed in serotype f and k (Nakano et al., 2007, J. Clin. Microbiol., 45: 2616-2625).
The studies by the inventors revealed that, for CBP of serotype k TW295 strain (DDBJ Accession No. AB102689, amino acids: SEQ ID NO. 3, nucleic acids: SEQ ID NO. 4), CBD (amino acids: SEQ ID NO. 5, nucleic acids: SEQ ID NO. 6) and LPXTG motif are highly conserved between strains, whereas the number of repeats in the B repeat domain varies between strains (Nomura et. al., 2009, J. Med. Microbiol., 58:469-75).
In one embodiment of the present invention, PA is defined as:
Preferably, PA comprises a polypeptide consisting of an amino acid sequence expressed by SEQ ID NOs. 1, 17, 19, 21 or 23. More preferably, PA comprises a polypeptide consisting of an amino acid sequence expressed by SEQ ID NO. 1.
PA that can be used in the method of the present invention may be a polypeptide comprising one or more amino acid mutations (deletions, substitutions and/or additions), as long as it comprises an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent condition with a nucleic acid sequence expressed by SEQ ID NOs. 2, 18, 20, 22 or 24 (nucleic acid encoding the PA protein sequence) or its complementary sequence or its fragment, and has a equal function as a polypeptide comprising an amino acid sequence expressed by SEQ ID NOs. 1, 17, 19, 21 or 23 (amino acid sequence of PA protein). Mutations may be naturally occurring mutations or mutations generated by any known procedures, e.g., cleavage or insertion of a nucleic acid by restriction enzyme, site-specific mutagenesis, or radiation or ultraviolet irradiation. Moreover, the number of mutated amino acids may be 1 to 20, 1 to 15, 1 to 10, or 1 to several, for example.
Furthermore, in one embodiment of the present invention, CBP is defined as:
CBP polypeptide that can be used in the method of the present invention may be a polypeptide comprising one or more, e.g., 1 to 20, 1 to 15, 1 to 10, or one or several amino acid mutations (deletions, substitutions and/or additions), as long as it comprises an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent condition with a nucleic acid sequence expressed by SEQ ID NOs. 6, 10, 28 or 32 (nucleic acid sequence encoding CBD of S. mutans TW295, TW871, SA53 or LJ32 strains) or its complementary sequence or its fragment, and has an equal function as a polypeptide comprising an amino acid sequence expressed by SEQ ID NOs. 5, 9, 27 or 31 (CBD amino acid sequence of S. mutans TW295, TW871, SA53 or LJ32 strain).
For instance, CBP polypeptide may be a polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that hybridizes under stringent condition with a nucleic acid sequence expressed by SEQ ID NOs. 4, 8, 26 or 30 (a nucleic acid sequence encoding CBP of S. mutans TW295 strain, TW871 strain (DDBJ Accession No. AB469914), SA53 strain (AB465299) or LJ32 strain (AB465263)) or its complementary sequence or its fragment, and has an equal function as a polypeptide comprising an amino acid sequence expressed by SEQ ID NOs. 3, 7, 25 or 29 (an amino acid sequence of CBP protein of S. mutans TW295, TW871, SA53 or LJ32 strain).
Preferably, CBP comprises a polypeptide consisting of an amino acid sequence expressed by SEQ ID NOs. 5, 9, 27 or 31.
Whether a PA or CBP mutant has an equal function as PA or CBP or not may be confirmed using any known means. For instance, the ability of PA mutant making the bacterial cell adhere to a hydroxyapatite substrate may be determined by raising a specific antibody against the mutant peptide by a known method, and assaying the inhibition of adhesion of bacteria to the hydroxyapatite by said antibody according to a method described in Kawato et al., 2008, Oral Microbiology and Immunology, 23:14-20. Alternatively, the biding ability of a CBP mutant to Type I collagen may be determined by collagen binding assay described in Nomura et al., 2009, J. Med. Microbiol., 58(4): 469-475. By such means, the ability of a mutant can be assessed in comparison with an appropriate negative control, or with PA or CBP as a positive control. For instance, certain mutant is considered as a functional mutant when at least one function described above is better, e.g., 10% or better, 25% or better, 50% or better, 75% or better, or even 100% or better, than the negative control, and/or when said function is 1/100 or less, 1/50 or less, 1/25 or less, 1/10 or less, ⅕ or less, or even ½ or less, than the positive control.
In the method of the present invention, the surface charge of a bacterial cell can be measured by any known method, e.g., zeta potential measuring method. Zeta potential, also called as electrokinetic potential, is a potential difference that arises on the interface between a solid and a liquid contacting to each other in a relative motion, which may be used as an index for the surface charge of a bacterial cell. Zeta potential can be calculated from electrophoretic mobility of bacterial cells using an equation of Smoluchowski:
ζ=ηu/ε0εr
wherein, ζ indicates the zeta potential, η indicates the viscosity of the solvent, u indicates the electrophoretic mobility, ε0 indicates the dielectric constant of a vacuum, εr indicates the dielectric constant of the solvent.
Methods of electrophoresis suitable for measuring zeta potential are not particularly limited as long as it can measure the migrating speed of bacterial cells, and include, for example, capillary electrophoresis, microscopic electrophoresis, rotating diffraction gating method and laser Doppler electrophoresis.
In the method of the present invention, a negative surface charge of the bacterial cell is an index for a highly virulent oral bacterium, and is a criterion for the presence of a hemorrhage-aggravating oral bacterium and a risk of hemorrhage aggravation. Namely, collagen fibers denuded within a damaged vessel are positively charged, and if bacterial cell surface is negatively charged, the bacterial cell may easily interact with denuded collagen fibers, thereby resulting in hemorrhage aggravation due to the inhibition of platelet aggregation. Typically, an oral bacterium is determined to be highly virulent when the surface charge measured as zeta potential is −1.0 mV or below, more preferably −3.0 mV or below, still more preferably −4.0 mV or below, even more preferably −5.0 mV or below, particularly preferably −8.0 mV or below.
In aforementioned methods of the present invention, oral bacterial PA, CBP and cell surface charge may be used either alone or in combination. Accordingly, either PA alone, CBP alone, or cell surface charge alone may be detected, or any combination of PA, CBP and cell surface charge, namely, both PA and CBP, both PA and cell surface charge, both CBP and cell surface charge, or, all of PA, CBP and cell surface charge may be detected. Furthermore, each of the criteria, i.e., that PA is not detected, that CBP is detected and that the cell surface charge is negative, may be used alone or in combination, according to the items to be detected.
Major bacteria species that are identified as hemorrhage-aggravating oral bacteria include mutans streptococci such as Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, Streptococcus downei, Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, and Streptococcus salivarius. Particularly, S. mutans TW295 strain, TW871 strain, SA53 strain, and LJ32 strain would cause severe hemorrhage aggravation.
Screening of other bacteria that could induce hemorrhage aggravation can be carried out utilizing databases such as NCBI GenBank®, DDBJ (DNA Data Bank of Japan, http://www.ddbj.nig.ac.jp/) and EMBL, and publicly available search tools such as BLAST.
The present invention provides, in one embodiment, a reagent for the detection of a hemorrhage-aggravating oral bacterium comprising an oral bacterial PA detecting agent and/or an oral bacterial CBP detecting agent.
In one embodiment, the PA detecting agent comprises an oral bacterial PA-specific antibody. Using the PA-specific antibody developed by the inventors, the presence or absence of a highly virulent S. mutans can rapidly and easily detected. The PA-specific antibody is preferably an antibody or its fragment induced from polypeptide comprising an amino acid sequence of SEQ ID NO. 1 or its immunogenic fragment. Alternatively, the PA-specific antibody may be an antibody or its fragment induced from a polypeptide having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology with an amino acid sequence of SEQ ID NOs. 1, 17, 19, 21 or 23, and having an immunogenicity to induce an antibody production against a polypeptide comprising an amino acid sequence of SEQ ID NOs. 1, 17, 19, 21 or 23. For example, a recombinant PA comprising the polypeptide (see, e.g., Nakano et al., 2006, Microbes and Infection, 8:114-121) may be used as an antigen to produce a monoclonal or polyclonal antibody.
In one embodiment, CBP detecting agent comprises a substrate (such as a microplate, test tube or slide glass) coated with Type I collagen. The binding affinity of CBP to Type I collagen (Nomura et al., 2009, J. Med. Microbiol., 58(4): 469-475) can be utilized to allow CBP-expressing bacterial cell to attach a substrate coated with Type I collagen, which can easily be detected.
In another embodiment, the CBP detecting agent comprises a specific antibody against an oral bacterial CBP. The CBP-specific antibody may be a specific antibody against the collagen binding domain of CBP, preferably, an antibody or its fragment induced from a polypeptide comprising an amino acid sequence of SEQ ID NOs. 5, 9, 27 or 31 or its immunogenic fragment. Alternatively, the CBP-specific antibody may be an antibody or its fragment induced from a polypeptide having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more homology with an amino acid sequence of SEQ ID NOs. 5, 9, 27 or 31, and having an immunogenicity to induce an antibody production against a polypeptide comprising an amino acid sequence of SEQ ID NOs. 5, 9, 27 or 31.
In the present invention, the antibody fragment comprises, for example, without limitation, various functional fragments such as Fab, Fab′, F(ab′)2, scFv, dsFv (disulfide-stabilized V region fragment), and CDR-containing fragment.
The present invention provides, in one embodiment, a kit for the detection of a hemorrhage-aggravating oral bacterium, and/or for the screening of a subject at a high risk of hemorrhage aggravation, and/or for the determination of the risk of hemorrhage aggravation in a subject. The kit comprises at least a PA-detecting reagent and a CBP-detecting reagent.
In one embodiment, the kit comprises as a PA-detecting reagent an oral bacterial PA-specific antibody.
In one embodiment, the kit comprises as a CBP-detecting reagent a substrate coated with Type I collagen (such as a microplate, test tube or slide glass).
In another embodiment, the kit comprises as a CBP-detecting reagent a CBP-specific antibody.
The kit of the present invention may further comprise one or more of the followings for culturing S. mutans:
The kit of the present invention may further contain one or more of the followings for detecting S. mutans:
The kit of the present invention may further comprise one or more of the followings for detecting PA-deleted S. mutans:
The kit of the present invention may further comprise one or more of the followings for detecting CBP-carrying S. mutans:
A skilled person in the art may appropriately adjust the concentration of above-mentioned component, e.g., antiserum, secondary antibody, formaldehyde or crystal violet, to be optimum depending on the experimental condition.
The method of the present invention for the detection of a hemorrhage-aggravating oral bacterium is carried out, specifically, for example in a scheme comprising following four steps as shown in
In Analysis 1, culturing of bacteria is carried out by following procedures using for example instruments and reagents in the aforementioned kit for culturing mutans streptococci.
The saliva of the subject is collected in a small amount using a spitz for collecting saliva. 10 μl of the saliva is taken from the spitz using a dropper, plated onto a S. mutans selection agar medium (e.g., above-mentioned Special Medium A), and cultured at 37° C. for 48 hours, preferably under an anaerobic condition. After culturing, the presence of bacterial colonies are grossly confirmed, colonies are picked up and added to a liquid medium (e.g., above-mentioned Special Medium B) and cultured for 37° C. for 18 hours, then used for the following Analysis 2, 3 and 4. Preferably, rough colonies are picked up, since S. mutans forms rough colonies, whereas S. sobrinus forms smooth colonies.
In Analysis 2, detection of S. mutans is carried out by following procedures using for example instruments and reagents in the aforementioned kit for detecting S. mutans.
10 μl of the bacterial solution cultured from the method of Analysis 1 is added to a medium (e.g., above-mentioned Special Medium C), incubated at 37° C. for 3 hours. The medium is washed with a wash buffer (e.g., above Wash Buffer A) for three times, then left still about 15 minutes with the last wash buffer. The wash buffer is removed, and again the medium is washed with Wash Buffer A for once, then a buffer containing a Gram-positive bacteria staining reagent (e.g., above Buffer 1) is added and left still for 1 minute. It is washed with the wash buffer for three times, and a buffer containing a mordanting agent (e.g., above Buffer 2) is added. If the color of the medium was changed, it is determined to be S. mutans-positive, if the color of the medium is unchanged, it is determined to be S. mutans-negative. A reagent in which a staining reagent and a mordanting agent are already combined may also be used.
In Analysis 3, detection of PA-deleted S. mutans is carried out by following procedures using for example instruments and reagents in the aforementioned kit for detecting PA-deleted S. mutans.
To the bacterial solution cultured by the method of Analysis 1 above a suitable buffer (e.g., above-mentioned Buffer 3) is added, which is then immersed in boiling water for 10 minutes, and frozen if it is to be stored.
In Analysis 4, detection of CBP-carrying S. mutans is carried out by following procedures using for example instruments and reagents in the aforementioned kit for detecting CBP-carrying S. mutans.
Wash Buffer C), bacterial solution cultured by'the method of Analysis 1 above is added, and incubated at 37° C. for 2 hours.
It is determined to be CBP-positive then the color of the solution is changed, and it is determined to be CBP-negative when the color of the solution is not changed.
In any of the detecting methods described above, the detection is possible if bacterial concentration is 1 CFU or more.
Moreover, a culture of e.g., S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius may be used as a control to confirm in Analysis 1 that any bacterium other than S. mutans and S. sobrinus grows; in Analysis 3 that any bacterium other than PA-carrying S. mutans shows a positive reaction; and in Analysis 4 that any bacterium other than CBP-carrying S. mutans shows a positive reaction, respectively
A skilled person in the art may appropriately modify the method of the present invention according to its object. For example, for detecting PA-deleted S. mutans, a substrate to which a specific antibody for PA or CBP is attached may be contacted with a bacterial solution, washed to remove the bacteria which are not attached to the substrate, then only the bacterial cells that are attached to the substrate can be detected by the Gram-positive bacteria staining reagent. Alternatively, primers or probes for a PA or CBP-coding nucleic acid may be used to detect whether the cultured bacterium has the gene of PA or CBP.
In preferred embodiment of the present invention, S. mutans MT8148 strain may be used as a positive control for detection of a PA-deleted oral bacterium, and/or as a negative control for detection of a CBP-carrying oral bacterium. As a positive control for detection of a PA-deleted oral bacterium, depending on the detection method, an isolated PA protein, a nucleic acid or vector comprising a DNA encoding PA or its fragment, a cell transformed with said vector may also be used. As a negative control for detection of a CBP-carrying oral bacterium, CND strain, which is a TW295 strain in which CBP-encoding gene has been knocked out, and a Gram-positive bacterium that does not express CBP may also be used.
The present invention provides, in one embodiment, a hemostatic agent comprising an oral bacterial PA protein or a nucleic acid encoding the PA protein. When the subject has been infected with a PA-deficient, highly virulent bacterium, a hemostatic effect through the induction of platelet aggregation will be provided by supplying PA protein or expressing PA in the subject or bacterium.
Accordingly, the present invention also provides a use of an oral bacterial PA protein or a nucleic acid encoding the PA protein for the production of a hemostatic agent, as well as a method of hemostatic method comprising a step of administering an oral bacterial PA protein or a nucleic acid encoding the PA protein.
The present invention provides, in another embodiment, an inhibitor of platelet aggregation caused by a PA-expressing oral bacterium, the inhibitor comprising a substance that binds to an oral bacterial PA protein or a nucleic acid encoding the PA protein. When the subject has been infected with a PA-expressing oral bacterium, PA in the bacterial cell surface layer may be blocked by a substance that binds to PA protein, or the production of PA by the bacterial cell may be inhibited by a substance that inhibits the expression of PA protein, thereby inhibiting the platelet aggregation effect of the bacterium can be inhibited.
Accordingly, the present invention also provides a use of a substance that binds to an oral bacterial PA protein or a nucleic acid encoding the PA protein for the production of an inhibitor of platelet aggregation caused by a PA-expressing oral bacterium, as well as a method of inhibiting platelet aggregation caused by a PA-expressing oral bacterium comprising a step of administering a substance that binds to an oral bacterial PA protein or a nucleic acid encoding the PA protein.
The present invention provides, in another embodiment, an inhibitor of hemorrhage aggravation comprising a substance that binds to an oral bacterial CBP or a nucleic acid encoding the CBP protein. When the subject has been infected with a CBP-expressing hemorrhage-aggravating oral bacterium, using a substance that binds to CBP, e.g., a CBP-specific antibody, the CBP protein in the bacterial cell surface layer may be blocked and the binding of the bacterial cell to collagen-denuded site (i.e., the damaged site of vascular endothelia) may be inhibited, thereby treating or preventing hemorrhage aggravation. Alternatively, by using a nucleic acid encoding a substance that binds to CBP protein (e.g., an siRNA, antisense nucleic acid), CBP production by a bacterial cell can be inhibited, thereby inhibiting the binding of the bacterial cell to collagen-denuded site.
Accordingly, the present invention also provides a use of a substance that binds to an oral bacterial CBP or a nucleic acid encoding the CBP protein for the production of a hemorrhage aggravation inhibitor, as well as a method of inhibiting hemorrhage aggravation comprising a step of administering a substance that binds to an oral bacterial CBP or a nucleic acid encoding the CBP protein.
The present invention provides, in another embodiment, an agent for detecting collagen-denuded site in tissue comprising CBP of an oral bacterium. When connective tissue collagen is denuded due to vascular endothelia injury, the damaged site can be detected using the detecting agent of the present invention. Particularly, the detecting agent of the present invention allows noninvasive detection of the damaged site even if the hemorrhage site is in an area difficult to be detected, e.g., in head. Various labels may be added to the detecting agent for the convenience of detection. The label may be selected from any known labels, e.g., any radioisotopes, magnetic bodies, a substance that binds to the above-mentioned components (e.g., an antibody), biotin, fluorescent substances, fluorophores, chemiluminescent substances, elements that induce nuclear magnetic resonance (e.g., hydrogen, phosphorus, sodium and fluorine) and enzymes.
Accordingly, the present invention also provides a use of oral bacterial CBP for the production of an agent for detecting collagen-denuded site in tissue, as well as a method of detecting a collagen-denuded site in tissue comprising a step of administering an oral bacterial CBP.
Furthermore, the present invention provides, in another embodiment, a carrier for delivering a substance to the collagen-denuded site comprising an oral bacterial CBP. The hemostatic agent of the present invention or other drugs (e.g., an antibiotic or an anti-inflammatory agent) can be incorporated into the delivering carrier and administering it to an organism to target the hemostatic agent and the drugs to the damaged site, thereby expecting a damaged site-specific therapy. The carrier may be, for example, a liposome fused with a CBP protein or its collagen binding domain (CBD). To the carrier of the present invention, the hemostatic agent of the present invention or other drugs may be incorporated. Alternatively, the carrier of the present invention may be the CBP protein itself, and in this case, the therapeutic agent can directly be bound to the CBP protein or CBD.
The present invention provides, in another embodiment, a therapeutic agent for hemorrhage comprising an oral bacterial CBP and a hemostatic agent. The therapeutic agent for hemorrhage of the present invention is particularly useful in a subject having low platelet sensitivity to collagen. A subject having low platelet sensitivity to collagen includes a subject suffering such as aplastic anemia, acute leukemia, thrombocytopenic purpura, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, systemic lupus erythematosus, thrombasthenia or storage pool syndrome. Also, the therapeutic agent for hemorrhage of the present invention is particularly useful in a subject having a disease caused by a disorder of coagulation factor, such as hemophilia.
The CBP to be used for the carrier for substance delivery to the collagen-denuded site and therapeutic agent for hemorrhage of the present invention may be obtained, for example, by incorporating a nucleic acid construct comprising CBP gene into a suitable expression vector, and expressing CBP protein in the suitable host cell. Such techniques are well known in the art. For example, a plasmid, cosmid, phage, virus, YAC or BAC vector system comprising CBP gene can be incorporated into a host cell by various nucleic acid introducing method, e.g., calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, microinjection method, liposome method (e.g., by cationic liposome), competent cell method or protoplast method to express CBP gene. CBP may also be the CBP-positive bacterium itself, or the CBP-containing component of the CBP-positive bacterium. Such component may be isolated by, for example, lysing and/or homogenizing CBP-positive bacteria and exposing to a substrate coated with Type I collagen. If the CBP-positive bacterium itself is to be used, said bacterium may be inactivated by a conventional method.
Moreover, the present invention relates to, in another embodiment, a prophylactic agent for hemorrhage aggravation comprising an agent for removing an oral bacterium.
According to the method of the present invention, in a case if a hemorrhage-aggravating oral bacterium has been detected, the hemorrhage-aggravating oral bacterium should be removed from the subject in order to alleviate the risk of hemorrhage aggravation and prevent it. As an oral bacterium-removing agent e.g., beta-lactam antibiotic may be used. A beta-lactam antibiotic includes, e.g., penicillin, methicillin, cephalosporin, cephamycin and carbapenems.
The hemostatic agent, platelet aggregation inhibitor, hemorrhage aggravation inhibitor, prophylactic agent for hemorrhage aggravation, therapeutic agent for hemorrhage, collagen-denuded site detecting agent and the carrier for substance delivery to the collagen-denuded site of the present invention may be administered by various routes encompasses oral and parenteral routes, such as, for example, oral, buccal, intravenous, intramuscular, subcutaneous, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary and intrauterine routes, and may be formulated into a dosage form suitable for each administration route. Any known dosage form and method for formulation may be employed as appropriate (see, e.g., Watanabe et al., eds., 2003, HYOJUN YAKUZAIGAKU, Nanzando).
For example, formulations suitable for oral administration include, without limitation, a powder, granule, tablet, capsule, liquid, suspension, emulsion, gel and syrup. Formulations suitable for parenteral administration include injections such as an injectable solution, injectable suspension, injectable emulsion, and preparation-at-use injection. A formulation for parenteral administration may be in a form of aqueous or nonaqueous isotonic sterile solution or suspension. Specifically, for example, it may be formulated into a suitable unit dosage form, by combining appropriately with a pharmacologically acceptable carrier or medium such as, in specific, sterile water or physiological saline, vegetable oil, emulsifier, surfactant, stabilizing agent, excipient, vehicle, preservative or a binder. The amount of the effective ingredient in these formulations may be determined as appropriate so that a therapeutically effective amount can be provided to the subject in the defined dosage frequency.
Injectable aqueous solutions include, for example, a physiological saline, an isotonic solution comprising glucose and other adjuvant, e.g., D-sorbitol, D-mannose, D-mannitol and sodium, chloride. Appropriate solubilizing agent such as alcohol, specifically ethanol, a polyalcohol such as propyleneglycol, polyethyleneglycol, or a nonionic surfactant such as polysorbate 80 or HCO-50 may be used in combination.
Oily solutions includes e.g., a sesame oil and soy bean oil, which may be used in combination with a solubilizer such as benzyl benzoate or benzyl alcohol. Moreover, a buffering agent, e.g., a phosphate buffer, sodium acetate buffer, soothing agent, e.g., procaine hydrochloride, stabilizing agent, e.g., benzyl alcohol, phenol or antioxidant may be mixed. The injection prepared is usually filled in an appropriate container such as an ampoule, vial, tube, bottle or a pack.
Administration of hemostatic agent, platelet aggregation inhibitor, hemorrhage aggravation inhibitor, prophylactic agent for hemorrhage aggravation, therapeutic agent for hemorrhage, collagen-denuded site detecting agent and the carrier for substance delivery to the collagen-denuded site of the present invention into the body of subject may be via any of the above-mentioned routes, though, preferably, it is parenteral administration, more preferably topical or intravenous administration, particularly preferably intraportal or intratumoral administration. The frequency of dosage is preferably at once, though plurality of dosage may be used depending on the situation. The duration of dosage may be short, or may be sustained for a long time. More specifically, the composition of the present invention may be administered by injection or transdermally. The examples of administration by injection include but not limited to, e.g., by local injection, intravenous injection, intra-arterial injection, selective arterial infusion, portal vein injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intratumoral injection, intrathecal injection, intra-articular injection, intraventricular injection. An intravenous injection allows an administration in a manner of an ordinal blood transfusion, requiring neither a surgical operation to the subject nor local anesthesia, thus enabling alleviating the burden of both the subject and the operator. Moreover, it is advantageous that administration can be carried out elsewhere out of an operation room.
Furthermore, the present invention relates to, in one embodiment, a method of treating hemorrhage comprising administering an effective amount of the hemostatic agent, hemorrhage aggravation inhibitor, prophylactic agent for hemorrhage aggravation and/or therapeutic agent for hemorrhage described above to a subject. The present invention also relates to, in one embodiment, a method of treating a disease condition caused by platelet aggregation comprising administering an effective amount of the platelet aggregation inhibitor described above to a subject. Disease conditions caused by platelet aggregation include thrombosis and disseminated intravascular coagulation.
Moreover, the present invention relates to, in one embodiment, a method for diagnosing the site of hemorrhage comprising administering the collagen-denuded site detecting agent described above to a subject. Furthermore, the present invention relates to, a method of treating a disease associated with hemorrhage comprising administering an effective amount of the carrier for delivering a substance to the collagen-denuded site to a subject.
In the method of treatment or diagnosis of the present invention, the administration of the composition for treatment or diagnosis of the present invention to a subject may appropriately performed according to, for example, above-mentioned administration method. Also, a physician or veterinarian may appropriately modify the administration method described above to administrate the agent of the invention to a subject. Here, an effective amount is an amount of the hemostatic agent, hemorrhage aggravation inhibitor and/or therapeutic agent for hemorrhage described above that inhibits, alleviates or prevents the hemorrhage, or an amount of the platelet aggregation inhibitor that decreases the onset of, alleviates the symptoms or preventing the progress of a disease condition caused by platelet aggregation. It is preferably an amount that does not cause an adverse effect that exceeds the benefit by the administration. Such amount may be determined as appropriate by an in vitro examination using cultured cell, etc., or an examination in an animal model of such as a mouse, rat, dog or pig.
Specific amount of the composition for treatment or diagnosis of the present invention to be administered in the method of treatment or diagnosis of the present invention may be determined in consideration of various conditions associated with the subject in need of such treatment, e.g., the severity of the symptom, general health conditions of the subject, age, body weight and sexuality of the subject, diet, timing and frequency of administration, combination therapies, reactivity to the treatment, and the compliance to the treatment, etc., and thus may differ from the general effective amount, though, even in such cases, these methods are still encompassed within the scope of the present invention.
Routes of administration include various routes encompassing both oral and parenteral routes, e.g., oral, buccal, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, intraportal, intraventricular (cardiac), transmucosal, transdermal, intranasal, intraperitoneal, intrathecal, intraarticular, intraventricular (brain), intrapulmonary and intrauterine routes.
The frequency of administration may vary depending on the characteristics of the composition to be used and the conditions of the subject as described above, though, for example, it may be plurality of times a day (namely, twice, three times, four times or five times or more a day), or once a day, once per several days (namely, e.g., every 2, 3, 4, 5, 6 or 7 days), once a week, once per several weeks (namely, e.g., every 2, 3 or 4 weeks).
Moreover, in the method of treating hemorrhage of the present invention, a drug other than the hemostatic agent, hemorrhage aggravation inhibitor and/or therapeutic agent for hemorrhage of the present invention which is effective for the treatment of a hemorrhage-associated disease described above may be used in combination. Also, in the method of treating a disease condition caused by platelet aggregation of the present invention, a drug other than the platelet aggregation inhibitor of the present invention which is effective for the treatment of a disease condition caused by platelet aggregation may be used in combination.
The term “subject” in the present invention means any living organism, preferably an animal, still more preferably a mammal, still more preferably a human individual.
Hereinafter, the present invention is more specifically illustrated by way of examples, though the present invention is not to be limited by these examples.
All animal experiments in the present study has been carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH), and approved by the Institutional Animal Care and Use Committee of the Graduate School of Dentistry Osaka University and Hamamatsu University School of Medicine.
Study protocols using human samples has been approved by the ethics committee of the Graduate School of Dentistry Osaka University, Hamamatsu University School of Medicine and Suita Municipal Hospital (Suita City, Osaka, Japan). Before entry, all subjects were asked to sign to a consent form after the explanation about the protocols.
Major S. mutans strains used in the present study are shown in Table 1 (Reference 11, 21, 24, 25 and 29). Furthermore, 58 clinical S. mutans strains (strains isolated from blood: n=13, strains isolated from oral cavity: n=45) were used in the present study. All strains were cultured in Brain Heart Infusion (BHI) broth (Difco Laboratories, Detroit, Mich., USA), and erythromycin was added for the selection of the mutant strains. For each assay, bacterial cells were washed with PBS, and diluted to adjust the cell number.
S. mutans used in this study.
The collagen binding properties of the mutant strain and parent strain were assessed by a modified version of the method of Reference 27 (Reference 22). The result for each strain was shown in a percentage relative to the binding of TW871.
Platelet aggregation assay were carried out using mouse whole blood by the impedance method with an aggregometer (Whole-blood aggregometer C540, Baxter Ltd., Tokyo, Japan). In brief, whole blood were taken from mice (ICR, male, 8 weeks old, body weight 35 to 40 g, CLEA Japan, Inc., Tokyo, Japan), and the mixture of the whole blood and various amount (103, 105 or 107 CFU) of the bacterial cells were incubated at 37° C. for 5 minutes, then 4.0 pg collagen (native collagen fibril (Type I), Chrono-log Co., Havertown, Pa., USA) were added. The aggregation rate for each strain were calculated by the impedance (Ω) values in the presence or absence of the bacterial cells, and expressed as a percentage to that of the vehicle (where only collagen were added). Also, the platelet aggregation properties of 58 clinical strains and 3 MT8148 isogenic mutant strains were analyzed in the presence of 107 bacterial cells.
The cell surface charge of the bacteria tested was measured using zeta potential analyzer (ELSZ-2, Otsuka Electronics, Co., Ltd., Hirakawa, Osaka, Japan). Said analyzer automatically calculates the zeta potential from the electrophoretic mobility using Smoluchowski equation. The bacterial cells cultured overnight were washed with PBS, adjusted to be 107 CFU, loaded onto the analyzer, which automatically measured the zeta potential of the cells at five standard points. The results are shown as the mean values.
In mice, an injury was induced in vascular endothelial cells of the middle cerebral artery using a modified version of the already-described photochemical method (References 12, 28,
Gelatin gel zymography was carried out by a modified version of already-described method (Reference 13). In brief, the tissue sample collected 24 hours after the administration of either tested bacteria or a vehicle was homogenized in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS and 0.1% deoxycholic acid, pH7.4, supplemented with a protease inhibitor. Subsequently, the sample was separated using gelatin-zymo electrophoresis kit (Cosmo Bio., Tokyo, Japan).
Three hours after the induction of cerebral hemorrhage, the brain tissue was resected from the mouse, and the region of cerebral hemorrhage was observed with an electron microscope. In brief, the brain having a hemorrhage was fixed with 2% glutaraldehyde and dissected so that the section included a part of the obstacle, which was then fixed again with 1% osmium tetraoxide and dehydrated through an ethanol series. The sample was frozen, fractured into 2 to 4 pieces using a freeze-fracturing device filled with liquid nitrogen. The torn surface was perpendicular to the cerebral surface and included the hemorrhage site. Fractured samples were desiccated with a freeze-drying apparatus using t-butyl alcohol, then attached to the sample stage using a conductive paste so that the section came on top, and coated with osmium in order to confer conductance. The samples were observed with SEM.
Three-Dimensioned Computerized Tomography of Bacterial Cells Using Transmission electron Microscopy
Bacterial cell membranes were compared using three-dimensioned reconstructed images generated by a TEM CT (JEM 1220: JEOL Co., Tokyo). The TEM images of the bacterial cells were taken at ×150,000 magnification, at every 1° in a tilt range from −60° to +60°. The three-dimensioned reconstructed CT images were generated using Radon transform software. These CT images can be displayed in any direction.
Detection of bacterial infection in several organs was carried out as follows using PCR. Total DNA was extracted from resected tissues such as the damaged and undamaged hemispheres of the brain, lung, liver and intestine, and examined by PCR method using S. mutans-specific primers (Reference 9) below.
S. mutans-specific primers:
The detection limit of bacteria was from 5 to 50 cells in each sample. In order to confirm the presence of the viable cells in tissue, each tissue resected was compressed in PBS, then the stock and diluted solutions were streak-cultured on a bacitracin (100 units/ml; Sigma-Aldrich, St. Louis, Mo. USA)-containing Mitis-Salivariusagar plate (Difco) which is an agar plate for selective culture.
TW295 strain cnm gene fragment was amplified using following primers designed based on the full length sequence of cnm gene encoding CBP of TW295 strain (SEQ ID NO. 4: DDBJ Accession No. AB469913)
Amplified fragment was incorporated into pGEM-T Easy vector (Promega, Madison, Wis., USA) to generate the plasmid pTN11. pTN11 was treated with the restriction enzyme BsmI to digest the middle part of the open reading flame of cnm and generated the plasmid pTN12, in which an erythromycin-resistant gene fragment obtained from the plasmid pKN100 was incorporated. pTN12 was disassembled into single strands using the restriction enzyme PstI, and homologously recombined into TW295 strain by a chemical procedures using horse serum. The screening of a strain having an erythromycin resistant gene in the middle part of cnm gene (CND strain) was carried out using an erythromycin-containing S. mutans-selection medium. Generated strain was confirmed by Southern hybridization and measurement of collagen binding ability.
According to the method described in Nakano et al. Microbes Infect. 2006 8(1)114-21, PD strain was generated and confirmed by a similar method as the CND strain above using primers based on the full sequence of pac gene encoding PA of MT8148 strain (SEQ ID NO. 2: DDBJ Accession No. X14490).
Statistical Analysis was performed using Prism 4 software (GraphPad Software Inc., San Diego, Calif., USA). Fisher's PLSD, Student's t-test, regression analysis and ANOVA was performed. The result was considered significant if p<0.05.
For 170 strains of S. mutans isolated from 170 child patients who consulted to Osaka University Graduate School of Dentistry, Department of Pediatric Dentistry from 2002 to 2003, frequencies of carrying PA and CBP. Furthermore, the malignancy of hemorrhage aggravation in representative bacterial strains was determined in mouse cerebral hemorrhage model.
At 24 hours after the onset of cerebral hemorrhage, in the control group which had not been given the bacteria at all, a mild cerebral hemorrhage was confirmed in the downstream of middle cerebral artery in the vessel-damaged hemisphere (4b). This cerebral hemorrhage induces cerebral infarction (Reference 12). In the group which had been infected with MT8148 strain, no exacerbation of cerebral hemorrhage was confirmed as compared to vehicle control (
Moreover, the effect of TW295 strain on activation of matrix-metalloprotease (MMP)-9 was investigated. Destruction of vascular obstacle by activated MMP-9 is an important amplifying route that causes further hemorrhage (References 12, 13). As shown in
In order to testify the hypothesis that the administered bacteria are localized specifically to the damaged site, the localization of S. mutans in the damaged tissue after bacterial administration was investigated. The transfer of the bacteria to each organ was examined by PCR method, and transfer of the administered TW295 strain was observed only the ipsilateral hemisphere of the vascular injury, but not in other parts of the brain or in other organs (
Accordingly, the inventors focused on the direct interaction of serotype k S. mutans and collagen fibers. It has been known that denuded collagen fibers are present in the vascular surface of the vessel damaged by the disruption of endothelial cells, and that the interaction of the collagen fibers and platelets is important for platelet aggregation. Recently, a cell surface collagen binding protein of 120 kDa on (CBP, also known as collagen binding adhisin and Cnm) has been identified in S. mutans, and its coding gene (cnm) has been cloned and its sequence has been disclosed (Reference 14). Among S. mutans clinical strain, about 10% are carrying CBP, and their distribution is dominant in serotype k or f strain (Reference 15 and 16). Interestingly, all of the highly virulent strain observed in the cases of human cerebral hemorrhage described hereinbelow (TW871, TW295, SA53 and LJ32, see,
The inventors generated a mutant strain (TW295CND, Table 1) that is deficient in expression of collagen binding adhisin, from TW295 strain. Suppression of platelet aggregation observed in TW295 strain was completely recovered in TW295CND strain (
Subsequently, the inventors administered TW295CND strain to a mouse cerebral hemorrhage model. As shown in
Platelet aggregation is the most important step to hemostasis after a vessel injury. Effects of S. mutans of various serotypes on platelet aggregation induced by collagen were examined using mouse whole blood. The standard strain MT8148 did not show any platelet aggregation inhibitory effect in whole blood as compared to the vehicle control (
Also, the effects of clinically isolated 58 other S. mutans strains on platelet aggregation were investigated. The platelet aggregation rate in the presence of a serotype k strain was significantly lower than other serotypes (p<0.05;
However, arachidonic acid-induced platelet aggregation was not inhibited by administration of TW295 strain (
The ionic charge of the platelet surface is an important factor that induces an interaction with the denuded collagen fibers of the damaged vessel. The anionicity of the platelet surface provides an interaction with the cation charge of collagen (References 17 to 19). Accordingly, the ionic charge of bacterial cell surface which may influence the interaction with collagen was measured. The mean value of the zeta potential (which is used as an index of the cell surface ionic charge) of MT8148 cells lysed in physiological saline was −0.75 mV, which is almost nonionic (
Studies have been done in order to elucidate the role of S. mutans surface protein antigen as a virulence factor of dental caries, and a 190 kDa protein antigen (PA) has been known to be relevant to the initial attachment to dental surface (Reference 20). It has been shown that a PA-knockout strain has a decreased antigenicity as compared to a strain with a normal expression of PA, and thus maintains a prolonged duration of bacteremia (Reference 21). The cell surface condition of the PA-knockout isogenic mutant strain generated from
MT8148 (MT8148PD, Reference 21) was measured. The mean value of the zeta potential of MT8148PD was much lower than that of MT8148 strain (
The transmission electron microscopy observation (TEM,
5. S. Mutans Strain Isolated from Human Stroke Patients
In order to prove the hypothesis that the infection of CBP gene-expressing S. mutans is a risk factor of stroke, the frequency of the occurrence of S. mutans carrying the collagen binding protein among stroke patients using oral cavity samples. The results are shown in Table 4.
S. mutans
Among 17 cases of stroke patients, S. mutans was isolated from the patients in 11 cases. 5 cases among those were infected with CBP gene-expressing S. mutans (5/11, 45.5%, Table 4). This is much higher than the frequency of detecting collagen binding protein-carrying S. mutans in healthy subjects (10%). These results suggest that the infection with CBP gene-expressing S. mutans is likely to be a risk factor of stroke.
Furthermore, the virulence of isolated CBP gene-expressing S. mutans was examined in mice. Among the CBP-expressing S. mutans strains isolated from stroke patients, two strains (SMH4 and SMH6,
Table 5 summarizes the results of the investigation of the frequency of carrying bacterial surface protein by S. mutans for 170 strains of S. mutans isolated from 170 child patients. Malignancy was estimated from the area of hemorrhage region caused by each bacterial strain in the mouse cerebral hemorrhage model.
S. mutans
Strains that do not express PA shared 3% of the overall, while strains that do not carry CBP occupy 90% of the total. Malignancy in cerebral hemorrhage was determined to be the highest in 1.8% of strains that do not carry PA and that carries CBP from the area of hemorrhage region caused by each bacterial strain, and which was defined as 100% malignancy. According to this definition, the malignancy of the strains that do not express PA and that do not carry CBP (frequency=about 1.2%) and the strains that express PA and that carry CBP (frequency=about 8.2%) were determined about 50 to 70% and about 40 to 60%, respectively. This result agrees to the experimental results using PA and CBP gene knockout strains described above.
In the present study, it is first shown that a CBP-expressing and/or PA-deficient S. mutans is potential risk factor of a disease associated with hemorrhage, especially hemorrhagic stroke.
In the present study, an aggravation of cerebral hemorrhage by serotype S. mutans strain was confirmed. Furthermore, since infectious bacteria were detected only in the vessel-damaged hemisphere but not in the contralateral hemisphere, it was shown that the interaction between the serotype k S. mutans and the damaged vessel is an important event in the onset of cerebral hemorrhage. These strains show the expression of the collagen binding protein (CBP) and/or the deficiency in the protein antigen (PA) as a common protein expression pattern, which are shown to be important in aggravation of cerebral hemorrhage (
Another potential virulent factor of cerebral hemorrhage is the deficiency in protein antigen (PA) expression. The highly virulent strains TW295, SA53 and LJ32 all were shown to be deficient in PA expression. On the other hand, TW871 expresses PA antigen (
In general, collagen is cationic under physiological conditions, and therefore the ionic properties of bacterial surface are considered to be important in their interaction with denuded collagen fibers. In fact, PA-deficient isogenic mutant shows the lowest zeta potential value, and other PA-knockout strains also tend to have a low zeta potential value. This indicates that PA influences zeta potential value. Because there was a positive correlation between the zeta potential value and collagen-induced platelet aggregation rate, a strain having a low zeta potential value can also be categorized as a highly virulent strain. From these results, it can be considered that a strain expressing S. mutans collagen binding protein possesses a high affinity to denuded collagen fibers, and a low level expression of PA in S. mutans inclines the cell surface condition to be anionic, which further increases the affinity with cationic collagen fibers. The synergic effect of the presence of the collagen binding protein and the deficiency in 190 kDa protein results in a strong bound to collagen fibers and an accumulation of highly virulent bacteria to collagen-denuded vessels. Bacterial accumulation subsequently leads the activation of MMP-9 and inhibition of platelet aggregation in the damaged vessels, resulting in an acceleration of hemorrhage and hemorrhagic infarction (
Among the patients infected with S. mutans, the rate of those who has been infected with strains expressing collagen binding protein is estimated to be 8 to 10% (Reference 16, 22). On the other hand, PA is normally expressed in most strains, and the strains as little as 4% do not express it (Reference 21). Accordingly, a S. mutans strain that expresses collagen binding protein and that is deficient in PA expression, i.e., a strain with an extremely high virulence is quite rare, and a limited number of strains become a potential risk factor of cerebral hemorrhage aggravation due to S. mutans bacteremia. Because the therapeutic approaches for cerebral hemorrhage are limited after its onset, prophylaxis is considered to be the most important approach (Reference 23). Accordingly, it is important to identify a patient who has been infected with a highly virulent S. mutans strain for the prevention of cerebral hemorrhage. In fact, the inventors has isolated CBP-expressing, highly virulent
TW295-type S. mutans from stroke patients with an extremely high frequency. Moreover, some of such strains also induced cerebral hemorrhage aggravation in a mouse model of hemorrhagic infarction, which indicates the relevance of a highly virulent S. mutans in the onset of hemorrhagic stroke.
From these results, it can be concluded that infection by a highly virulent, stroke-inducing S. mutans is a potential risk factor of stroke. Two important virulent factors of cerebral hemorrhage are the presence of collagen binding protein and the deficiency in PA expression, which are the common features shared by many of clinically isolated serotype k strains. Accordingly, the possession or deficiency of PA and/or CBP by a S. mutans strain can be an index for the determination of the risk at the hemorrhage in a carrier, which can be useful in prevention of cerebral hemorrhage.
Tested Bacteria: Following Bacteria were Used in the Establishment of the Detection System.
S. mutans
S. sobrinus
S. sanguinis
S. oralis
S. gordonii
S. salivarius
Culturing of S. Mutans Employs Following Things:
Culturing of S. Mutans is Carried Out as Follows:
The saliva of the subject is collected in a small amount using the spitz for collecting saliva. 10 μl of the saliva is taken from the spitz using the special dropper, plated onto Special Medium A, then cultured at 37° C. for 48 hours, preferably in an anaerobic condition. After culturing, the presence of bacterial colonies is confirmed on gloss, colonies (rough colonies are desirable) are picked up and added into Special Medium B, cultured at 37° C. for 18 hours, and used in following Analyses 2, 3 and 4. Cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius are used as controls, and in Analysis 1, it is confirmed that no bacterium other than S. mutans and S. sobrinus grows.
Although the method of culturing mutans streptococci of above Analysis 1 is provided with conditions in which the mutans streptococci group (S. mutans/S. sobrinus) can preferably grow, a bacterium having bacitracin-resistance other than mutans streptococci may grow. Therefore, confirmation is done in this step.
Detection Employs Following Things:
Detection is Carried out as Follows:
10 μl of the bacterial solution cultured according to the method of Analysis 1 is added to Special Medium C, incubated at 37° C. for 3 hours. The Special Medium C is washed 3 times with Wash Buffer A, then left still for approximately 15 minutes after the last Wash Buffer A is added. Wash Buffer A is removed, and the Special Medium C is washed once again with the Wash Buffer A, then 100 μl Buffer 1 is added to the Special Medium C, left still for 1 minute. This is washed 3 times with Wash Buffer A, and 200 μl of Buffer 2 is added thereto.
It is determined to be S. mutans-positive if the color of the medium is changed, S. mutans-negative if the color of the medium is unchanged.
Detection of PA-Deleted S. Mutans Employs Following Things:
Detection of PA-Deleted S. Mutans is Carried Out as Follows:
To 100 μl of the bacterial solution cultured according to the method of Analysis 1 above, Buffer 3 is added, and immersed in boiling water for 10 minutes, and frozen if it is to be stored.
Detection of CBP-Carrying S. Mutans Employs the Followings:
Detection of CBP-Carrying S. Mutans is Carried Out as Follows:
It is determined to be CBP-positive if the color of the solution is changed, CBP-negative if the color of the solution is not changed. Cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius are used as controls, and in Analysis 4, it is confirmed that no bacterium other than CBP-carrying S. mutans shows a positive reaction.
Subsequently, in steps in Analysis 2, the bacterial solution cultured in Analysis 1 was added to Special Medium C, incubated at 37° C. for 3 hours, washed with Wash Buffer A, then stained with Buffer 1 containing crystal violet. Since the buffer was changed to blue-violet in the medium in which samples A and B has been cultured, the presence of S. mutans was determined. As the buffer remained transparent in the medium in which sample C has been cultured, no presence of S. mutans was determined.
In steps in Analysis 3, Buffer 3 was added to each of the bacterial solutions of the samples A and B cultured in Analysis 1 and boiled for 10 minutes, and stored frozen. This was added to Special Plate (96-well plate MICROTEST U-Bottom(BECTON DICKINSON)), left still overnight at 4° C. After washing with Wash Buffer B, Buffer 4 was added and blocked at room temperature for 1 hour, then Buffer 5 containing rabbit anti-PA antiserum was added and reacted at room temperature for 1 hour. After washing with Wash Buffer B, Buffer 6 containing porcine anti-rabbit immunoglobulin antibody was added and reacted at room temperature for 1 hour. After washing with Wash Buffer B, Buffer 7 which contained an alkaline phosphatase reaction-detecting reagent was added, and after 15 minutes changes in the color of the solution were observed. Since the solution was changed to pink in the plate of the sample A, the presence of PA-carrying S. mutans was determined. As the color of the solution remained transparent for the sample B, no presence of PA-carrying S. mutans was determined. Similar analysis was performed using cultures of S. sobrinus, S. sanguinis, S. oralis, S. gordonii, and S. salivarius as controls, confirming that no bacterium other than the PA-carrying S. mutans showed a positive reaction.
In steps in Analysis 4, Buffer 8 containing 5% bovine albumin was added to the Special Medium D coated with Type I collagen (Sigma), and left still at 37° C. for 1 hour. After washing with Wash Buffer C, bacterial solution cultured in Analysis 1 was added and incubated at 37° C. for 2 hours. After washing with Wash Buffer A, Buffer 9 containing 25% formaldehyde was added, left still at room temperature for 30 minutes. After washing with Wash Buffer A, Buffer 1 was added and left still for 1 minute. After washing with Wash Buffer A, Buffer B was added and changes in the color of the solution were observed. Since the color of the solution remained transparent in the plate containing the sample A, no presence of CBP-carrying S. mutans was determined. As the color of the solution changed to blue-violet in the plate containing the sample B, the presence of CBP-carrying S. mutans was determined. Similar analysis was performed using cultures of S. sobrinus, S. sanguinis, S. rails, S. gordonii, and S. salivarius as controls, confirming that no bacterium other than the CBP-carrying S. mutans showed a positive reaction.
In order to obtain a determination with higher accuracy in Analyses 2 to 4 above, it is considered to be important to culture S. mutans as many as possible in Analysis 1 and to ensure the contamination of bacteria other than S. mutans as little as possible. As conditions for culturing, (1) culturing in an aerobic condition/anaerobic condition, (2) antibiotics (bacitracin) concentration, and (3) nutrient (sucrose) concentration were investigated.
We investigated the stock period of saliva usable for detection of a virulent S. mutans under the optimal conditions shown in Example 4 using saliva that has been kept for a certain time after being sampled.
The sequences of the protein, polypeptide and nucleic acid used herein are described in the attached sequence listings, as follows:
S. mutans MT8148
S. mutans MT8148
S. mutans TW295
S. mutans TW295
S. mutans TW871
S. mutans TW871
S. mutans TW871
S. mutans TW871
S. mutans-primer F
S. mutans-primer R
S. mutans-CBD-primer F (cnm1F)
S. mutans-CBP-primer R (cnm1R)
S. mutans-PAC-primerF (pac-F)
S. mutans-PAC-primer R (pac-R)
S. mutans LJ23
S. mutans LJ23
S. mutans SA98
S. mutans SA98
S. mutans
S. mutans
Neisseria meningitidis
Neisseria meningitidis
S. mutans SA53
S. mutans SA53
S. mutans SA53
S. mutans SA53
S. mutans LJ32
S. mutans LJ32
S. mutans LJ32
S. mutans LJ32
Lancet 371, 1612-1623 (2008).
Cardiovasc. Surg. 110, 1745-1755 (1995).
mutans strains isolated from patients with bacteremia.
Eur. J. Oral Sci. 109, 330-334 (2001).
Streptococcus mutans in infective endocarditis patients.
J. Med. Microbiol. 56, 1413-1415 (2007).
Br. Med. J. 1, 1074-1077 (1966).
B Biol. Sci. 196, 471-474 (1977).
Infect. 8, 114-121 (2006).
Streptococcus mutans. J. Med. Microbiol. 58, 469-475
Streptococcus mutans strains containing the cnm gene encoding
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-088239 | Mar 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/054364 | 3/15/2010 | WO | 00 | 9/28/2011 |
