Protective properties of recombinant IgA1 protease from meningococcus

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form has shown that the active form of IgA1 protease exhibited species- and type-specificity for mouse and human immunoglobulins. A mutant form, lacking enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C; it protected mice from lethal infection by live virulent cultures of heterologous serogroups of meningococcus. The results obtained in this study suggest that IgA1 protease may be considered as a perspective preparation at the stages of development of a polyvalent vaccine for protection of human against meningococcal infections of various etiology.     

Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections

Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.     

Cloning and Expression of <Emphasis Type="Italic">H. influenzae</Emphasis> 49247 IgA Protease in <Emphasis Type="Italic">E. coli</Emphasis>

IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H. influenzae 49247 IgA protease showed unique DNA and amino acid sequence but with typical endopeptidase domain and beta transporter domain compared with known IgA proteases from the same species. To facilitate expression and purification, the H. influenzae 49247 IgA protease gene was sub-cloned into the pET28-A(+) vector with insertion of a 6xHis tag downstream of the endopeptidase domain and upstream of the potential autocleavage site. The recombined IgA protease can be constitutively expressed in E. coli and secreted into the culture medium. With a simple nickel affinity binding, the secreted IgA protease can be purified with high purity (95%) and a molecular weight of about 130 kDa. The identity of the IgA protease was validated by the presence of 6xHis tag in the purified protein by western blotting and its ability to cleave human IgA1 molecule. Collectively, the successful cloning, expression and purification of H. influenzae 49247 IgA protease will augment its therapeutic study in IgAN treatment.     

B群脑膜炎球菌疫苗的研制策略

脑膜炎奈瑟菌主要引起儿童细菌性脑脊髓膜炎和败血症,有较高的发病率和病死率。现用疫苗能够控制A、C、W135和Y群脑膜炎球菌引起的感染,而由于B群荚膜多糖免疫原性弱,外膜蛋白变异性高等原因,仍无安全和具有广泛保护性的疫苗用于控制B群脑膜炎球菌的感染。目前,B群脑膜炎球菌大多已成为引起发达国家侵袭性脑膜炎疾病的主要病原体。随着研究的不断深入,B群脑膜炎球菌疫苗的研究已经取得了很大的进展,外膜囊(Out membrane vesicles,OMV)疫苗已经在控制特异性菌株爆发流行中取得了成功。然而,人们对具有广泛保护性的B群脑膜炎球菌疫苗的探索仍在继续。本文对近年来B群脑膜炎球菌基于不同型抗原疫苗的各种研制策略及其存在的问题进行了综述。     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Bacterial Protease ECP32 Specifically Hydrolyzing Actin and Its Effect on Cytoskeleton in vivo

A procedure for isolation of bacterial protease ECP32 yielding 100 µg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.     

流感嗜血杆菌ATCC49247来源的IgA酶理化性质及其对低糖基化IgA1的分解作用

【背景】近年来,能够特异性切割人IgA1分子的IgA蛋白酶(IgA酶)被认为是治疗IgA肾病(IgAN)的潜在药物,但各种物理化学因素均可能影响其生物学活性。【目的】研究流感嗜血杆菌(Haemophilus influenzae) ATCC49247 IgA酶的理化性质,确定IgA酶最适的作用条件后观察IgA酶对低糖基化IgA1的作用。【方法】从细菌培养液中分离纯化IgA酶,SDS-PAGE电泳后银染法分别检测多种理化条件下IgA酶的水解活性及其对低糖基化IgA1的消化作用。【结果】H. influenzae ATCC49247IgA酶可耐受的反应温度较广,最适温度为50°C;在高于60°C的环境中,IgA酶便不可逆地丧失了稳定性;IgA酶在pH 6.0-9.0的环境下能够保持完整催化活性;1 mmol/L的PMSF和高于10 mmol/L的SDS能够强烈地抑制IgA酶的活性,DTT和EDTA对IgA酶活性无明显影响;所有浓度的Al~(3+)、Fe~(3+)和高浓度的Cu~(2+)、Zn~(2+)、Fe~(2+)对IgA酶均表现出强烈的抑制作用,同时Co~(2+)、Mn~(2+)、Ca~(2+)、Ni~(2+)和Mg~(2+)都对IgA酶活性没有明显影响。选择了最适宜的IgA酶作用条件,发现IgA酶能够有效降解低糖基化IgA1底物。【结论】确定IgA酶最适宜的作用条件能保持良好的酶活性,更好地发挥了IgA酶对低糖基化IgA1的降解作用,为IgA酶的临床研究和药用价值的进一步开发提供一定的依据。     

Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis

Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci. but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to lgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.     

Purification, characterization, and comparison of the immunoglobulin A1 proteases of Neisseria gonorrhoeae.

Each isolate of Neisseria gonorrhoeae produces one of two distinct immunoglobulin A1 (IgA1) proteases, type 1 or type 2, which are known to possess different cleavage specificities for peptide bonds in the hinge region of human IgA1. Both proteases were secreted into the culture medium throughout exponential growth; however, the activity level of the type 2 protease was 10-fold that observed for the type 1 enzyme. The type 2 protease was quite stable and resistant to a variety of inhibitors. In contrast, the type 1 enzyme was highly unstable and inhibited by low concentrations of metal chelators, salts, and thiol- or serine-specific chemical reagents. Both types of gonococcal IgA1 protease were purified from broth culture supernatants by a combination of anion-exchange, chromatofocusing, and molecular sieve chromatography techniques. The stable type 2 enzyme comprised a 114-kilodalton (kDa) peptide which converted to a still active 109-kDa peptide during isolation. In contrast, the type 1 protease possessed a 112-kDa peptide which did not convert to a smaller form and which could not be dissociated from peptides of 34 and 31 kDa without complete loss of enzyme activity.     

Inhibition of Induced Mutagenesis in Salmonella typhimurium by the Protein of Propionibacterium freudenreichii subsp. shermanii

Culture liquid and cells of Propionibacterium freudenreichii subsp. shermanii VKM-103 exerted a strong antimutagenic effect on mutations induced by 4-nitroquinoline-1-oxide, N-methyl-N′-nitro-N′-nitrosoguanidine, sodium azide (base pair substitutions) and 9-aminoacridine (frameshift mutations). No inhibitory effect was observed against mutagenesis induced by 2-nitrofluorene (frameshift mutations). The highest antimutagenic activity was found in the culture liquid of cells grown for 24 h. Acetic and propionic acids of the culture liquid produced by propionibacteria made no observable contribution to the antimutagenicity. Antimutagenic activity of the culture liquid was considerably reduced by protease treatment and by heating at 92°C for 10 min. Upon dialysis, the culture liquid lost almost all of its inhibitory activity. Cell wash solution also contained high antimutagenic activity which was lost upon protease treatment and dialysis. According to the exclusion limit of the dialysis bag, the molecular weight of the antimutagenic factor, presumably a protein, is less than 1.5 kDa. In addition, the cells of P. shermanii were capable of binding or modifying the mutagens, thereby decreasing their mutagenicity.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M_w-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

New approach to the diagnosis of meningococcal infection: latex-erythrocyte agglutination

A principal possibility and advantages of the diagnosis of meningococcal infection by means of agglutination of sensibilized latex particles with erythrocytes have been demonstrated. Polysterene carboxylated latex has been sensibilized by rabbit JgG to meningococcus of serogroup A. Dynamics of absorption of meningococcal polysaccharide on erythrocytes in vivo has been studied in mice.     

Cloning and expression of the IGA-specific endopeptidase genes from Neisseria meningitidis

IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.     

IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product

Gonococcal virulence is thought to rely on multiple characteristics including the production of an extracellular protease specific for human IgA1. Using a sensitive filter assay we have isolated an Escherichia coli clone which harbours the gene of Neisseria gonorrhoeae MS11 IgA protease on a multicopy number plasmid. This clone secrets IgA protease activity to an extent similar to that of the parental MS11 strain. By exonucleolytic digestion of the cloned insert we obtained a fragment of 4.6 kb which could not be shortened further without loss of IgA protease expression. Compared with the cloned IgA protease gene from N. gonorrhoeae F62, this minimal gene segment shows marked differences in the arrangement of restriction sites. We suppose that these differences determine strain-specific variations of N. gonorrhoeae IgA proteases and also affect the secretory properties of the enzyme when produced in E. coli. A novel purification procedure developed for IgA protease of N. gonorrhoeae allowed us to correlate the enzyme activity with a distinct protein band in SDS acrylamide gels. By comparison with the enzyme prepared from the E. coli clone, we identified a 105-kd protein as the extracellular form of gonococcal IgA protease.     

Role of Factor H Binding Protein in Neisseria meningitidis Virulence and Its Potential as a Vaccine Candidate To Broadly Protect against Meningococcal Disease

SUMMARY

Neisseria meningitidis is a Gram-negative microorganism that exists exclusively in humans and can cause devastating invasive disease. Although capsular polysaccharide-based vaccines against serogroups A, C, Y, and W135 are widely available, the pathway to a broadly protective vaccine against serogroup B has been more complex. The last 11 years has seen the discovery and development of the N. meningitidis serogroup B (MnB) outer membrane protein factor H binding protein (fHBP) as a vaccine component. Since the initial discovery of fHBP, a tremendous amount of work has accumulated on the diversity, structure, and regulation of this important protein. fHBP has proved to be a virulence factor for N. meningitidis and a target for functional bactericidal antibodies. fHBP is critical for survival of meningococci in the human host, as it is responsible for the primary interaction with human factor H (fH). Binding of hfH by the meningococcus serves to downregulate the host alternative complement pathway and helps the organism evade host innate immunity. Preclinical studies have shown that an fHBP-based vaccine can elicit serum bactericidal antibodies capable of killing MnB, and the vaccine has shown very encouraging results in human clinical trials. This report reviews our current knowledge of fHBP. In particular, we discuss the recent advances in our understanding of fHBP, its importance to N. meningitidis, and its potential role as a vaccine for preventing MnB disease.     

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase)

A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol.     

Evolutionary and genomic insights into meningococcal biology

Epidemic disease caused by Neisseria meningitidis, the meningococcus, has been recognized for two centuries, but remains incompletely controlled and understood. There have been dramatic reductions in serogroup A and C meningococcal disease following the introduction of protein-polysaccharide conjugate vaccines, but there is currently no comprehensive vaccine against serogroup B meningococci. Genetic analyses of meningococcal populations have provided many insights into the biology, evolution and pathogenesis of this important pathogen. The meningococcus, and its close relative the gonococcus, are the only pathogenic members of the genus Neisseria, and the invasive propensity of meningococci varies widely, with approximately a dozen 'hyperinvasive lineages' responsible for most disease. Despite this, attempts to identify a 'pathogenome', a subset of genes associated with the invasive phenotypes, have failed; however, genome-wide studies of representative meningococcal isolates using high-throughput sequencing are beginning to provide details on the relationship of invasive phenotype and genotype in this fascinating organism and how this relationship has evolved.     

The impact of protein-conjugate polysaccharide vaccines: an endgame for meningitis?

The development and implementation of conjugate polysaccharide vaccines against invasive bacterial diseases, specifically those caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, has been one of the most effective public health innovations of the last 25 years. These vaccines have resulted in significant reductions in childhood morbidity and mortality worldwide, with their effectiveness due in large part to their ability to induce long-lasting immunity in a range of age groups. At the population level this immunity reduces carriage and interrupts transmission resulting in herd immunity; however, these beneficial effects can be counterbalanced by the selection pressures that immunity against carriage can impose, potentially promoting the emergence and spread of virulent vaccine escape variants. Studies following the implementation of meningococcal serogroup C vaccines improved our understanding of these effects in relation to the biology of accidental pathogens such as the meningococcus. This understanding has enabled the refinement of the implementation of conjugate polysaccharide vaccines against meningitis-associated bacteria, and will be crucial in maintaining and improving vaccine control of these infections. To date there is little evidence for the spread of virulent vaccine escape variants of the meningococcus and H. influenzae, although this has been reported in pneumococci.