kDa Protease Research Articles

C-ter100 peptide derived from Vibrio vEP-45 protease acts as a pathogen-associated molecular pattern to induce inflammation and innate immunity.

The bacterium Vibrio vulnificus causes fatal septicemia in humans. Previously, we reported that an extracellular metalloprotease, vEP-45, secreted by V. vulnificus, undergoes self-proteolysis to generate a 34 kDa protease (vEP-34) by losing its C-terminal domain to produce the C-ter100 peptide. Moreover, we revealed that vEP-45 and vEP-34 proteases induce blood coagulation and activate the kallikrein/kinin system. However, the role of the C-ter100 peptide fragment released from vEP-45 in inducing inflammation is still unclear. Here, we elucidate, for the first time, the effects of C-ter100 on inducing inflammation and activating host innate immunity. Our results showed that C-ter100 could activate NF-κB by binding to the receptor TLR4, thereby promoting the secretion of inflammatory cytokines and molecules, such as TNF-α and nitric oxide (NO). Furthermore, C-ter100 could prime and activate the NLRP3 inflammasome (NLRP3, ASC, and caspase 1), causing IL-1β secretion. In mice, C-ter100 induced the recruitment of immune cells, such as neutrophils and monocytes, along with histamine release into the plasma. Furthermore, the inflammatory response induced by C-ter100 could be effectively neutralized by an anti-C-ter100 monoclonal antibody (C-ter100Mab). These results demonstrate that C-ter100 can be a pathogen-associated molecular pattern (PAMP) that activates an innate immune response during Vibrio infection and could be a target for the development of antibiotics.

Characterization of a thermostable protease from Bacillus subtilis BSP strain

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Attenuation of Oxidative Damage via Upregulating Nrf2/HO-1 Signaling Pathway by Protease SH21 with Exerting Anti-Inflammatory and Anticancer Properties In Vitro.

Oxidative damage and inflammation are among the very significant aspects interrelated with cancer and other degenerative diseases. In this study, we investigated the biological activities of a 25 kDa protease (SH21) that was purified from Bacillus siamensis. SH21 exhibited very powerful antioxidant and reactive oxygen species (ROS) generation inhibition activity in a dose-dependent approach. The mRNA and protein levels of antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPx-1) were enhanced in the SH21-treated sample. SH21 also increased the transcriptional and translational activities of NF-E2-related factor 2 (Nrf2) with the subsequent development of detoxifying enzyme heme oxygenase-1 (HO-1). In addition, SH21 showed potential anti-inflammatory activity via inhibition of nitric oxide (NO) and proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. At concentrations of 60, 80, and 100 μg/mL, SH21 potentially suppressed nitric oxide synthase (iNOS) and cytokine gene expressions. Furthermore, SH21 significantly released lactate dehydrogenase (LDH) enzyme in cancer cell supernatant in a concentration-dependent manner and showed strong activity against three tested cancer cell lines, including HL-60, A549, and Hela. Our results suggest that SH21 has effective antioxidant, anti-inflammatory, and anticancer effects and could be an excellent therapeutic agent against inflammation-related diseases.

Mechanistic study on the anti-proinflammatory activity of Kunitz type inhibitor from Caesalpinia decapetala seeds.

The study reports the biochemical characterization and mechanism of action of a novel 19.6 kDa protease inhibitor (PIs) isolated from the seeds of Caesalpinia decapetala belonging to the Fabaceae family. A systematic study was performed to ascertain the purity, specificity, biochemical and structural characterization, and its potential in curbing inflammation in vitro conditions. A two-step chromatography technique was used to purify the PIs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flightwere employed to detect the molecular mass of the protein. N-terminal sequence analysis of the inhibitor showed sequence similarity with the Kunitz family PIs. The in vitro test tube assay was performed for determining the anti-inflammatory activity and the inhibitor is antiproliferative against macrophage (RAW264.7) and lung cancer cell lines (A549). An effective decrease in the release of inflammatory mediators (NO, IL-6, TNF-α) and on the activity of elastase was observed in macrophage cell lines (RAW264.7) which were treated with PIs. The purified inhibitor shows promising results against inflammation.

Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines.

From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg2+ and Ca2+ enhanced its enzymatic activity, whereas Zn2+ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM-cancer-cell interactions.

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183mg/mL and 4.904U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Exploration of Compost Soil for the Production of Thermo-Stable Bacillus Protease to Synthesize Bioactive Compounds through Soy Protein Hydrolysis

Application of bioactive peptides (BAPs) is promising due to their potential antimicrobial, antioxidant, agonistic, and ACE inhibition properties. To achieve a stable and active peptide at relatively high pH and temperatures by microbial fermentation, a wide variety of microorganisms need to be explored from diverse habitats, and compost is the excellent source. In an attempt to isolate potent protease-producing bacteria, gelatin-supplemented DM agar medium was used. Out of 140 pure cultures, initial protease production selects isolate D3L/1 (26 U/mL), and 16S rDNA sequencing confirmed it as Bacillus subtilis. Protease production was increased to 55.55 U/mL, with pH 7.5, 1% glucose, 1% casein, 1% ammonium sulfate, for 96 h of fermentation, at 37 °C under 140 rpm of shaking. Ion-exchange, and size-exclusion chromatography, 30 KDa protease was purified up to 4.1-fold (specific activity 3448.62 U/mL; 67.66% yield). The enzyme was active under broad temperatures (60 °C optimum), organic solvents, and pH variations. A total of 5% H2O2 can only reduce 40% of enzyme activity. However, 1 mM, Fe2+, and Cu2+ increased enzyme activity by five times. Soy hydrolysis (SPI) byD3L/1 protease produces bioactive compound (<3 KDa), which confirmed the peptide bond in the far UV region (205 nm, 215 nm, 225 nm, and 280 nm). The compound was ineffective towards Serratia marcescens but active against Escherechia coli (47%), Staphylococcus aureus (28%), and Pseudomonas aeruginosa (12%).

Antigens of Three Medically Important Schistosoma spp

The present study was carryout to review the antigens of three (3) medically important Schistosomes namely: Schistosoma mansoni, S. haematobium, and S. japonicum. The parasites-host relationship, the antigens, the species that produce the antigens, and the functions of the antigens were discussed. Identified antigens include: Sm-24 kDa, Sm108 kDa, Polymorphic mucins, 28/30 kDa Protease, 47 kDa protease, 60 kDa protease, Cercarial Elastase (CE) also called 28/30 kDa, Cathepsin, 22.6 kDa, 23 kDa, 16 kDa/SLP/SPO-1, Prostaglandin E2 (PGE2), SmEnolase, SmCalp1, 28-kD GST (rSh28GST), 29-kDa, Calpain (Sp80) and SG3PDH. The above mention antigens of the parasites were found to be much important since they enable them to compete with body immunity while carrying out their metabolic activities, reproduction, growth, defense, resistance, and so many other things within the host. Therefore, these antigens are very important in immunological studies; hence, it was recommended that, Since some parasite antigens were found to be promising candidates for developing a vaccine to protect against Schistosoma infection, more parasite antigens should be searched for the best planning and development of numerous strategies that aid in the prevention and control of schistosomiasis.

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7–11) and temperature (40–80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Generation of antioxidant peptides from soy protein isolate through psychrotrophic Chryseobacterium sp. derived alkaline broad temperature active protease

Antioxidant peptides confer numerous health benefits. Unexplored niches provide bioprospection opportunities for potential protease producing microorganisms that are useful in releasing antioxidant peptides from protein sources impacting human health. A cold-adapted bacterium Chryseobacterium polytrichastri ERMR1:04 produced an extracellular protease enzyme optimally at 20 °C, pH 8 with 1.25 g/100 mL skim milk inducer. The purified 22.24 kDa protease was active over a broad temperature 5–65 °C (optimum at 37 °C) and pH 6–10 (optimum at pH 8) range, inhibited by EDTA and 1, 10-phenanthroline, enhanced by Ca2+, Mn2+ and hexane, and retained its activity with surfactants and detergents. ERMR1:04 protease-mediated hydrolysis of soy protein generated antioxidant peptides of <6 kDa, with successful identification, showing radical scavenging activities of 77% DPPH, 96% ABTS, 93% ferrous ion chelation, and Abs700 0.159 for reducing power assay. Such promising properties qualify the ERMR1:04 protease as a potent catalyst for high-value functional food production.

Stigma receptivity with pollen in sunflower accompanies novel histochemical and biochemical changes in both male and female reproductive structures

Pollen–pistil interaction is one of the widely studied cell–cell communication events in angiosperms. Pollination of the receptive pistil with compatible pollen and the subsequent steps that determines the success of fertilization depend on the cooperative events between pollen and pistil. Before the onset of these interactive events between pollen and pistil, both the participants prepare themselves for the upcoming complex cellular events. Receptive surface of stigma is characterized by the accumulation of reactive oxygen species and expression of specific biomolecules and enzymes, such as non-specific esterases and peroxidases. Pollen grains produce nitric oxide that scavenge reactive oxygen species generated on stigma surface after pollination and just prior to pollen germination. Present work reports accumulation of glycoproteins, lipids and phospholipids at the base of stigmatic papillae in mature receptive stigma. A 31 kDa glycoprotein has been detected in stigma homogenate. Stigma exhibit enhanced expression of peroxidase isoforms relative to buffer soluble fractions of pollen, i.e. intact pollen, internal pollen and tryphine fractions. A 54 kDa protease is expressed in pollen grains as well as stigma. Biochemical analyses of these biomolecules in pollen and stigma relative to other vegetative (corolla of ray and disc floret, bracts, young leaves) and reproductive (anther wall and ovary) parts highlight the implications of these biomolecules in pollen–stigma interaction.

Macromolecule-Network Electrostatics Controlling Delivery of the Biotherapeutic Cell Modulator TIMP-2.

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is an endogenous 22 kDa proteinase inhibitor, demonstrating antitumorigenic, antimetastatic and antiangiogenic activities in vitro and in vivo. Recombinant TIMP-2 is currently undergoing preclinical testing in multiple, murine tumor models. Here we report the development of an inert, injectable peptide hydrogel matrix enabling encapsulation and sustained release of TIMP-2. We studied the TIMP-2 release profile from four β-hairpin peptide gels of varying net electrostatic charge. A negatively charged peptide gel (designated AcVES3) enabling encapsulation of 4 mg/mL of TIMP-2, without effects on rheological properties, facilitated the slow sustained release (0.9%/d) of TIMP-2 over 28 d. Released TIMP-2 is structurally intact and maintains the ability to inhibit MMP activity, as well as suppress lung cancer cell proliferation in vitro. These findings suggest that the AcVES3 hydrogel will be useful as an injectable vehicle for systemic delivery of TIMP-2 in vivo for ongoing preclinical development.

Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry.

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Purification, Characterization, and Cloning of a Cold-Adapted Protease from Antarctic Janthinobacterium lividum.

In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and 40°C. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

Characterization of protease activity from the digestive tract and tentacles of Isostichopus fuscus sea cucumber

Sea cucumbers possess evisceration mechanisms and regeneration capacity. The function of tentacles is to collect food particles. From our results, we suggest these organs could also be part of the digestive system. Therefore proteases in the digestive tract and tentacles of Isostichopus fuscus were partially characterized by histological and biochemical methods. Digestive cells and regions, and secretory granules were observed by histological methods in both organs. Proteolytic extracts of the digestive tract and tentacles showed peak activity at pH 6 and 8. The digestive tract extract had peak activity at 40 and 70 °C, whereas the tentacle extract peak activity was at 60 °C. Both extracts showed activity at 0 to 10 °C. The extracts retained 67 to 75 % residual activity when incubated at 60 °C for 1 h. The effect of different ions and specific inhibitors suggested the presence of cysteine- and metallo-proteases in both organs. SDS-PAGE showed 6 proteins of approximately 40, 43, 49, 76, 106, and 147 kDa in the digestive tract extract, and 5 proteins of approximately 44, 60, 81, 108, and 150 kDa in the tentacle extract. Native-PAGE and zymography assays confirmed the presence approximately 100 kDa proteases in both extracts. The tentacle extract had the highest proteolytic activity, suggesting that this organ could contribute to the digestion process of I. fuscus. Key Words: ; ; ; ;

Serine protease Bm-SP142 was differentially expressed in resistant and susceptible Bombyx mori strains, involving in the defence response to viral infection.

Bm-SP142 is a 35 kDa protease in the silkworm, but its exact functions remain unknown. In this study, sequence alignment revealed that the His-Asp-Ser catalytic triad is embedded in the TAAHC-DIAL-GDSGGP sequence motif, establishing Bm-SP142 as a serine protease. Soluble recombinant GST-BmSP142 was expressed and purified, and serine protease activity was confirmed in vitro. RT-qPCR results indicated that Bm-SP142 was mainly expressed in the middle part of the silkworm midgut, and Bm-SP142 transcripts were significantly up-regulated at 24 hours post infection (hpi) in BmBDV-resistant strains (798) inoculated with BmBDV and BmNPV-resistant strains (NB) inoculated with BmNPV, but not in BmBDV-susceptible strains (306). Surprisingly, transcripts were significantly down-regulated at 12 hpi in BmNPV-susceptible strains (HuaBa 35) inoculated with BmNPV, compared with healthy silkworms. Recombinant BmNPV treated with purified Bm-SP142 effectively impaired its ability to infect BmN cells, and Bm-SP142 decreases the efficiency of BmNPV and BmBDV propagation in silkworms. Furthermore, overexpression of Bm-SP142 in BmN cells inhibited viral propagation.

Purification and enzymatic properties of a neutral metalloprotease produced from the cold-adapted Vibrio species Pr21 isolated from deep seawater in Sagami Bay

To identify new biopolymer-degrading enzymes produced from cold-adapted microorganisms, we searched for such enzymes in bag filters used for the purification of deep seawater pumped up in Sagami Bay, Japan. We isolated a casein- and azocoll-degrading cold-adapted bacterium, and it was identified as Vibrio species Pr21. The bacterium grew between 0 and 25 °C, optimally at 20–22 °C. An extracellular 36-kDa protease, which we named PR protease, was purified from the bacterial medium. PR protease is a neutral metalloprotease that degrades azocoll (optimum temperature 40 °C). When bovine skin type I collagen (acid soluble) was used as a substrate, β chains and larger multimers decreased and α1 and α2 chains accumulated with PR protease at 10, 20, and 30 °C. This indicates that PR protease cleaves telopeptide regions of collagen molecules. PR protease accelerated the liquefaction of live moon jellyfishes Aurelia aurita at 10 and 25 °C. The present findings suggest that the bag filters used in deep seawater-pumping plants are useful for the isolation of cold-adapted microorganisms, and that PR protease has potential for applications in food processing and wastewater treatment.

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice.

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non-Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H2 O2 and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN-γ, CD4 and CD8 double-positive T cells and Th1 cells (CD3, CD4 and Tim3-positive cells), and a decrease in the ratio of CD8-positive CD4-negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K-sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.