[Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes].

OBJECTIVE: To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. METHODS: Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. RESULTS: A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3 -phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. CONCLUSION: Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.

[Construction and characterization of ompH gene knockout mutant of avian Pasteurella multocida C48-3].

OBJECTIVE: To study the role of the outer membrane protein H (OmpH) in pathogenicity of avian Pasteurella multocida. METHODS: The ompH knock-out mutant of avian P. multocida C48-3 was constructed by homologous recombination. The DNA replacement was confirmed by PCR, RT-PCR and Western blot. We compared the differences of biological characteristics such as growth rate, capsular structure, adhesion ability and virulence between the ompH knockout mutant of C48-3 Delta ompH and parent strain C48-3, as well as the complemented strain C48-3C. RESULTS: C48-3 Delta ompH was successfully constructed. Electron microscopy examination of C48-3 Delta ompH shows the absence of capsular material compared to the parent strain C48-3 and complemented strain C48-3C. The adhesion assay shows that the number of C48-3 Delta ompH adhered to CEF cells was significantly lower than that of C48-3 and C48-3C. C48-3 Delta ompH was relatively attenuated in mice by intraperitoneal injection. CONCLUSION: The construction of C48-3 Delta ompH would facilitate further study on pathogenesis of avian Pasteurella multocida.

[Cross-protective effect of the Cp39 adhesive proteins against avian Pasteurella multicida in mice].

OBJECTIVE: We evaluated the cross-protective effect of a 39-kDa adhesive protein (Cp39), recombinant adhesive protein (rCp39), and capsular proteins from in vitro and in vivo grown avian P. multocida in mice. METHODS: The Cp39 was purified by electroelution from capsular proteins of in vivo grown strain C48-3. The rCp39 was expressed in E. coli B21 as a soluble protein by IPTG inducing, and purified with pMAL protein fusion and purification system. Mice of each group were subcutaneously immunized twice with 100 microg of rCp39, Cp39 or capsular proteins from in vivo or in vitro grown strain C48-3 with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of avian P. multocida strain C48-3 or rabbit P. multocida strain C51-3 two weeks after the second immunization, while the antibody response specific for rCp39 was determined by ELISA. RESULTS: SDS-PAGE indicated that the Cp39 protein was expressed in vitro and in vivo grown P. multocida. Mice immunized with rCp39, native Cp39 or capsular proteins from in vivo and in vitro grown strain C48-3 were protected completely against the challenge with the homologous strain C48-3, while 60 - 80% protection was demonstrated in the mice against the challenge with the heterologous strain C51-3. CONCLUSION: In conclusion, rCp39 is a cross immunoprotective antigen of P. multocida capsular serogroup A strains, and might be a useful vaccine candidate against fowl cholera.

[Immunity of native SpaA and recombinant SpaA-N against Erysipelothrix rhusiopathiae infection in mice].

OBJECTIVE: We evaluated the effects of surface protective antigen A (SpaA) and its N-teminal protective domain (rSpaA-N) against Erysipelothrix rhusiopathiae infection in mice. METHODS: The SpaA was purified by electroelution from NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain C43311. The rSpaA-N was expressed in E. coli BL21 as a soluble protein by IPTG inducing, and purified with GST affinity chromatography. Mice of each group were subcutaneously immunized three times with 50 microg or 100 microg of native SpaA, rSpaA-N or NaOH-extracted antigen with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD50 of Erysipelothrix rhusiopathiae virulent strain C43065 two weeks after the third immunization, and the specific antibody responses for SpaA was determined by indirect ELISA. RESULTS: Mice immunized with 50 microg or 100 microg of native SpaA, rSpaA-N, or NaOH-extracted antigen were protected completely against the challenge with strain C43065. There was no significant difference (P < 0.05) between the antibody responses observed for native SpaA, rSpaA-N and NaOH-extracted antigen at any dosages. Western blot results indicated that the native SpaA and rSpaA-N were recognized specifically by an antiserum against the native SpaA of strain C43311. CONCLUSION: These results demonstrated that the rSpaA-N is a protective antigen of Erysipelothrix rhusiopathiae serotype 2 strains, and might be a useful vaccine candidate against swine erysipelas.

Alteromonas halophila sp. nov., a new moderately halophilic bacterium isolated from a sea anemone.

A pale yellow-colored, moderately halophilic, Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, motile, aerobic bacterium, designated strain JSM 073008(T), was isolated from a sea anemone (Anthopleura xanthogrammica) collected from Naozhou Island, Leizhou Bay, South China Sea. The organism was able to grow with 1-20% (w/v) total salts (optimum, 5-10%), at pH 6.0-10.0 (optimum, pH 7.5) and 10-40 degrees C (optimum, 25-30 degrees C). The major cellular fatty acids were C(16:0), C(16:1) omega7c/iso-C(15:0) 2-OH and C(18:1) omega7c. The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid. The predominant respiratory quinone was Q-8 and the genomic DNA G + C content was 47.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 073008(T) should be assigned to the genus Alteromonas, being most closely related to Alteromonas hispanica F-32(T) (sequence similarity 96.9%), followed by Alteromonas genovensis LMG 24078(T) (96.6%) and Alteromonas litorea TF-22(T) (96.4%). The sequence similarities between the novel isolate and the type strains of other recognized Alteromonas species ranged from 95.9% (with Alteromonas stellipolaris ANT 69a(T)) to 94.5% (with Alteromonas simiduii BCRC 17572(T)). The combination of phylogenetic analysis, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 073008(T) represents a new species of the genus Alteromonas, for which the name Alteromonas halophila sp. nov. is proposed. The type strain is JSM 073008(T) (=CCTCC AA 207035(T) = KCTC 22164(T)).

Halobacillus naozhouensis sp. nov., a moderately halophilic bacterium isolated from a sea anemone.

A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071068(T), was isolated from a sea anemone (Anthopleura xanthogrammica) collected from the Naozhou Island on the Leizhou Bay in the South China Sea. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071068(T) was able to grow with 1-20% (w/v) total salts (optimum, 6-9%), at pH values of 6.0-10.0 (optimum, pH 7.5) and a temperature range of 10-35 degrees C (optimum, 25 degrees C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0) and iso-C(15:0). The genomic DNA G + C content was 42.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 071068(T) belonged to the genus Halobacillus. The 16S rRNA gene sequence similarities between strain JSM 071068(T) and the type strains of the recognized Halobacillus species ranged from 97.9% (with Halobacillus alkaliphilus) to 95.3% (with Halobacillus kuroshimensis). The levels of DNA-DNA relatedness between the new isolate and the type strains of H. alkaliphilus, Halobacillus campisalis, Halobacillus halophilus and Halobacillus seohaensis were 25.6, 22.1, 10.8 and 13.2%, respectively. The combination of phylogenetic analysis, DNA-DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 071068(T) represents a new species of the genus Halobacillus, for which the name Halobacillus naozhouensis sp. nov. is proposed, with JSM 071068(T) (=DSM 21183(T) =KCTC 13234(T)) as the type strain.

Combinational phototherapy and hypoxia-activated chemotherapy favoring antitumor immune responses.

Background: Tumor metastasis is responsible for most cancer death worldwide, which lacks curative treatment. Purpose: The objective of this study was to eliminate tumor and control the development of tumor metastasis. Methods: Herein, we demonstrated a smart nano-enabled platform, in which 2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2h-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (IR780) and tirapazamine (TPZ) were co-loaded in poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL) to form versatile nanoparticles (PEG-PCL-IR780-TPZ NPs). Results: The intelligence of the system was reflected in the triggered and controlled engineering. Specially, PEG-PCL not only prolonged the circulation time of IR780 and TPZ but also promoted tumor accumulation of nanodrugs through enhanced permeability and retention (EPR) effect. Moreover, reactive oxygen species (ROS) generated by IR780 armed by an 808 nm laser irradiation evoked a cargo release. Meanwhile, IR780, as a mitochondria-targeting phototherapy agent exacerbated tumor hypoxic microenvironment and activated TPZ for accomplishing hypoxia-activated chemotherapy. Most significantly, IR780 was capable of triggering immunogenic cell death (ICD) during the synergic treatment. ICD biomarkers as a "danger signal" accelerated dendritic cells (DCs) maturation, and subsequently activated toxic T lymphocytes. Conclusion: Eventually, antitumor immune responses stimulated by combinational phototherapy and hypoxia-activated chemotherapy revolutionized the current landscape of cancer treatment, strikingly inhibiting tumor metastasis and providing a promising prospect in the clinical application.

[Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311].

The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the previously reported different serotype strains of E. rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fusion protein of GST-SpaA-N possessed high immunoreactivity.

Tailor-made PEG-DA-CuS nanoparticles enriched in tumor with the aid of retro Diels-Alder reaction triggered by their intrinsic photothermal property.

INTRODUCTION: In recent years, near-infrared laser-induced photothermal therapy is being considered as a promising approach to kill tumors owing to its noninvasive nature and excellent antitumor efficiency. However, the lack of ideal photothermal agents hinders further development of this technology. MATERIALS AND METHODS: Aiming at solving this long-standing obstacle, we report here about the polyethylene glycol (PEG)-DA modified copper sulfide (CuS) nanoparticles (NPs) (PEG-DA-CuS NPs), a kind of semiconductor photothermal agents that show excellent photothermal stability and high heat conversion efficiency. RESULTS AND DISCUSSION: Owing to the surrounding PEG, the water solubility of CuS NPs was significantly improved when circulating in blood in the body. When the NPs reached the tumors and were irradiated by a 1,064 nm laser (1 W/cm2, 10 minutes), the local temperature increased above 90°C, triggering the retro Diels-Alder reaction. After the release of PEG chain, CuS NPs soon formed aggregates and enriched the tumor via the enhanced permeability and retention effect, promoting the efficacy of photothermal therapy. CONCLUSION: Therefore, we believe PEG-DA-CuS NPs are able to serve as a kind of cytotoxic and efficient photothermal agent to kill cancer.

Role of surface protective antigen A in the pathogenesis of Erysipelothrix rhusiopathiae strain C43065.

To clarify the role of surface protective antigen A (SpaA) in the pathogenesis of Erysipelothrix rhusiopathiae C43065 (serotype 2), the spaA deletion mutant of E. rhusiopathiae ΔspaA was constructed by homologous recombination. The virulence of the ΔspaA mutant decreased more than 76-fold compared with that of the wild-type strain C43065 in mice. The mutant strain was sensitive to the bactericidal action of swine serum, whereas the wild-type strain was resistant. The adhesion of wild-type strain to MEF cells was inhibited significantly by treatment with rabbit antiserum against recombinant SpaA (rSpaA) as compared with the treatment with normal rabbit serum, but the mutant strain was not affected. The mutant strain was readily taken up by mouse peritoneal macrophages in the normal rabbit serum, whereas the wild-type strain was resistant. Whereas the rabbit antiserum against rSpaA promoted the phagocytosis of wild-type strain by macrophages, the mutant strain was not affected. In addition, mice vaccinated with the formalin-killed mutant strain were provided 40% protection against challenge by the homologous virulent strain as compared with those with wild-type strain, NaOH-extracted antigen, or rSpaA, which provided more than 80% protection against the same infection. These suggested that SpaA has an important role in the pathogenesis of E. rhusiopathiae infection and could be a target for vaccination against swine erysipelas.

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens.

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells.

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.

[Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.