Immunological Identification and Characterization

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The passenger domain of EspP is released to the extracellular milieu after cleavage; thus, the processed EspP is detected in the culture supernatant but not in ...

FULL PAPER  Bacteriology

Immunological Identification and Characterization of Extracellular Serine ProteaseLike Protein Encoded in a Putative espP2 Gene of Haemophilus parasuis Nian-Zhang ZHANG1)#, Yue-Feng CHU1)#, Peng-Cheng GAO1), Ping ZHAO1), Ying HE1) and Zhong-Xin LU1)* 1)State

Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Epizootic Diseases of Grazing Animals of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

(Received 13 June 2011/Accepted 12 March 2012/Published online in J-STAGE 26 March 2012) Abstract. Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESPlike protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 109 bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection. KEY WORDS: cloning, ESP-like protein, expression, Haemophilus parasuis, immunogenicity.

doi: 10.1292/jvms.11-0260; J. Vet. Med. Sci. 74(8): 983–987, 2012

Haemophilus parasuis is a Gram-negative bacterium belonging to the family Pasteurellaceae and a commensal organism of the upper respiratory tract of healthy pigs [3, 8]. Under appropriate conditions, some strains can be invasive and cause severe systemic disease characterized by fibrinous polyserositis, arthritis and meningitis [8]. Recently, large numbers of virulence genes have been screened. However, most of them are yet to be understood [12, 20]. The autotransporter (AT) protein family is the largest family of Gram-negative bacterial extracellular proteins [13]. In this family, there are more than 700 members [7]. These proteins are synthesized as precursor proteins with three common functional domains, an N-terminal signal peptide, a passenger domain and a C-terminal translocator domain, which is embedded in the outer membrane as the pore-forming structure to facilitate delivery of the internal passenger domain to the bacterial surface [3, 19]. The trimeric autotransporters, a subfamily of the autotransporter protein family named VtaAs (virulence-associated trimeric autotransporters) of H. parasuis, have been reported by Pina et al. [14], while the existence of many other autotransporter protein family members has been suggested. *Correspondence to: Lu, Z.-X., State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Epizootic Diseases of Grazing Animals of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China. e-mail: [email protected] #These authors contributed equally to this work. ©2012 The Japanese Society of Veterinary Science

In this study, we selected a putative extracellular serine protease (ESP) gene, espP2, annotated in a published genome sequence of H. parasuis [17]. This putative AT protein of H. parasuis was cloned from a serotype 5 field strain, expressed and then characterized immunologically. The recombinant ESP-like protein-based vaccine conferred partial protection when compared with a formalin-inactivated bacterin. MATERIALS AND METHODS Bacterial strains and growth conditions: The reference strain of H. parasuis serovar 5 (strain Nagasaki) was kindly presented by Dr. Pat Blackall, Qld DPI, Animal Research Institute, Australia. H. parasuis strain HPS0819 originated from a diseased pig on a farm in Northwest China and was serotyped as serovar 5 by the gel diffusion (GD) and indirect hemagglutination (IHA) tests as described by Cai et al. [5]. Tryptone soya agar (TSA) and tryptone soya broth (TSB) medium were used with the addition of a final concentration of 10% horse serum, 5% yeast extract (Becton, Dickinson and Co., San Jose, CA, U.S.A.) and 0.05% NAD (Roche, Shanghai, China) to culture H. parasuis at 37°C in 5% CO2. When the OD600 reached 0.6–1.0, the bacteria were centrifuged and were suspended in sterile PBS. E. coli strains were cultured in Luria-Bertani medium (LB) at 37°C. When required, the LB was supplemented with kanamycin at a final concentration of 50 µg/ml. Sera preparation from pigs experimentally infected with H. parasuis: Sera were obtained from three eight-week-old pigs, which were reared in accordance with Blanco et al. [4], and were experimentally infected with H. parasuis. Each

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animal was intratracheally inoculated with 4-ml inoculums containing 106 CFU (subclinical dose) of H. parasuis strain HPS0819. Sera were collected at 7 days pre-infection and 7, 14, 22 and 42 days post infection. When the OD450 value of collected sera was above 0.40 as measured by ELISA as described by Martín et al. [9], the sera were used to analyze the immunogenicity of ESP-like protein by western blotting in this study. Cloning of the putative espP2 gene: Extraction of bacterial genomic DNA of HPS0819 was carried out as reported previously [6]. Forward primer espF (5’-GGCGAATTCATGAAACCTTATCTTTCGAA-3’, restriction sites are shown in italics) and reverse primer espR (5’- CCCTCGAGTTAGAACGAGTATCTTACAT-3’), designed in this study based on the nucleotide sequence from the H. parasuis SH0165 strain (locus _tag in SH0165 genome: HAPS_1381), were used for PCR amplification of the putative espP2 gene. The primers were synthesized by TaKaRa, Dalian, China. PCR reactions were carried out with Premix Taq version 2.0 (TaKaRa) according to the manufacturer’s instructions, and the reaction conditions were 5 min at 94°C; 35 cycles of 60 sec at 94°C, 50 sec at 48°C and 2 min 50 sec at 72°C; and a final extension at 72°C for 10 min. The PCR product was then cloned into pMD19-T vector (TaKaRa). The recombinant plasmid, named pMD-ESP, was transformed into E. coli strain JM109 (Invitrogen, Carlsbad, CA, U.S.A.) for sequencing by TaKaRa. Sequence analysis: The obtained sequence was analyzed by the BLAST program (http://blast.ncbi.nlm.nih.gov/) [18] and MEME program (http://meme.sdsc.edu/meme4_6_1/ cgi-bin/meme.cgi) [2]. Plasmid construction and expression of the putative espP2 gene: The recombinant plasmid pMD-ESP was purified and digested by Eco RI and Xho I (TaKaRa). Then, the putative espP2 gene was cloned into the expression vector pET-30a (Invitrogen) to yield the recombinant plasmid pET-ESP. The plasmid pET-ESP was transformed into E. coli BL21 (DE3) (Invitrogen) for expression of the target protein. BL21 (DE3)/pET-ESP was cultured at 37°C in LB supplemented with kanamycin until the OD600 reached 0.6–1.0. The optimized conditions for expression were at a final concentration of 0.8 mM IPTG (TaKaRa) and shaking for 8 hr at 32°C. Whole cell proteins from E.coli BL21 (DE3)/pET-ESP were separated by SDS-PAGE and stained with Coomassie brilliant blue. Purification and immunoblotting analysis of the ESPlike protein: The recombinant protein was purified using Ni-NTA His Bind resin (Invitrogen) according to the manufacturer’s instructions. Purified protein, the total protein of E.coli BL21(DE3)/pET30a and BL21 (DE3)/pET-ESP were separated on SDS-PAGE gel and electrotransferred onto nitrocellulose (NC) membranes (Pall, Pensacola, FL, U.S.A.), respectively. Nonspecific binding sites of the NC membranes were blocked in TBS (150 mM NaCl, 10 mM Tris-HCl) containing 5% bovine serum albumin (BSA) for 1 hr at room temperature (RT). The NC membranes were then incubated with the sera obtained from the pigs experimentally infected with H. parasuis (dilution of 1:2,000) for 1 hr at

RT. After being washed 3 times with TBST (0.5% Tween 20 in TBS), the membranes were incubated with goat anti-pig IgG-alkaline phosphatase antibody (Sigma, St. Louis, MO, U.S.A.) for 1 hr at RT. After washing off unbound second antibody, the specific antigen-bound antibody was visualized using a BCIP/NBT kit (Invitrogen). Vaccine formulations and experimental challenge: Two vaccine formulations were compared. Formulation I consisted of 100 ng/ml of recombinant ESP-like protein from the HPS0819 strain. The concentration of the antigen was referenced from the TbpB-vaccine reported by Martín et al. [9]. Formulation II contained the inactivated HPS0819 strain, which was manufactured as described by Olvera et al. [12]. Both formulations were adjuvanted using ISA206 emulsion (SEPPIC, Paris, France). Thirty specific pathogen free (SPF) guinea pigs, obtained from the animal farm of Lanzhou Veterinary Research Institute [Licence number: SYXK (Gan) 2004–2005], were randomly allotted into three experimental groups. The animals in group I (n=10) were immunized through dorsal subcutaneous injection with 1.0 ml of formulation I, and the group II animals (n=10) were immunized with formulation II. A booster immunization was administered 7 days later in both groups and every 7 days thereafter for a total of 4 times. The group III animals (n=10) were inoculated with an identical volume of saline as a control. Seven days after the last immunization, all groups were challenged intraperitoneally with 5 × 109 CFU of the HPS0819 strain. Clinical symptoms were observed for 48 hr. Animals that survived for 48 hr were euthanized. Serum collection and detection: Blood samples were collected by cardiocentesis from two randomly selected guinea pigs in each group at day 0 (before the first immunization) and day 35 (before the challenge). Sera were obtained after centrifugation and stored at −20°C until use. Specific antibodies to ESP-like protein were detected in sera from immunized guinea pigs by western blotting, which was performed using anti-guinea pig IgG-alkaline phosphatase antibody (Sigma) as the second antibody, and ELISA [9]. Clinical and pathological examinations: Clinical signs were monitored for 48 hr after challenge. All the animals were necropsied, and the macroscopic lesions were recorded. Spleen specimens from all dead and surviving guinea pigs were cultured on TSA plates to isolate H. parasuis. The cultured colonies were then identified by the PCR method as described by Angen et al. [1]. RESULTS Cloning of the putative espP2 gene and sequence analysis: A fragment of putative serine protease gene was amplified by PCR. The agarose gel electrophoresis analysis showed that the size of the PCR product was approximately 2,300 bp, which coincided with the expected length of the target gene (Fig. 1). The nucleotide sequence analysis showed that the cloned gene fragment codes for a polypeptide of 780 amino acids containing a complete open reading frame and having 100% identity with the putative espP2 gene sequence of H.

IMMUNOLOGICAL CHARACTERIZATION OF ESP-LIKE PROTEIN OF HAEMOPHILUS PARASUIS

Fig. 1. PCR amplification of the esp gene of H. parasuis strain HPS0819. M, DNA marker; lane 1, HPS0819; lane 2: negative control.

Fig. 2. SDS-PAGE of the recombinant ESP of H. parasuis. M, protein molecular weight marker; lane 1, BL21(DE3)/pET-ESP; lane 2, non-induced control; lane 3, Purifying Ni-Denature-6.0 washing solution with Ni column; lane 4, Purifying Ni-Denature-4.0 washing solution with Ni column.

parasuis SH0165 (GenBank No. ACL32961). The amino acid sequence of the predicted ESP-like protein was shown to have homology with E. coli IgA protease (Protein Data Bank accession: 2QOM_A) at the C-terminal putative translocation domain (ESP-like protein residues 507–780 and IgA protease residues 1–277) with 44% similarity. MEME analysis results demonstrated a motif sequence, EMNNLNKRMGELRG, at ESP-like protein residues 504–518, which is similar to the sequence, EVNNLNKRMGDLRD, conserved among members of the serine protease family. The ESP-like protein was shown to have no sequence similarity with the known ATs of H. parasuis (VtaAs). Immunological analysis of the ESP-like protein: SDSPAGE of the induced BL21(DE3)/pET-ESP showed that the molecular weight of the recombinant ESP-like protein

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Fig. 3. Western blot analysis of H. parsuis ESP-like protein. Lane 1, protein molecular weight marker; lane 2, the purified recombinant ESP-like protein. Lanes show the reaction with a selected serum from pig infection sera. Lane 3, E.coli BL21 (DE3) carrying the plasmid PET30a; lane 4, BL21(DE3)/pET-ESP; lane 5, whole cell proteins of H. parasuis.

was approximately 85 kilodaltons (kDa) (Fig. 2), which coincided with the theoretical value. SDS-PAGE of the purified ESP-like protein showed the abundant target protein appeared in elution buffer B (pH 6.0). In Western blot analysis, ESP-like protein reacted with infection in selected serum from pigs with experimental H. parasuis infection (Fig. 3), whereas no band at the predicted size was detected in the E.coli without expression of ESPlike protein. Meanwhile, one positive band at the position of approximately 85 kDa from the whole cell proteins of H. parasuis was recognized with the serum from guinea pigs immunized with the purified recombinant ESP-like protein (Fig. 3). Clinical and pathological signs: Eight non-immunized guinea pigs (group III) died within 12 hr after challenge. Two guinea pigs in group II and four from group I also died in 24 hr post infection. At 48 hr, the end of the experiment, one guinea pig in group II and two in group III died. All the dead guinea pigs had shown trembling, prostration, rough hair, anorexia, dyspnea and depression. The pathological analysis showed the typical pericarditis and peritonitis. None of the survivors showed any remarkable clinical signs or lesions. Bacteriological examination and antibody detection results: H. parasuis was obligate to the dead animals (Table 1). The two non-immunized control guinea pigs (group III) remained seronegative until day 35. The antibody response in the recombinant protein vaccinated group (group I) at day 35 increased when compared with that at day 0 (mean OD of 0.29 versus 0.09). The mean antibody level also rose from 0.12 (day 0) to 0.41 (day 35) in group II (Table 1). DISCUSSION H. parasuis produces a group of virulence-associated AT proteins known as VtaAs [12, 14]. This pathogen has been shown to possess several genes encoding other putative

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Table 1. Results of challenge and antibody detection in guinea pigs from groups immunized with different vaccine formulations Numbers of guinea pigs Group (n=10) Group I Group II Group III

Death 4 3 10

H. parasuis re-isolation 4 3 10

OD450 of antibody Day 0

Day 35

0.09 ± 0.04 0.12 ± 0.06 0.11 ± 0.03

0.29 ± 0.01 0.41 ± 0.02 0.08 ± 0.04

AT proteins [17]; however, none of these genes have been characterized yet, as far as we know. In this study, we first obtained the purified recombinant ESP-like protein to clarify the fact that ESP-like protein was expressed from the putative espP2 gene annotated previously in the same manner as a putative extracellular serine protease of H. parasuis. With antiserum to the purified ESP-like protein, the protein product from the putative espP2 gene was detected as an 85 kDa antigen in H. parasuis HPS0819 cultured bacteria. Although we have not yet examined it, we need to perform a further analysis in order to know whether the 85 kDa antigen is detected among various H. parasuis strains. Also, the fact that the purified ESP-like protein reacted with antibodies in the serum collected from pigs with experimental H. parasuis infection indicates that H. parasuis expresses the putative espP2 gene product during the infection process in pigs. These results mean that the putative espP2 gene can be expressed both in vitro and in vivo. The function of H. parasuis ESP-like protein remains to be clarified. It has analyzed homology at the C-terminal region mostly with serine protease family protein, IgA protease of E. coli H252 [16]. However, the unique catalytic motifs for serine protease activity found among serine protease autotransporters of Enterobacteriaceae were absent in ESP-like protein of H. parasuis. Whether this protein may have serine protease activity or some other unknown function should be further studied. The ESP-like protein of H. parasuis was shown to contain a sequence, EMNNLN, that is similar to the known cleavage sequence, EVNNLN, of EspP of E. coli [19]. The passenger domain of EspP is released to the extracellular milieu after cleavage; thus, the processed EspP is detected in the culture supernatant but not in the cell pellet from E. coli culture. In the case of H. parasuis, the ESP-like protein was detected in the cell pellet from bacterial culture, but the size of the detected protein was the same as the full size of ESP-like protein. Whether the possible cleavage sequence of ESP-like protein is an active site or not should also be elucidated in our future study. Cesarean-derived, colostrum-deprived (CDCD) pigs [15] and snatch-farrowed colostrum-deprived pigs [11] have both been used successfully to reproduce clinical disease caused by H. parasuis. However, the high cost and workload involved in obtaining both kinds of animals make these models unsuitable for most studies. Alternative experimental animal models for Glasser’s disease, such as guinea pigs, have been evaluated by Morozumi et al. [10]. Therefore, specific pathogen free (SPF) guinea pigs were used as experimental animals to evaluate the immune efficacy of recombinant protein

in this study. The results of antibody detection showed that the ESP-like protein could induce an antibody response to H. parasuis in guinea pigs. The results of challenge clearly suggested that the ESP was effective in preventing experimental H. parasuis infection, though the protection rate (6/10) was a little lower than that of the bacterin (7/10). Therefore, the recombinant ESP-like protein could partially protect animals from H. parasuis infection and could be a candidate antigen to improve H. parasuis vaccines. ACKNOWLEDGMENTS. This work was supported by the open fund of the Guangdong Common Lab of Veterinary Public Health (GSKJ090202). It was also supported in part by the 973 Project of China (2006CB504403) and by the State Key Laboratory of Veterinary Etiological Biology (SKLVEB2008ZZKT009). REFERENCES 1. Angen, O., Oliverira, S., Ahrens, P., Svensmark, B. and Leser, T. D. 2007. Development of an improved species specific PCR test for detection of Haemophilus parasuis. Vet. Microbiol. 119: 266–276. [Medline] [CrossRef] 2. Bailey, T. L. and Elkan, C. 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2: 28–36. [Medline] 3. Bigas, A., Garrido, M. E., de Rozas, A. M., Badiola, I., Barbé, J. and Llagostera, M. 2005. Development of a genetic manipulation system for Haemophilus parasuis. Vet. Microbiol. 105: 223–228. [Medline] [CrossRef] 4. Blanco, I., Galina-Pantoja, L., Oliveira, S., Pijoan, C., Sánchez, C. and Canals, A. 2004. Comparison between Haemophilus parasuis infection in colostrums-deprived and sow-reared piglets. Vet. Microbiol. 103: 21–27. [Medline] [CrossRef] 5. Cai, X., Chen, H., Blackall, P. J., Yin, Z., Wang, L., Liu, Z. and Jin, M. 2005. Serological characterization of Haemophilus parasuis isolates from China. Vet. Microbiol. 111: 231–236. [Medline] [CrossRef] 6. Chu, Y. F., Gao, P. C., Zhao, P., He, Y., Zhang, N. Z., Liu, Y. S., Liu, J. X. and Lu, Z. X. 2011. Genotyping of Haemophilus parasuis isolated from the northwest of China using PCR-RFLP based on ompA gene. J. Vet. Med. Sci. 73: 337–343. [Medline] [CrossRef] 7. Desvaux, M., Parham, N. J. and Henderson, I. R. 2004. The autotransporter secretion system. Res. Microbiol. 155: 53–60. [Medline] [CrossRef] 8. Hoefling, D. C. 1991. Acute myositis associated with Haemophilus parasuis in primary SPF sows. J. Vet. Diagn. Invest. 3: 354–355. [Medline] [CrossRef] 9. Martín de la Fuente, A. J., Rodríguez-Ferri, E. F., Frandoloso, R., Martínez, S., Tejerina, F. and Gutiérrez-Martín, C. B. 2009. Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis. Res. Vet. Sci. 86: 248–253. [Medline] [CrossRef] 10. Morozumi, T., Hiramune, T. and Kobayashi, K. 1982. Experimental infection of mice and guinea pigs with Haemophilus parasuis. Natl. Inst. Anim. Health Q (Tokyo) 22: 23–31. 11. Oliveira, S., Galina, L., Blanco, I., Canals, A. and Pijoan, C. 2003. Naturally-farrowed, artificially-reared pigs as an alternative model for experimental infection by Haemophilus parasuis. Can. J. Vet. Res. 67: 146–150. [Medline] 12. Olvera, A., Pina, S., Pérez-Simó, M., Oliveira, S. and Bensaid,

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