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The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3
Veterinary Microbiology 196 (2016) 67–71
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3 Lingqiang Ding, Xintian Wen, Lvqin He, Xuefeng Yan, Yongping Wen, Sanjie Cao, Xiaobo Huang, Rui Wu, Yiping Wen* Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
A R T I C L E I N F O
Article history: Received 22 March 2016 Received in revised form 25 August 2016 Accepted 9 October 2016 Keywords: H. parasuis arcA gene Serum resistance Virulence
A B S T R A C T
As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (DarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the DarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis. ã 2016 Published by Elsevier B.V.
1. Introduction Currently, Glässer’s disease, which is characterized by polyserositis and fibrinopurulent polyarthritis (Cai et al., 2005), is causing a significant increase with respect to mortality and morbidity in swine, leading to major economic losses in the pig industry (Zhang et al., 2014a). H. parasuis, a member of the family Pasteurellaceae (Biberstein and White, 1969), is the causative agent of Glässer’s disease. As a commensal colonizing the upper respiratory tract of pigs, under certain circumstances, H. parasuis can breach the mucosal barrier and enter the blood stream, causing severe systemic disease (Huang et al., 2016). To date, several virulence factors have been identified in H. parasuis, including lipopolysaccharide (LPS), capsular polysaccharide, fimbriae, outer membrane proteins, neuraminidase and iron acquisition systems (Zhang et al., 2014a). However, the virulence factors and the mechanisms of the entire infection process remain largely unclear (Oliveira and Pijoan, 2004). In natural environments, bacteria are challenged with a variety of rapidly changing conditions that have to be sensed in order for the bacteria to respond appropriately. To this end, most bacteria
* Corresponding author at: Huimin Road 211# Wenjiang District, Chengdu, Sichuan, 611130, China. E-mail addresses: [email protected] (L. Ding), [email protected] (X. Wen), [email protected] (L. He), [email protected] (X. Yan), [email protected] (Y. Wen), [email protected] (S. Cao), [email protected] (X. Huang), [email protected] (R. Wu), [email protected], [email protected] (Y. Wen). http://dx.doi.org/10.1016/j.vetmic.2016.10.011 0378-1135/ã 2016 Published by Elsevier B.V.
employ two-component signal transduction system (TCSTS) comprising a sensor histidine kinase (HK) and a response regulator (RR) (Wuichet et al., 2010). Three TCSTS, encoded by cpxAR, arcAB, and qseBC, were identified in the genome of H. parasuis. The ArcA/B consists of the sensor kinase ArcB which accepts extracellular stimulating signal, and the DNA-binding response regulator ArcA which regulates the expression of many genes. As a global transcriptional factor, ArcA relates to bacterial aerobic and anaerobic metabolism, anabolic and catabolic regulation, iron transport and energy generation pathways in Escherichia coli (Bai et al., 2014; Federowicz et al., 2014). For H. influenzae, the virulence of the arcA mutant for Balb/C mice was significantly reduced. The H. influenzae arcA mutant also markedly enhanced sensitivity to serum (De Souza-Hart et al., 2003). H. influenzae utilizes ArcA to appropriately modify its immune evasion strategies in different environments encounter during infection (Wong et al., 2011). Compared with wild-type, the arcA mutant has poor biofilms in A. pleuropneumoniae, which likely resulted in the reduction of virulence (Buettner et al., 2008). Salmonella arcA mutant have been shown to grow more slowly than the wild-type strains in both aerobic and microaerobic conditions (Šev9 cı’k et al., 2001). However, less functional description of this gene has been reported, and its specific roles during H. parasuis infection have been little understood. In this study, we generated an arcA deletion mutant of H. parasuis EP3 strain to investigate its role in growth characteristic, serum resistance, biofilm formation and virulence, aiming to provide some data for the further study about ArcA/B in H. parasuis.
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2. Materials and methods
2.4. Animal infection studies
2.1. Bacterial strains, plasmids and growth conditions
The animal experiments were conducted in strict accordance with the recommendations in the China Regulations for the Administration of Affairs Concerning Experimental Animals (1988) and had been approved by the Institutional Animal Care and Use Committee of Sichuan Agriculture University (Approval Number YP2014-039), Sichuan, China. Twenty-nine 6-week-old female specific pathogen free (SPF) Balb/C mice (Sichuan Province Dashuo Experimental Animal Centre, Chengdu, China) were allocated randomly to EP3, DarcA and C-arcA groups, each group contained eight mice. The remaining five were allocated to the PBS control group. The mice were injected intraperitoneally with bacteria at a dose of 1.4 109 CFU/animal (0.5 mL) or with 0.5 mL PBS. All surviving mice were euthanized 7 days post infection.
The bacterial strains and plasmids used in this study were described in Table S1. E.coli DH5a was grown in LB medium at 37 C. The H. parasuis serovar 13 clinical strain EP3, which was isolated from lungs of 4-month-old diseased pigs in China’s Sichuan province, was cultivated in Trypticase Soy Agar (TSA) or Trypticase Soy Broth (TSB) (Difco, Detroit, USA) supplemented with 0.001% (w/v) nicotinamide adenine dinucleotide (NAD) (Sigma Aldrich, Missouri, USA) and 5% (v/v) inactivated bovine serum at 37 C. When necessary, the media were supplemented with kanamycin (50 mg/mL) or streptomycin (50 mg/mL).
2.2. Construction and complementation of the arcA deletion mutant
2.5. Serum bactericidal assay
The primers used for amplification are listed in Table S2. The 1 kb upstream and downstream homologous arms were amplified from either side of the arcA sequence of EP3 strain by primers arcAUp-F/R and arcA-Down-F/R, respectively. A kanamycin resistant (kanr) cassette was amplified from pKD4. Three PCR fragments (upstream, kanamycin and downstream) were integrated by overlap PCR with primers arcA-UD-F/R. Then the fusion segment was digested by EcoRI and BamHI, and subsequently cloned into pK18mobsacB which was also digested by EcoRI and BamHI to construct plasmid pAD. Both DNA fragments (upstream homologous arm and downstream homologous arm) included a 9-bp core DNA uptake signal sequence (USS) of 50 - ACCGCTTGT 30 (Zhang et al., 2012). Eventually, the plasmid was mobilized into H. parasuis EP3 strain by natural transformation. The 717-bp PCR product, which contains the entire arcA open reading frame (ORF), was amplified by the primers arcA-F/R and cloned into the pLS88 plasmid to construct plasmid pCA (Xie et al., 2013). Then the pCA was introduced into DarcA mutant by electroporation. The complemented DarcA mutant (C-arcA) was selected on TSA containing NAD, bovine serum and streptomycin. For western blotting analysis, the whole-cell extract of EP3 strains, DarcA mutant strains and C-arcA strains were analyzed by 12% SDS-PAGE and electrotransferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in PBST (phosphate-buffered saline containing 0.05% Tween 20) at room temperature (RT) for 30 min, the membrane was incubated at RT for 1 h with mice antiserum against recombinant ArcA as the primary antibody. Horseradish peroxidase-conjugated goat antimice IgG (Bioss, China) were used as the secondary antibody. The membrane was developed with Immun-Star Western C Kit (biorad, USA) according to the manufacturer’s instructions (Zhang et al., 2015).
The fresh sera were collected from healthy piglets and were filtered by the filtration membrane of 0.22 mm. A part of the sera were treated at 56 C for 30 min to inactivate the complement. 80 mL bacterial cultures were mixed with 20 mL fresh sera or heattreated sera. The mixtures were incubated at 37 C for 1 h with gentle agitation (130 rpm/min), subsequently were diluted with 10-fold serial and treated with TSA containing 5% bovine serum and 0.01% NAD. Then they were incubated at 37 C for 24 h. The assay was performed according to the previous literature (Zou et al., 2013). The survival ratio was calculated by determining the ratio of colonies in the fresh serum to those in the heat-treated serum. All experiments were performed in triplicate. 2.6. Biofilm formation assay Biofilm formation assay was performed according to the previous literature (Wang et al., 2013). 1 mL TSB medium containing 5% inactivated bovine serum and 0.01% NAD in borosilicate glass tubes were inoculated with 10 mL bacterial culture, which was incubated for 16 h at 37 C with agitation (150 rpm/min). The bacterial suspension was removed and the tubes were washed rigorously with water. Then each tube was stained with 2 mL 1% crystal violet solution at room temperature for 5 min. Next, the tubes were washed rigorously with water and then dried. 1 mL 33% (v/v) acetic acid was added into each tube. Finally, the optical density (OD) was measured at a wavelength of 630 nm. TSB without bacterial cultures was designed as a negative control in this experiment. All experiments were performed in triplicate. 3. Results 3.1. Verification of the arcA gene deletion and complementation
2.3. Genetic stability and growth characteristic of the mutant strains The DarcA mutant strains were cultured and passaged for 10 times continuously in TSB supplemented with 5% inactivated bovine serum and 0.01% NAD (Liu et al., 2013). The corresponding fragments were amplified by PCR to identify the genetic stability of each generation of the mutant strains. The wild-type strains EP3, DarcA strains and C-arcA strains were grown respectively in TSB supplemented with serum and NAD, then they were subcultured 1:100 into fresh medium at 37 C under aerobic or anaerobic conditions. The optical density at 600 nm (A600) was measured at 1 h intervals. All experiments were performed in triplicate.
The arcA gene mutant was constructed by homologous recombination and was confirmed by PCR (Fig. 1A). The arcA fragment (732 bp) was amplified from both EP3 strains (Lane 1) and C-arcA strains (Lane 7) by primers arcA-F/R, but not from the DarcA strains (Lane 4). The 2753bp fragment (upstream-arcAdownstream) was amplified from the EP3 strains (Lane 2), while 2971bp fragment (upstream-Kanr-downstream) was amplified from both DarcA strains (Lane 5)and C-arcA strains (Lane 8)by the primers arcA-UD-F/R. The kanamycin fragment (935bp) was amplified from both DarcA strains (Lane 6) and C-arcA strains (Lane 9) by primers arcA-F/R, but not from the EP3 strains (Lane 3). Additionally, Western blotting (Fig. 1B) showed that the ArcA was detected in the lysates of EP3 (Lane 1) and C-arcA strains (Lane 3),
L. Ding et al. / Veterinary Microbiology 196 (2016) 67–71
Fig. 1. (A) PCR identification of the arcA deletion and complementation. Primers arcA-F/R were used to amplify the arcA gene fragment from EP3 strain (Lane 1), DarcA strain (Lane 4) and C-arcA strain (Lane 7); primers arcA-UD-F/R were used to amplify arcA upstream to downstream from EP3 strain (Lane 2), DarcA strain (Lane 5) and C-arcA strain (Lane 8); primers Kan-F/R were used to amplify the Kanr gene fragment from EP3 strain (Lane 3), DarcA strain (Lane 6) and C-arcA strain (Lane 9); lane M shows DNA molecular marker. (B) Western blotting analysis of the EP3 strain. DarcA strain and C-arcA strain. The lysates of the EP3, DarcA and C-arcA strains were detected using anti-ArcA antibodies. The C-arcA strain displayed the similar band as that of the parent strain EP3, while DarcA strain did not. Lane 1: EP3 strain, Lane 2: DarcA strain, Lane 3: C-arcA strain, Lane 4: positive control (ArcA protein).
while not in the lysates of DarcA strains (Lane 2). Together, these results indicated the successful generation of an arcA gene deletion mutant and its complemented strain. 3.2. Growth characteristics of DarcA mutant strains Compared with the wild-type EP3 strains, the DarcA strains did not display significantly growth defects in vitro under aerobic condition (Fig. 2A). However, the growth curve of the DarcA strains significantly (p < 0.05) decreased under anaerobic condition. The absorbance reading at 600 nm (OD600) in the 12 h of DarcA strains was 0.480, whereas the EP3 strains achieved 1.168 at the same time (Fig. 2B).The results showed that deletion of the arcA gene in H. parasuis EP3 strain led to significant (p < 0.05) growth defects under anaerobic condition, but not under aerobic condition.
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Fig. 2. The growth curves of the H. parasuis EP3 under aerobic and anaerobic conditions. Overnight cultures of the wild-type EP3 (D), DarcA () and C-arcA (&) strains were diluted into TSB/V/S and then incubated under either aerobic (A) or anaerobic conditions (B). Bacterial growth was monitored by measurement of optical density at 600 nm. Data points represent the mean values of three replicates, and error bars indicate standard deviations.
the PBS control group throughout the entire virulence experiments. These findings indicated that arcA was involved in bacterial virulence. 3.4. Serum resistance phenotype The serum-resistance assay showed that EP3 strain presented better survival ability when incubated with 20% porcine serum at
3.3. Virulence attenuation of the DarcA mutant in Balb/C mice The animal infection experiment showed that mice in the EP3 group presented severe clinical signs of infection, such as weight loss, arthrocele, rough coat, lethargy and shivering, while the eight mice challenged with DarcA mutant showed slightly clinical signs. Moreover, only two mice from the EP3 group survived after day 4 post-infection, while the seven mice from the DarcA mutant group survived after day 4 post-infection and had recovered from the infection symptoms by day 6. In the C-arcA group, only three mice survived after day 4 post-infection. No anomalies were observed in
Fig. 3. Survival of H. parasuis strains treated with 20% porcine serum. The DarcA strain showed significantly increased susceptibility to serum compared with the wild type strain EP3, while the C-arcA strains restored the serum resistant phenotype. Error bars represent the standard deviation from three independent experiments.
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60 min (Fig. 3). Compared with EP3 strain, loss of arcA resulted in significantly increased sensitivity to serum killing. The survival rate of the DarcA mutant in 20% serum was 10.87% which was less than that of the wild strain EP3 (76.9%). However, the C-arcA strains largely corrected this defective phenotype of serum resistance. These results revealed that the DarcA mutant was more sensitive to serum killing than the wild-type EP3 strains, indicating that arcA may be associated with serum resistance. 3.5. Biofilm formation assay The biofilm formation assay indicated that the DarcA mutant significantly weakened the ability to form biofilm compared with the wild-type EP3 strain (Fig. S). Biofilm production in borosilicate glass tubes was characterized by the absorbance (630 nm) of crystal violet solution. The absorbance was 0.280 for the wild-type EP3 strain and 0.182 for the DarcA mutant. The C-arcA strains restored biofilm formation to wild-type levels (Fig. 4). The results revealed that the biofilm production of wild strain was apparently more than DarcA mutant, indicating that arcA was involved in bacterial biofilm formation. 4. Discussion H. parasuis is a commensal organism found in the upper respiratory tract of swine. However, under certain circumstances, virulent strains can breach the mucosal barrier and enter the blood stream, causing severe systemic disease (Huang et al., 2016). The serum-resistance is a virulence mechanism in H. parasuis (CerdaCuellar and Aragon, 2008). In this study, the sensitivity of the DarcA mutant strain to bactericidal activities in porcine serum strongly increased compared with wild-type strain. However, the complementation recovered the phenotype. Moreover, the DarcA mutant exhibited highly attenuated virulence compared to the wild-type strain in the Balb/C mice, which was similar to the results of the deletion of the arcA gene in H. influenzae (De SouzaHart et al., 2003). The LOS core oligosaccharide of H. parasuis participates in pathogenesis of the disease process, including serum resistance, adhesion and invasion (Zhang et al., 2014b). For H. influenzae, the ArcA positively regulates transcription of glycosyltransferase gene, lic2B, which is responsible for a galactose addition to the LOS outer core. For H. parasuis, we assumed that ArcA also activates LOS biosynthesis genes which are required for serum resistance. These indicated definitely that arcA gene plays important roles in the serum-resistance and virulence of H.parasuis EP3.
Alteration of the cellular redox environment has been shown to affect a broad range of biological processes including energy metabolism, protein folding, signal transduction and stress responses. Under anaerobic conditions, ArcA activated the expression of operons encoding enzyms that are important for adapting to anaerobic environments in Escherichia coli, which maintained redox balance for cell survival (Park et al., 2013). In this study, under anaerobic condition, deletion of arcA from H. parasuis wild-type EP3 resulted in significant (p < 0.05) growth defects, the growth curve of the arcA mutant strain observably decreased compared with the wild-type EP3 strain, but the DarcA mutant and its parental strain EP3 showed similar growth rates under aerobic conditions. These indicated that arcA may play an important role for anaerobic metabolism in H.parasuis. The biofilm formation by microorganisms is a mechanism that allows them to become persistent colonies, resist clearance by the host immune system, enhance resistance to antibiotics and exchange genetic materials. Many veterinary pathogens are capable to form biofilm, which are essential to cause persistent infection (Zhang et al., 2014c). The most serovars of H. parasuis can form biofilm at different levels in vitro (Jin et al., 2006). In this study, we observed that deletion of arcA gene reduced the ability of biofilm formation compared with wile-type in H. parasuis. For A. pleuropneumoniae (App), a difference in threadlike extracellular material was observed between parent strains and DarcA. This extracellular matrix appears to not be appropriately anchored to DarcA (Buettner et al., 2008). For H. influenzae, ArcA may modulate the expression level of genes involved in metabolic pathways to regulate biofilm (Post et al., 2014). More research is needed, though, to investigate whether these effects would also apply to H. parasuis. In conclusion, we successfully constructed the arcA deletion mutant of H. parasuis and preliminary investigated the effect of arcA gene in the H. parasuis EP3 strain on growth characteristic, serum resistance, biofilms and virulence. We found that the DarcA mutant strain exhibited growth defects under anaerobic condition, weakened virulence to mice, increased sensitivity to serum killing and reduced ability of biofilm formation, which indicated that the arcA gene was involved in pathogenesis of H. parasuis EP3 strain. In addition, further studies are needed to determine the role of arcA gene in regulating the expression of specific genes under aerobic or anaerobic conditions. Acknowledgements This work was supported by Haemophflussuis and Porcine Contagious Pleuropneumonia Prevention Technology Research and Demonstration Project (201303034), which was financed by public service (Agriculture) research project special funds. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2016.10.011. References
Fig. 4. Quantification of biofilm production in H. parasuis EP3, DarcA and C-arcA strains. Formed at the air–liquid interface of glass tubes, stained with crystal violet solution. The optical density (OD) was measured at a wavelength of 630 nm. Error bars represent the standard deviation from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Bai, H., Rolfe, M.D., Jia, W., Coakley, S., Poole, R.K., Green, J., Holcombe, M., 2014. Agent-based modeling of oxygen-responsive transcription factors in Escherichia coli. PLoS Comput. Biol. 10, e1003595. Biberstein, E.L., White, D.C., 1969. A proposal for the establishment of two new Haemophilus species. J. Med. Microbiol. 2, 75–78. Buettner, F.F., Maas, A., Gerlach, G.F., 2008. An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation. Vet. Microbiol. 127, 106–115. Cai, X., Chen, H., Blackall, P., Yin, Z., Wang, L., Liu, Z., Jin, M., 2005. Serological characterization of isolates from China. Vet. Microbiol. 111, 231–236.
L. Ding et al. / Veterinary Microbiology 196 (2016) 67–71 Cerda-Cuellar, M., Aragon, V., 2008. Serum-resistance in Haemophilus parasuis is associated with systemic disease in swine. Vet. J. 175, 384–389. De Souza-Hart, J.A., Blackstock, W., Di Modugno, V., Holland, I.B., Kok, M., 2003. Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance. Infect. Immun. 71, 163–172. Federowicz, S., Kim, D., Ebrahim, A., Lerman, J., Nagarajan, H., Cho, B.K., Zengler, K., Palsson, B., 2014. Determining the control circuitry of redox metabolism at the genome-scale. PLoS Genet. 10, e1004264. Huang, J., Wang, X., Cao, Q., Feng, F., Xu, X., Cai, X., 2016. ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis. Vet. Microbiol. 182, 141–149. Jin, H., Zhou, R., Kang, M., Luo, R., Cai, X., Chen, H., 2006. Biofilm formation by field isolates and reference strains of Haemophilus parasuis. Vet. Microbiol. 118, 117–123. Liu, Q., Gong, Y., Cao, Y., Wen, X., Huang, X., Yan, Q., Huang, Y., Cao, S., 2013. Construction and immunogenicity of a (apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Onderstepoort J. Vet. Res. 80, E1–E6. Oliveira, S., Pijoan, C., 2004. Computer-based analysis of Haemophilus parasuis protein fingerprints. Can. J. Vet. Res. 68, 71–75. Park, D.M., Akhtar, M.S., Ansari, A.Z., Landick, R., Kiley, P.J., 2013. The bacterial response regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally. PLoS Genet. 9, e1003839. Post, D.M., Held, J.M., Ketterer, M.R., Phillips, N.J., Sahu, A., Apicella, M.A., Gibson, B. W., 2014. Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry. BMC Microbiol. 14, 329. Šev9 cı’k, M.r., Šebková, A., Volf, J., Rychlı’k, I., 2001. Transcription of arcA and rpoS during growth of Salmonella typhimurium under aerobic and microaerobic conditions. Microbiology 147, 701–708. Wang, X., Xu, X., Wu, Y., Li, L., Cao, R., Cai, X., Chen, H., 2013. Polysaccharide biosynthesis protein CapD is a novel pathogenicity-associated determinant of
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Haemophilus parasuis involved in serum-resistance ability. Vet. Microbiol. 164, 184–189. Wong, S.M., St Michael, F., Cox, A., Ram, S., Akerley, B.J., 2011. ArcA-regulated glycosyltransferase lic2B promotes complement evasion and pathogenesis of nontypeable Haemophilus influenzae. Infect. Immun. 79, 1971–1983. Wuichet, K., Cantwell, B.J., Zhulin, I.B., 2010. Evolution and phyletic distribution of two-component signal transduction systems. Curr. Opin. Microbiol. 13, 219–225. Xie, F., Zhang, Y., Li, G., Zhou, L., Liu, S., Wang, C., 2013. The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae. PLoS One 8, e53600. Zhang, B., Feng, S., Xu, C., Zhou, S., He, Y., Zhang, L., Zhang, J., Guo, L., Liao, M., 2012. Serum resistance in Haemophilus parasuis SC096 strain requires outer membrane protein P2 expression. FEMS Microbiol. Lett. 326, 109–115. Zhang, B., Tang, C., Liao, M., Yue, H., 2014a. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors. Vet. Microbiol. 168, 1–7. Zhang, B., Yu, Y., Zeng, Z., Ren, Y., Yue, H., 2014b. Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance, adhesion and invasion. Microb. Pathog. 74, 33–37. Zhang, J., Xu, C., Shen, H., Li, J., Guo, L., Cao, G., Feng, S., Liao, M., 2014c. Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance, serotype and genetic typing. Res. Vet. Sci. 97, 171–175. Zhang, L., Li, Y., Dai, K., Wen, X., Wu, R., Huang, X., Jin, J., Xu, K., Yan, Q., Huang, Y., Ma, X., Wen, Y., Cao, S., 2015. Establishment of a successive markerless mutation system in haemophilus parasuis through natural transformation. PLoS One 10, e0127393. Zou, Y., Feng, S.X., Xu, C.G., Zhang, B., Zhou, S.M., Zhang, L.Y., He, X.H., Li, J.Y., Yang, Z., Liao, M., 2013. The role of galU and galE of Haemophilus parasuis SC096 in serum resistance and biofilm formation. Vet. Microbiol. 162, 278–284.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3 Lingqiang Ding, Xintian Wen, Lvqin He, Xuefeng Yan, Yongping Wen, Sanjie Cao, Xiaobo Huang, Rui Wu, Yiping Wen* Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
A R T I C L E I N F O
Article history: Received 22 March 2016 Received in revised form 25 August 2016 Accepted 9 October 2016 Keywords: H. parasuis arcA gene Serum resistance Virulence
A B S T R A C T
As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (DarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the DarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis. ã 2016 Published by Elsevier B.V.
1. Introduction Currently, Glässer’s disease, which is characterized by polyserositis and fibrinopurulent polyarthritis (Cai et al., 2005), is causing a significant increase with respect to mortality and morbidity in swine, leading to major economic losses in the pig industry (Zhang et al., 2014a). H. parasuis, a member of the family Pasteurellaceae (Biberstein and White, 1969), is the causative agent of Glässer’s disease. As a commensal colonizing the upper respiratory tract of pigs, under certain circumstances, H. parasuis can breach the mucosal barrier and enter the blood stream, causing severe systemic disease (Huang et al., 2016). To date, several virulence factors have been identified in H. parasuis, including lipopolysaccharide (LPS), capsular polysaccharide, fimbriae, outer membrane proteins, neuraminidase and iron acquisition systems (Zhang et al., 2014a). However, the virulence factors and the mechanisms of the entire infection process remain largely unclear (Oliveira and Pijoan, 2004). In natural environments, bacteria are challenged with a variety of rapidly changing conditions that have to be sensed in order for the bacteria to respond appropriately. To this end, most bacteria
* Corresponding author at: Huimin Road 211# Wenjiang District, Chengdu, Sichuan, 611130, China. E-mail addresses: [email protected] (L. Ding), [email protected] (X. Wen), [email protected] (L. He), [email protected] (X. Yan), [email protected] (Y. Wen), [email protected] (S. Cao), [email protected] (X. Huang), [email protected] (R. Wu), [email protected], [email protected] (Y. Wen). http://dx.doi.org/10.1016/j.vetmic.2016.10.011 0378-1135/ã 2016 Published by Elsevier B.V.
employ two-component signal transduction system (TCSTS) comprising a sensor histidine kinase (HK) and a response regulator (RR) (Wuichet et al., 2010). Three TCSTS, encoded by cpxAR, arcAB, and qseBC, were identified in the genome of H. parasuis. The ArcA/B consists of the sensor kinase ArcB which accepts extracellular stimulating signal, and the DNA-binding response regulator ArcA which regulates the expression of many genes. As a global transcriptional factor, ArcA relates to bacterial aerobic and anaerobic metabolism, anabolic and catabolic regulation, iron transport and energy generation pathways in Escherichia coli (Bai et al., 2014; Federowicz et al., 2014). For H. influenzae, the virulence of the arcA mutant for Balb/C mice was significantly reduced. The H. influenzae arcA mutant also markedly enhanced sensitivity to serum (De Souza-Hart et al., 2003). H. influenzae utilizes ArcA to appropriately modify its immune evasion strategies in different environments encounter during infection (Wong et al., 2011). Compared with wild-type, the arcA mutant has poor biofilms in A. pleuropneumoniae, which likely resulted in the reduction of virulence (Buettner et al., 2008). Salmonella arcA mutant have been shown to grow more slowly than the wild-type strains in both aerobic and microaerobic conditions (Šev9 cı’k et al., 2001). However, less functional description of this gene has been reported, and its specific roles during H. parasuis infection have been little understood. In this study, we generated an arcA deletion mutant of H. parasuis EP3 strain to investigate its role in growth characteristic, serum resistance, biofilm formation and virulence, aiming to provide some data for the further study about ArcA/B in H. parasuis.
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2. Materials and methods
2.4. Animal infection studies
2.1. Bacterial strains, plasmids and growth conditions
The animal experiments were conducted in strict accordance with the recommendations in the China Regulations for the Administration of Affairs Concerning Experimental Animals (1988) and had been approved by the Institutional Animal Care and Use Committee of Sichuan Agriculture University (Approval Number YP2014-039), Sichuan, China. Twenty-nine 6-week-old female specific pathogen free (SPF) Balb/C mice (Sichuan Province Dashuo Experimental Animal Centre, Chengdu, China) were allocated randomly to EP3, DarcA and C-arcA groups, each group contained eight mice. The remaining five were allocated to the PBS control group. The mice were injected intraperitoneally with bacteria at a dose of 1.4 109 CFU/animal (0.5 mL) or with 0.5 mL PBS. All surviving mice were euthanized 7 days post infection.
The bacterial strains and plasmids used in this study were described in Table S1. E.coli DH5a was grown in LB medium at 37 C. The H. parasuis serovar 13 clinical strain EP3, which was isolated from lungs of 4-month-old diseased pigs in China’s Sichuan province, was cultivated in Trypticase Soy Agar (TSA) or Trypticase Soy Broth (TSB) (Difco, Detroit, USA) supplemented with 0.001% (w/v) nicotinamide adenine dinucleotide (NAD) (Sigma Aldrich, Missouri, USA) and 5% (v/v) inactivated bovine serum at 37 C. When necessary, the media were supplemented with kanamycin (50 mg/mL) or streptomycin (50 mg/mL).
2.2. Construction and complementation of the arcA deletion mutant
2.5. Serum bactericidal assay
The primers used for amplification are listed in Table S2. The 1 kb upstream and downstream homologous arms were amplified from either side of the arcA sequence of EP3 strain by primers arcAUp-F/R and arcA-Down-F/R, respectively. A kanamycin resistant (kanr) cassette was amplified from pKD4. Three PCR fragments (upstream, kanamycin and downstream) were integrated by overlap PCR with primers arcA-UD-F/R. Then the fusion segment was digested by EcoRI and BamHI, and subsequently cloned into pK18mobsacB which was also digested by EcoRI and BamHI to construct plasmid pAD. Both DNA fragments (upstream homologous arm and downstream homologous arm) included a 9-bp core DNA uptake signal sequence (USS) of 50 - ACCGCTTGT 30 (Zhang et al., 2012). Eventually, the plasmid was mobilized into H. parasuis EP3 strain by natural transformation. The 717-bp PCR product, which contains the entire arcA open reading frame (ORF), was amplified by the primers arcA-F/R and cloned into the pLS88 plasmid to construct plasmid pCA (Xie et al., 2013). Then the pCA was introduced into DarcA mutant by electroporation. The complemented DarcA mutant (C-arcA) was selected on TSA containing NAD, bovine serum and streptomycin. For western blotting analysis, the whole-cell extract of EP3 strains, DarcA mutant strains and C-arcA strains were analyzed by 12% SDS-PAGE and electrotransferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in PBST (phosphate-buffered saline containing 0.05% Tween 20) at room temperature (RT) for 30 min, the membrane was incubated at RT for 1 h with mice antiserum against recombinant ArcA as the primary antibody. Horseradish peroxidase-conjugated goat antimice IgG (Bioss, China) were used as the secondary antibody. The membrane was developed with Immun-Star Western C Kit (biorad, USA) according to the manufacturer’s instructions (Zhang et al., 2015).
The fresh sera were collected from healthy piglets and were filtered by the filtration membrane of 0.22 mm. A part of the sera were treated at 56 C for 30 min to inactivate the complement. 80 mL bacterial cultures were mixed with 20 mL fresh sera or heattreated sera. The mixtures were incubated at 37 C for 1 h with gentle agitation (130 rpm/min), subsequently were diluted with 10-fold serial and treated with TSA containing 5% bovine serum and 0.01% NAD. Then they were incubated at 37 C for 24 h. The assay was performed according to the previous literature (Zou et al., 2013). The survival ratio was calculated by determining the ratio of colonies in the fresh serum to those in the heat-treated serum. All experiments were performed in triplicate. 2.6. Biofilm formation assay Biofilm formation assay was performed according to the previous literature (Wang et al., 2013). 1 mL TSB medium containing 5% inactivated bovine serum and 0.01% NAD in borosilicate glass tubes were inoculated with 10 mL bacterial culture, which was incubated for 16 h at 37 C with agitation (150 rpm/min). The bacterial suspension was removed and the tubes were washed rigorously with water. Then each tube was stained with 2 mL 1% crystal violet solution at room temperature for 5 min. Next, the tubes were washed rigorously with water and then dried. 1 mL 33% (v/v) acetic acid was added into each tube. Finally, the optical density (OD) was measured at a wavelength of 630 nm. TSB without bacterial cultures was designed as a negative control in this experiment. All experiments were performed in triplicate. 3. Results 3.1. Verification of the arcA gene deletion and complementation
2.3. Genetic stability and growth characteristic of the mutant strains The DarcA mutant strains were cultured and passaged for 10 times continuously in TSB supplemented with 5% inactivated bovine serum and 0.01% NAD (Liu et al., 2013). The corresponding fragments were amplified by PCR to identify the genetic stability of each generation of the mutant strains. The wild-type strains EP3, DarcA strains and C-arcA strains were grown respectively in TSB supplemented with serum and NAD, then they were subcultured 1:100 into fresh medium at 37 C under aerobic or anaerobic conditions. The optical density at 600 nm (A600) was measured at 1 h intervals. All experiments were performed in triplicate.
The arcA gene mutant was constructed by homologous recombination and was confirmed by PCR (Fig. 1A). The arcA fragment (732 bp) was amplified from both EP3 strains (Lane 1) and C-arcA strains (Lane 7) by primers arcA-F/R, but not from the DarcA strains (Lane 4). The 2753bp fragment (upstream-arcAdownstream) was amplified from the EP3 strains (Lane 2), while 2971bp fragment (upstream-Kanr-downstream) was amplified from both DarcA strains (Lane 5)and C-arcA strains (Lane 8)by the primers arcA-UD-F/R. The kanamycin fragment (935bp) was amplified from both DarcA strains (Lane 6) and C-arcA strains (Lane 9) by primers arcA-F/R, but not from the EP3 strains (Lane 3). Additionally, Western blotting (Fig. 1B) showed that the ArcA was detected in the lysates of EP3 (Lane 1) and C-arcA strains (Lane 3),
L. Ding et al. / Veterinary Microbiology 196 (2016) 67–71
Fig. 1. (A) PCR identification of the arcA deletion and complementation. Primers arcA-F/R were used to amplify the arcA gene fragment from EP3 strain (Lane 1), DarcA strain (Lane 4) and C-arcA strain (Lane 7); primers arcA-UD-F/R were used to amplify arcA upstream to downstream from EP3 strain (Lane 2), DarcA strain (Lane 5) and C-arcA strain (Lane 8); primers Kan-F/R were used to amplify the Kanr gene fragment from EP3 strain (Lane 3), DarcA strain (Lane 6) and C-arcA strain (Lane 9); lane M shows DNA molecular marker. (B) Western blotting analysis of the EP3 strain. DarcA strain and C-arcA strain. The lysates of the EP3, DarcA and C-arcA strains were detected using anti-ArcA antibodies. The C-arcA strain displayed the similar band as that of the parent strain EP3, while DarcA strain did not. Lane 1: EP3 strain, Lane 2: DarcA strain, Lane 3: C-arcA strain, Lane 4: positive control (ArcA protein).
while not in the lysates of DarcA strains (Lane 2). Together, these results indicated the successful generation of an arcA gene deletion mutant and its complemented strain. 3.2. Growth characteristics of DarcA mutant strains Compared with the wild-type EP3 strains, the DarcA strains did not display significantly growth defects in vitro under aerobic condition (Fig. 2A). However, the growth curve of the DarcA strains significantly (p < 0.05) decreased under anaerobic condition. The absorbance reading at 600 nm (OD600) in the 12 h of DarcA strains was 0.480, whereas the EP3 strains achieved 1.168 at the same time (Fig. 2B).The results showed that deletion of the arcA gene in H. parasuis EP3 strain led to significant (p < 0.05) growth defects under anaerobic condition, but not under aerobic condition.
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Fig. 2. The growth curves of the H. parasuis EP3 under aerobic and anaerobic conditions. Overnight cultures of the wild-type EP3 (D), DarcA () and C-arcA (&) strains were diluted into TSB/V/S and then incubated under either aerobic (A) or anaerobic conditions (B). Bacterial growth was monitored by measurement of optical density at 600 nm. Data points represent the mean values of three replicates, and error bars indicate standard deviations.
the PBS control group throughout the entire virulence experiments. These findings indicated that arcA was involved in bacterial virulence. 3.4. Serum resistance phenotype The serum-resistance assay showed that EP3 strain presented better survival ability when incubated with 20% porcine serum at
3.3. Virulence attenuation of the DarcA mutant in Balb/C mice The animal infection experiment showed that mice in the EP3 group presented severe clinical signs of infection, such as weight loss, arthrocele, rough coat, lethargy and shivering, while the eight mice challenged with DarcA mutant showed slightly clinical signs. Moreover, only two mice from the EP3 group survived after day 4 post-infection, while the seven mice from the DarcA mutant group survived after day 4 post-infection and had recovered from the infection symptoms by day 6. In the C-arcA group, only three mice survived after day 4 post-infection. No anomalies were observed in
Fig. 3. Survival of H. parasuis strains treated with 20% porcine serum. The DarcA strain showed significantly increased susceptibility to serum compared with the wild type strain EP3, while the C-arcA strains restored the serum resistant phenotype. Error bars represent the standard deviation from three independent experiments.
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60 min (Fig. 3). Compared with EP3 strain, loss of arcA resulted in significantly increased sensitivity to serum killing. The survival rate of the DarcA mutant in 20% serum was 10.87% which was less than that of the wild strain EP3 (76.9%). However, the C-arcA strains largely corrected this defective phenotype of serum resistance. These results revealed that the DarcA mutant was more sensitive to serum killing than the wild-type EP3 strains, indicating that arcA may be associated with serum resistance. 3.5. Biofilm formation assay The biofilm formation assay indicated that the DarcA mutant significantly weakened the ability to form biofilm compared with the wild-type EP3 strain (Fig. S). Biofilm production in borosilicate glass tubes was characterized by the absorbance (630 nm) of crystal violet solution. The absorbance was 0.280 for the wild-type EP3 strain and 0.182 for the DarcA mutant. The C-arcA strains restored biofilm formation to wild-type levels (Fig. 4). The results revealed that the biofilm production of wild strain was apparently more than DarcA mutant, indicating that arcA was involved in bacterial biofilm formation. 4. Discussion H. parasuis is a commensal organism found in the upper respiratory tract of swine. However, under certain circumstances, virulent strains can breach the mucosal barrier and enter the blood stream, causing severe systemic disease (Huang et al., 2016). The serum-resistance is a virulence mechanism in H. parasuis (CerdaCuellar and Aragon, 2008). In this study, the sensitivity of the DarcA mutant strain to bactericidal activities in porcine serum strongly increased compared with wild-type strain. However, the complementation recovered the phenotype. Moreover, the DarcA mutant exhibited highly attenuated virulence compared to the wild-type strain in the Balb/C mice, which was similar to the results of the deletion of the arcA gene in H. influenzae (De SouzaHart et al., 2003). The LOS core oligosaccharide of H. parasuis participates in pathogenesis of the disease process, including serum resistance, adhesion and invasion (Zhang et al., 2014b). For H. influenzae, the ArcA positively regulates transcription of glycosyltransferase gene, lic2B, which is responsible for a galactose addition to the LOS outer core. For H. parasuis, we assumed that ArcA also activates LOS biosynthesis genes which are required for serum resistance. These indicated definitely that arcA gene plays important roles in the serum-resistance and virulence of H.parasuis EP3.
Alteration of the cellular redox environment has been shown to affect a broad range of biological processes including energy metabolism, protein folding, signal transduction and stress responses. Under anaerobic conditions, ArcA activated the expression of operons encoding enzyms that are important for adapting to anaerobic environments in Escherichia coli, which maintained redox balance for cell survival (Park et al., 2013). In this study, under anaerobic condition, deletion of arcA from H. parasuis wild-type EP3 resulted in significant (p < 0.05) growth defects, the growth curve of the arcA mutant strain observably decreased compared with the wild-type EP3 strain, but the DarcA mutant and its parental strain EP3 showed similar growth rates under aerobic conditions. These indicated that arcA may play an important role for anaerobic metabolism in H.parasuis. The biofilm formation by microorganisms is a mechanism that allows them to become persistent colonies, resist clearance by the host immune system, enhance resistance to antibiotics and exchange genetic materials. Many veterinary pathogens are capable to form biofilm, which are essential to cause persistent infection (Zhang et al., 2014c). The most serovars of H. parasuis can form biofilm at different levels in vitro (Jin et al., 2006). In this study, we observed that deletion of arcA gene reduced the ability of biofilm formation compared with wile-type in H. parasuis. For A. pleuropneumoniae (App), a difference in threadlike extracellular material was observed between parent strains and DarcA. This extracellular matrix appears to not be appropriately anchored to DarcA (Buettner et al., 2008). For H. influenzae, ArcA may modulate the expression level of genes involved in metabolic pathways to regulate biofilm (Post et al., 2014). More research is needed, though, to investigate whether these effects would also apply to H. parasuis. In conclusion, we successfully constructed the arcA deletion mutant of H. parasuis and preliminary investigated the effect of arcA gene in the H. parasuis EP3 strain on growth characteristic, serum resistance, biofilms and virulence. We found that the DarcA mutant strain exhibited growth defects under anaerobic condition, weakened virulence to mice, increased sensitivity to serum killing and reduced ability of biofilm formation, which indicated that the arcA gene was involved in pathogenesis of H. parasuis EP3 strain. In addition, further studies are needed to determine the role of arcA gene in regulating the expression of specific genes under aerobic or anaerobic conditions. Acknowledgements This work was supported by Haemophflussuis and Porcine Contagious Pleuropneumonia Prevention Technology Research and Demonstration Project (201303034), which was financed by public service (Agriculture) research project special funds. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2016.10.011. References
Fig. 4. Quantification of biofilm production in H. parasuis EP3, DarcA and C-arcA strains. Formed at the air–liquid interface of glass tubes, stained with crystal violet solution. The optical density (OD) was measured at a wavelength of 630 nm. Error bars represent the standard deviation from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Bai, H., Rolfe, M.D., Jia, W., Coakley, S., Poole, R.K., Green, J., Holcombe, M., 2014. Agent-based modeling of oxygen-responsive transcription factors in Escherichia coli. PLoS Comput. Biol. 10, e1003595. Biberstein, E.L., White, D.C., 1969. A proposal for the establishment of two new Haemophilus species. J. Med. Microbiol. 2, 75–78. Buettner, F.F., Maas, A., Gerlach, G.F., 2008. An Actinobacillus pleuropneumoniae arcA deletion mutant is attenuated and deficient in biofilm formation. Vet. Microbiol. 127, 106–115. Cai, X., Chen, H., Blackall, P., Yin, Z., Wang, L., Liu, Z., Jin, M., 2005. Serological characterization of isolates from China. Vet. Microbiol. 111, 231–236.
L. Ding et al. / Veterinary Microbiology 196 (2016) 67–71 Cerda-Cuellar, M., Aragon, V., 2008. Serum-resistance in Haemophilus parasuis is associated with systemic disease in swine. Vet. J. 175, 384–389. De Souza-Hart, J.A., Blackstock, W., Di Modugno, V., Holland, I.B., Kok, M., 2003. Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance. Infect. Immun. 71, 163–172. Federowicz, S., Kim, D., Ebrahim, A., Lerman, J., Nagarajan, H., Cho, B.K., Zengler, K., Palsson, B., 2014. Determining the control circuitry of redox metabolism at the genome-scale. PLoS Genet. 10, e1004264. Huang, J., Wang, X., Cao, Q., Feng, F., Xu, X., Cai, X., 2016. ClpP participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis. Vet. Microbiol. 182, 141–149. Jin, H., Zhou, R., Kang, M., Luo, R., Cai, X., Chen, H., 2006. Biofilm formation by field isolates and reference strains of Haemophilus parasuis. Vet. Microbiol. 118, 117–123. Liu, Q., Gong, Y., Cao, Y., Wen, X., Huang, X., Yan, Q., Huang, Y., Cao, S., 2013. Construction and immunogenicity of a (apxIC/ompP2 mutant of Actinobacillus pleuropneumoniae and Haemophilus parasuis. Onderstepoort J. Vet. Res. 80, E1–E6. Oliveira, S., Pijoan, C., 2004. Computer-based analysis of Haemophilus parasuis protein fingerprints. Can. J. Vet. Res. 68, 71–75. Park, D.M., Akhtar, M.S., Ansari, A.Z., Landick, R., Kiley, P.J., 2013. The bacterial response regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally. PLoS Genet. 9, e1003839. Post, D.M., Held, J.M., Ketterer, M.R., Phillips, N.J., Sahu, A., Apicella, M.A., Gibson, B. W., 2014. Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry. BMC Microbiol. 14, 329. Šev9 cı’k, M.r., Šebková, A., Volf, J., Rychlı’k, I., 2001. Transcription of arcA and rpoS during growth of Salmonella typhimurium under aerobic and microaerobic conditions. Microbiology 147, 701–708. Wang, X., Xu, X., Wu, Y., Li, L., Cao, R., Cai, X., Chen, H., 2013. Polysaccharide biosynthesis protein CapD is a novel pathogenicity-associated determinant of
71
Haemophilus parasuis involved in serum-resistance ability. Vet. Microbiol. 164, 184–189. Wong, S.M., St Michael, F., Cox, A., Ram, S., Akerley, B.J., 2011. ArcA-regulated glycosyltransferase lic2B promotes complement evasion and pathogenesis of nontypeable Haemophilus influenzae. Infect. Immun. 79, 1971–1983. Wuichet, K., Cantwell, B.J., Zhulin, I.B., 2010. Evolution and phyletic distribution of two-component signal transduction systems. Curr. Opin. Microbiol. 13, 219–225. Xie, F., Zhang, Y., Li, G., Zhou, L., Liu, S., Wang, C., 2013. The ClpP protease is required for the stress tolerance and biofilm formation in Actinobacillus pleuropneumoniae. PLoS One 8, e53600. Zhang, B., Feng, S., Xu, C., Zhou, S., He, Y., Zhang, L., Zhang, J., Guo, L., Liao, M., 2012. Serum resistance in Haemophilus parasuis SC096 strain requires outer membrane protein P2 expression. FEMS Microbiol. Lett. 326, 109–115. Zhang, B., Tang, C., Liao, M., Yue, H., 2014a. Update on the pathogenesis of Haemophilus parasuis infection and virulence factors. Vet. Microbiol. 168, 1–7. Zhang, B., Yu, Y., Zeng, Z., Ren, Y., Yue, H., 2014b. Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance, adhesion and invasion. Microb. Pathog. 74, 33–37. Zhang, J., Xu, C., Shen, H., Li, J., Guo, L., Cao, G., Feng, S., Liao, M., 2014c. Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance, serotype and genetic typing. Res. Vet. Sci. 97, 171–175. Zhang, L., Li, Y., Dai, K., Wen, X., Wu, R., Huang, X., Jin, J., Xu, K., Yan, Q., Huang, Y., Ma, X., Wen, Y., Cao, S., 2015. Establishment of a successive markerless mutation system in haemophilus parasuis through natural transformation. PLoS One 10, e0127393. Zou, Y., Feng, S.X., Xu, C.G., Zhang, B., Zhou, S.M., Zhang, L.Y., He, X.H., Li, J.Y., Yang, Z., Liao, M., 2013. The role of galU and galE of Haemophilus parasuis SC096 in serum resistance and biofilm formation. Vet. Microbiol. 162, 278–284.