lgtF effects of Haemophilus parasuis LOS induced inflammation through regulation of NF-κB and MAPKs signaling pathways

Microbial Pathogenesis 110 (2017) 380e384

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lgtF effects of Haemophilus parasuis LOS induced inflammation through regulation of NF-kB and MAPKs signaling pathways Ze Zeng, Bin Zhang*, Huan He, Xinnuo Chen, Yupeng Ren, Hua Yue, Cheng Tang** College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 November 2016 Received in revised form 2 June 2017 Accepted 22 June 2017 Available online 14 July 2017

The lgtF gene encodes a glucosyltransferase responsible for adding a glucose to the first sugar of heptose I in the synthesis of lipooligosaccharides (LOS). To study the function of lgtF, we constructed an lgtF mutant (DlgtF) from Haemophilus parasuis SC096 using a natural transformation system. A highly purified preparation of LOS from DlgtF (DlgtF-LOS) exhibited an obvious truncation in structure compared to the LOS of the wild-type SC096 strain (WT-LOS). The DlgtF-LOS also displayed a significantly reduced ability to induce inflammatory cytokine mRNA expression of tumor necrosis factor alpha (TNF-a), interleukin-1a (IL-1a), IL-1b, IL-6 and IL-8 in porcine alveolar macrophages (PAMs) in comparison with the WT-LOS. Furthermore, we also found that DlgtF-LOS-treated cells had significantly decreased phospho-p65 and phospho-p38, and inhibited IkBa degradation. These findings suggested that the lgtF gene mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-kB and MAPKs signaling pathways during H. parasuis infection. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Haemophilus parasuis lgtF LOS Pro-inflammatory factors Signaling pathways

1. Introduction Haemophilus parasuis is a Gram-negative bacterium of the family Pasteurellaceae and is the etiologic agent of Gl€ asser's disease in pigs, which is characterized by serofibrinous to fibrinopurulent polyserositis, arthritis, and meningitis. H. parasuis infection produces significant morbidity and mortality in swine herds, giving rise to important economic losses in the pig industry [1,2]. Multiplication of H. parasuis in the host induces a strong inflammatory reaction mediated by inflammatory cytokines, including IL-1a, IL1b, IL-6, IL-8 and macrophage inflammatory protein-1b (MIP-1b/ CCL4) [3,4]. Furthermore, it has been reported that H. parasuis activates nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinases (MAPKs) pathways mediated by toll-like receptors in host cells [5,6]. Lipooligosaccharides (LOS) are the major constituents of Gram-

* Corresponding author. College of Life Science and Technology, Southwest University for Nationalities, No.16, South 4th Section 1st Ring Road, Chengdu 610041, China. ** Corresponding author. College of Life Science and Technology, Southwest University for Nationalities, No.16, South 4th Section 1st Ring Road, Chengdu 610041, China. E-mail addresses: [email protected] (B. Zhang), [email protected] (C. Tang). http://dx.doi.org/10.1016/j.micpath.2017.06.035 0882-4010/© 2017 Elsevier Ltd. All rights reserved.

negative bacteria the outer membranes and greatly influence pathogenesis [7,8]. The LOS generally contains lipid A, kdo, heptoses and other glycosyl. In H. parasuis, the monosaccharide analysis of LOS showed the presence of glucose, galactose, galactose-N, L-glycero-D-manno-heptose, and D-glycero-D-manno-heptose, with glucose as the main component [9]. Further studies found that the LOS of H. parasuis is able to upregulate the expression of IL-1a, IL1b, IL-6 and IL-8 in host cells [10,11]. The lgtF gene encodes glucosyltransferase enzymes responsible for adding the first glycose to heptose I and is involved in the assembly of LOS [12]. The lgtF gene was known as a virulence factor in Gram-negative pathogenic bacteria [7,12,13]. Nevertheless, the role of the lgtF gene in the pathogenesis of H. parasuis was not known. In this study, we constructed an lgtF mutant of H. parasuis SC096 and showed that its LOS significantly reduced inflammatory cytokine expression by regulating NF-kB and MAPKs signaling pathways in porcine alveolar macrophages. 2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli plasmids were propagated in E. coli DH5a grown in Luria-Bertani medium at 37  C. The H. parasuis strain

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Table 1 Strains and plasmids used in this study. Strains or plasmids Strains H.parasuisSC096 DlgtF Plasmids pMD-19T pK18mobsacB pHV1249 pZZ1 pZZ2 pZZ3

Description

Reference

Serovar 4 clinical isolate SC096 DlgtF::EmR

[14] This study

T-vector, AmpR Suicide and narrow-broad-host vector, KanR Em resistance cassette-carrying vector, EmR A fragment containing the upstream and downstream sequences of the lgtF gene in pMD-19T, AmpR A fragment containing the upstream and downstream sequences of the lgtF gene inpK18mobsacB,KanR A fragment containing EmR, the upstream and downstream sequences of the lgtF gene in pK18mobsacB, KanREmR

Takara [22] [23] This study

SC096 was cultivated in Trypticase Soy Agar (TSA) or Trypticase Soy Broth (TSB) (Oxoid, Hampshire, UK) supplemented with 0.002% (w/ v) nicotinamide adenine dinucleotide (NAD) (Sigma Aldrich, Missouri, USA) and 5% (v/v) inactivated bovine serum at 37  C in a 5% CO2 enriched atmosphere. When required, the media were supplemented with kanamycin (30 mg/mL), ampicillin (100 mg/mL) or erythromycin (30 mg/mL). 2.2. Construction of the lgtF mutant The primers used in this study are listed in Table S1. A 481 bp DNA fragment region upstream lgtF was PCR amplified using the primer pairs Pa and Pb, and the 484 bp downstream DNA fragment region was PCR amplified using the primer pairs Pc and Pd. These two fragments were connected by overlapping PCR with primers Pa and Pd, then purified, and cloned into pMD19-T (TaKaRa, Japan) for plasmid pZZ1. The plasmid pZZ1 was digested with HindIII and EcoRI, purified, and ligated into pK18mobsacB for plasmid pZZ2. An 863 bp erythromycin resistant fragment was cloned into the plasmid pZZ2 and digested with BamHI and SalI for plasmid pZZ3. The pZZ3 plasmid was mobilized into E. coli DH5a during a CaCl2mediated transformation. Natural transformation was used to introduce pZZ3 into the SC096 strain following a previously described method [14]. 2.3. LOS extraction and SDS-PAGE LOS was extracted from H. parasuis using the hot-phenol method [15], and quantified using the anthrone-sulfuric acid method [11]. LOS preparations were treated with sodium dodecyl sulfate (SDS) loading buffer (100 mM Tris-HCl pH 8.0, 2% b-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 0.2% xylene cyanole, 20% glycerol), separated by SDS-polyacrylamide gel electrophoresis (PAGE; 5% stacking gel and 18% separating gel) by electrophoresis at 100 V for 1 h and visualized with silver staining. 2.4. Quantitative real-time PCR (qRT-PCR) Porcine alveolar macrophages (PAMs, cell line 3D4/2) were obtained from ATCC. PAMs were seeded in 12-well tissue culture trays, incubated in RPMI 1640 medium (Invitrogen, USA) supplemented with 10% (V/V) heat-inactivated fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin. Cells were stimulated with LOS from H. parasuis SC096 and lgtF mutant strains at concentrations of 5 and 10 mg/mL, respectively. Cell pellets were collected at 6 h, 12 h and 24 h after incubation and stored at 20  C. Total RNA was isolated from PAMs using TRIzol (Invitrogen), and cDNA was synthesized with the PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). Real-time PCR was performed with the

This study This study

primer pairs (Table S1) using SYBR® Premix Ex Taq™ II (TaKaRa) and an Applied Biosystems 7300 Real-time PCR System (ABI, USA). The data were analyzed using the 2-△△CT method in triplicates for three independent experiments. A LPS preparation from E. coli O111: B4 (10 mg/mL, Sigma Aldrich) was used as a positive control, and the unstimulated PAMs cells was used as mock-stimulus. The ribosomal protein L4 (RPL4), stably expressed in PAMs, was used as a reference gene for normalization gene expression results detected by the real-time PCR assay [16]. 2.5. Western blot analysis Western blot analysis was performed with anti-NF-kB p65, antiphospho-NF-kB p65, anti-IkBa and anti-GADPH monoclonal antibodies, as well as anti-p38 and anti-phospho-p38 polyclonal antibodies were obtained from Cell Signaling Technology (CST, USA). The HRP-conjugated goat anti-mouse or goat anti-rabbit IgG were obtained from Abbkine (USA), respectively. Densitometry values of immunoblot signals were obtained from three separate experiments using FusionCapt Advance software (Vilber Lourmat, Germany). 2.6. Statistical analysis The comparison of several test series was performed using oneway analysis of variance (ANOVA) with SPSS version 16.0 software (SPSS Inc., USA). A student's t-test was used to identify differences among groups. P values < 0.05 were considered to be statistically significant. 3. Result and discussion 3.1. Characterization of the H. parasuis lgtF mutant LOS was confirmed as an important virulence factor in H. parasuis [2,10,17]. To further investigate the association of pathogenesis with LOS residues, we constructed an lgtF mutant of H. parasuis SC096 by natural transformation. To obtain the lgtF mutant, the pZZ3 plasmid was used as donor DNA, and was introduced by natural transformation into the wild-type SC096 strain. Colony PCR was used to check the erythromycin resistant transformants. As expected, the primers Pa and Pd amplified an 1828 bp fragment from the lgtF mutant, whereas a 1974 bp fragment was observed in the wild-type SC096 strain (Fig. 1A and Fig. 1B). The primers Pe and Pf amplified an 863 bp erythromycin resistant fragment from the lgtF mutant, whereas no erythromycin resistant fragment was observed in the wild-type SC096 strain. To investigate the variation of LOS synthesis, the LOS profiles of the wild-type SC096 strain and lgtF mutant were assayed by SDS-PAGE. LOS from

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Fig. 1. (A) Characterization of the lgtF mutant and wild-type SCO96 strain. Part 1 showed the map of the lgtF gene for wild-type SC096. Part 2 showed the map of the DlgtF:EmR insertion mutant. (B) PCR analysis of genomic DNA extracted from the strains. Primers Pa and Pd were used to amplify wild-type SC096 (Lane 1) and DlgtF mutant (Lane 2), and Primers Pe and Pf were used to amplify wild-type SC096 (Lane 3) and DlgtF mutant (Lane 4). Lane M, DNA molecular maker. (C) LOS profiles of the wild-type SC096 strain and lgtF mutant. Lane 1, wild-type SC096 strain; Lane 2, lgtF mutant.

the lgtF mutant (DlgtF-LOS) migrated faster than the LOS from the wild-type SC096 strain (WT-LOS) (Fig. 1B). The results showed that the lgtF mutant strain expressed truncated LOS structure, indicating that the DlgtF-LOS carried fewer glycosyl residues compared with the WT-LOS, which was consistent with results in other bacteria [12,13]. The lgtF encodes a glucosyltransferase responsible for adding a glucose to heptose I [12]. In H. parasuis, the heptose I was confirmed participating in the synthesis of LOS [17]. Deletion of the lgtB and lex-1 genes, which encode the two glycosyltransferases, produced severely truncated LOS structures in H. parasuis SC096, and both genes were involved in serum resistance, adhesion and invasion [18]. Therefore, we speculated that the lgtF gene was most probably involved in the synthesis of heptose I and loss of the gene caused severe truncation in the LOS chain of the H. parasuis SC096 strain.

3.2. DlgtF-LOS decreased pro-inflammatory cytokine expression in PAMs In gram-negative bacteria, LOS is a highly efficient proinflammatory response factor that activates the release of numerous pro-inflammatory cytokines (IL-1b, IL-6, IL-8, and TNF-a) through the NF-kB and MAPKs signaling pathways [19]. Macrophages, critical effector cells of innate and adaptive immunity during inflammatory responses, played a major role in this process by releasing pro-inflammatory cytokines [20]. In this study, proinflammatory cytokine expressions of IL-1a, IL-1b, IL-6, IL-8 and TNF-a in PAMs were measured following 6 h, 12 h and 24 h of stimulation at concentrations of 5 and 10 mg/mL for DlgtF-LOS and WT-LOS via real-time PCR (Fig. 2). A LPS preparation from E. coliLOS was used as a positive control and the unstimulated PAMs cells

Fig. 2. mRNA expression of pro-inflammatory cytokines in LOS-stimulated PAMs. PAMs were stimulated with DlgtF-LOS, WT-LOS and E. coli-LPS (5 and 10 mg/mL, respectively) for 6 h, 12 h, and 24 h. The level of IL-1a (A), IL-1b (B), IL-6 (C), IL-8 (D), and TNF-a (E) mRNAs were measured by qRT-PCR. E. coli-LPS preparation was used as positive control, and the unstimulated PAMs were used as mock-stimulus. Values were presented as the mean ± SD of three independent experiments and data were analyzed using one-way ANOVA. *p < 0.05; **p < 0.001 compared with the WT-LOS-treated PAMs.

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was used as mock-stimulus. The results showed that the stimulation of PAMs with LOS up-regulated mRNA expression of proinflammatory factors in a dose-dependent manner (Fig. 2). The WT-LOS induced a significant up-regulation of mRNA expression of pro-inflammatory factors in PAMs. However, the mRNA expression levels of IL-1a, IL-1b, IL-6, IL-8 and TNF-a significantly decreased in DlgtF-LOS-treated PAMs for 6 h, 12 h and 24 h compared with the WT-LOS-treated PAMs (5 and 10 mg/mL) (p < 0.05). Therefore, these results demonstrated that loss of the lgtF gene in H. parasuis SC096 resulted in decreased expressions of LOS-mediated pro-inflammatory cytokines in PAMs, indicating that lgtF gene played important roles in the LOS-induced PAMs model of inflammation. 3.3. lgtF mediated H. parasuis LOS-induced NF-kB and MAPKs signaling pathways in PAMs NF-kB and MAPKs inflammatory signaling pathways, which are common downstream signal transduction pathways, regulate the expression of cytokines, chemokines, anti-apoptotic factors and cell growth factors, which are essential mediators of inflammation and immune responses [21]. H. parasuis infections could activate the NF-kB and MAPKs pathways and induce the up-regulation of proinflammatory cytokines [5,6]. However, the signaling molecular mechanisms underlying induction of the inflammatory response have remained largely unknown for LOS in H. parasuis. In this study, we measured the signaling molecule expression of p65, IkBa, p38 and phosphorylation of p65 and p38 with western blotting, and the

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unstimulated PAMs were used as mock-stimulus (Fig. 3). PAMs were treated with WT-LOS and DlgtF-LOS (5 and 10 mg/mL, respectively) for 6 h and 12 h. The results showed that the DlgtFLOS-treated cells had no obvious decreased p65 and p38 compared to the WT-LOS-treated group, however, the signaling molecules of phosphorylation of p65 and p38 were noticeably decreased in a dose-dependent manner in the DlgtF-LOS-treated PAMs compared with the WT-LOS-treated group. In addition, the DlgtF-LOS resulted in a higher concentration of IkBa with a dose-dependent manner in stimulated PAMs compared with WT-LOS. Analysis of densitometry values showed that the relative ratios of IkBa/GAPDH significantly increased (p < 0.05), while the phosphorylation of p65/GAPDH and p38/GAPDH significantly decreased in the DlgtF-LOS-treated PAMs compared with the WT-LOS-treated group (p < 0.05). Nevertheless, there was no obvious difference in the relative rations of p65/ GAPDH and p38/GAPDH between WT-LOS-treated and DlgtF-LOStreated groups. Therefore, our data indicated that the lgtF was involved in LOS synthesis and mediated LOS induction of proinflammatory cytokines in PAMs, which was dependent on both the NF-kB and MAPKs signaling pathways. This study may bring new insights into the molecular pathogenesis of LOS-induced inflammation and aid in understanding the complex mechanisms of LOS on host immunity responses during H. parasuis infection. 4. Conclusion In this study, the DlgtF from H. parasuis SC096 was constructed

Fig. 3. DlgtF-LOS and WT-LOS activated NF-kB and MAPK pathways in PAMs. PAMs were stimulated with DlgtF-LOS and WT-LOS (5 and 10 mg/mL, respectively) for 6 h and 12 h, and the unstimulated PAMs was used as mock-stimulus. Western blots of p65, phospho-p65, IkBa, p38, phospho-p38, and GADPH are shown (A). Densitometry values showed relative protein expression of p65 (B), phospho-p65 (C), IkBa (D), p38 (E) and phospho-p38 (F) from three separate experiments. Values were presented as the mean ± SD and data were analyzed using one-way ANOVA. *p < 0.05 compared with WT-LOS-treated PAMs.

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and a highly purified preparation of LOS was extracted. Compared to WT-LOS, the DlgtF-LOS exhibited a significantly reduced ability to induce inflammatory cytokine mRNA expression in PAMs, and the DlgtF-LOS-treated cells also were significantly decreased phospho-p65 and phospho-p38, and inhibited IkBa degradation. Therefore, the above results suggested that the lgtF gene mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-kB and MAPKs signaling pathways during H. parasuis infection. Acknowledgement This work was supported by the 13th Five-year National Key Research and Development Program of China (2016YFD0500705), the National Natural Science Foundation of China (31302119), the Innovative Research Team Program of Department of Education of Sichuan Province (13TD0057) and Veterinary Medicine Discipline Program of Southwest University for Nationalities (2014XWDS0906).

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2017.06.035.

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