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Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp. paratuberculosis
Microbial Pathogenesis 142 (2020) 104055
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Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp. paratuberculosis
T
Hongxiu Liu1, Guanghui Dang1, Xinxin Zang, Zhuming Cai, Ziyin Cui, Ningning Song, Siguo Liu∗ State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Street, Harbin, 150069, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Mycobacterium avium subsp. paratuberculosis MAP3292c Serine protease Mycobacterium smegmatis Pathogenicity
Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.
1. Introduction Serine proteases, the best-known class of proteases, are involved in blood coagulation, cell signaling, inflammation, protein processing, and protein digestion [1,2]. In addition, they are reportedly found in many pathogenic microorganisms as a virulence factor of pathogens. Streptococcus pneumoniae serine protease HtrA promotes the dissemination of S. pneumoniae and lung inflammation in mice [3]. Staphylococcus aureus serine glutamyal endopeptidase SspA degrades cell surface protein A and clumping factor B to promote the release of S. aureus from colonization sites and spread infection [4,5]. Mycobacterium tuberculosis (MTB) serine proteases MycP1 and Rv3610c are important virulence factors and have important functions in the pathogenesis of MTB [6]. PepD, an HtrA-like serine protease of MTB, plays an important role in the stress response network by altering the expression of stress-responsive determinants [7]. The Rv3668c serine protease in MTB can promote the interaction between bacteria and host cells by activating the extracellular signal-regulated kinase-nuclear factor kappa B pathway, which mediates the secretion of inflammatory factors, such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) [8]. Paratuberculosis, also known as Johne's disease (JD), is a chronic irreversible consumptive disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), that is characterized by chronic
diarrhea, progressive extenuation, and hyperplastic enteritis [9,10]]. MAP is mainly infected with ruminants such as cattle and sheep [11], but it has also been found in other animals, including deer, hare, wild boar, and camel [12]. JD is dominated by sporadic herds, and largescale cattle farms are dominated by regional epidemics [13]. JD is widespread worldwide and causes significant economic losses to animal husbandry and severely threatens the healthy development of dairy farming [14,15]. Some reports have shown that MAP can be isolated from patients with Crohn's disease and type 1 diabetes [16,17]. In addition, MAP has a potential connection with rheumatoid arthritis, Hashimoto's thyroiditis, and Sardinian [18–20]. Kugadas [21] found that MAP0403 serine protease is upregulated in infected macrophages and bovine mammary alveolar cells associated with phagocytic acidification, suggesting that it may regulate bacterial survival in acidic environments via an acid stress response network. However, there have been few studies on the pathogenic mechanisms of proteases in MAP. MAP3292c belongs to the SdrC superfamily, 234–338 amino acid residues are predicted archaeal serine protease, S18 family domain. In this work, we identified and characterized MAP3292c, a serine protease from MAP. We showed that this protease was highly active in degrading casein at pH 9.0 and 41 °C and had autoproteolytic activity. In addition, MAP3292c serine protease activity promoted the colonization of Mycobacterium smegmatis (MS) in mice and induced pathological injury
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Corresponding author. E-mail address: [email protected] (S. Liu). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.micpath.2020.104055 Received 6 November 2019; Received in revised form 8 January 2020; Accepted 10 February 2020 Available online 11 February 2020 0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
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2.5. MAP3292c autocleavage activity and N-terminal sequencing
and inflammation.
The purified MAP3292c protein dissolved in 20 mM PB (pH 6.0) was incubated with 20 mM Tris-HCl (pH 8.0) overnight at 37 °C. Then the MAP3292c protein was resolved by 4–12% SDS-PAGE and transferred to a PVDF membrane. The target protein was stained with Ponceau red, and the excess liquid was removed by filter paper. The membrane was stored in a dry ziplock bag at 4 °C and sent to Shanghai Applied Protein Technology Co. Ltd. (Shanghai, China) for N-end sequencing using the Edman degradation method to determine the autocleavage site of the MAP3292c protein.
2. Materials and methods 2.1. Animals, bacterial strains, and plasmids The BALB/c mice (6–8 weeks old, female) used in this study were purchased from Vital River (Beijing, China). All of the experiments involving animals were conducted according to the instructions by the Heilongjiang Animal Ethics Committee at the Heilongjiang Science and Technology government agency (Harbin, China). The study was approved and supervised by the Commissioner for Animal Welfare at the Harbin Veterinary Research Institute, representing the Institutional Animal Care and Use Committee. The MAP K-10 reference strain, Mycobacterium smegmatis mc2 155, pAIC shuttle vector (acetamide-inducible expression vector constructed based on pMV261 in this experiment, the nucleotide sequence of pAIC is in Supplementary Materials), and pET-28a vector were stored in our laboratory. The Escherichia coli DH5α host strain was used for plasmid preparation, and the E. coli BL21 host strain was used for protein expression.
2.6. Determination of MAP3292c serine protease activity To measure MAP3292c serine protease activity, 15 μg casein (C7078; Sigma-Aldrich, St. Louis, MO, USA) was incubated with 10 μg purified MAP3292c protein in 20 μL reaction buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 8.0) at 37 °C. The casein in buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 8.0) served as a negative control. After 1 h, 20 μL reaction solution was mixed with 5 μL of 5 × SDS loading buffer and analyzed by 12% SDS-PAGE. All of the assays were conducted in triplicate.
2.2. Cloning and expression 2.7. Effects of divalent cations on MAP3292c serine protease activity The amino acid sequence of MAP3292c was predicted by the online server SignalP 4.0. The results showed that 1–23 was its signal peptide sequence. According to the MAP3292c gene sequence published in GeneBank, the signal peptide sequence was removed, and primers (Supplementary Materials Tables S1 and S2) were designed to amplify the corresponding fragments from the MAP K-10 reference strain genomic DNA, followed by cloning into the pET28a and pAIC vectors. Then pET28a-MAP3292c was transformed into BL21(DE3) E. coli, and MAP3292c protein was expressed as an inclusion body. The recombinant plasmid MAP3292c-pAIC was transformed into the MS strain. Recombinant MS (rMS) was grown in 7H9 medium (0.05% Tween 80, 0.2% glycerol, 100 μg/mL kanamycin) at 37 °C until the optical density at 600 nm was 0.6–1.0, and then was induced with 0.4% acetamide inducer at 37 °C for 12 h. Protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
To test whether the proteolytic activity of MAP3292c was divalent cation-dependent, an enzymatic assay using casein as a substrate (https://www.sigmaaldrich.com/catalog/product/sigma/c7078? lang=zh®ion=CN) was used to quantitatively determine MAP3292c serine protease activity. With casein as the reactant, 10 μg purified MAP3292c protein was incubated with different divalent salts (5 mM MgCl2, ZnCl2, CaCl2, BaCl2, NiCl2, CuCl2, and MnCl2) at 37 °C for 30 min. The results were measured at 660 nm using a multimode microplate reader (EnSpire; PerkinElmer, Waltham, MA, USA). 2.8. Effects of temperature and pH on MAP3292c serine protease activity The influence of pH on MAP3292c activity was detected with optimal divalent cations in 40 mM Britton-Robinson buffer solution (pH 3.0–12.0, interval of 1.0) at 37 °C for 1 h. The impact of temperature on MAP3292c activity was identified with optimal divalent cations and optimal pH at temperatures of 31 °C to 51 °C (interval of 2 °C) for 1 h. All of the results were measured using a multimode microplate reader at 660 nm, and all of the experiments were repeated three times. One enzymatic activity unit was defined as the quantity of enzyme hydrolyze casein to produce color equivalent to 1.0 μM tyrosine per min at pH 9.0 at 41 °C.
2.3. Purification of MAP3292c Bacterial culture (1 L) was collected, and the cell pellet was resuspended in 50 mL lysis buffer A (20 mM PB, 150 mM NaCl, 10% glycerol, pH 6.0) and sonicated in an ice bath. The precipitate was collected by centrifugation at 15,000 rpm for 30 min at 4 °C, and the pellet was re-suspended in 50 mL buffer B (20 mM PB, 150 mM NaCl, 6 M urea, 10% glycerol, pH 6.0). The dissolved supernatant was dialyzed in 1 L phosphate-buffered saline (PBS) (pH 6.0). After 12 h, the dialyzed protein was centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was loaded onto a Ni Sepharose 6 Fast Flow resin (GE Healthcare, Chicago, IL, USA). Unbound proteins were washed with 40 mL buffer C (20 mM PB, 150 mM NaCl, 0.6% OB-2, 10% glycerol, pH 6.0) and 40 mL buffer D (20 mM PB, 150 mM NaCl, 20 mM imidazole, 10% glycerol, pH 6.0), and the target protein was sequentially eluted with 5 mL buffer E (20 mM PB, 150 mM NaCl, 10% glycerol, 500 mM imidazole, pH 6.0). The collected proteins were analyzed by SDS-PAGE.
2.9. Kinetic studies of MAP3292c serine protease activity Casein was used as a substrate to determine the kinetic parameters of MAP3292c serine protease by an enzymatic assay using casein as a substrate. Under conditions of optimal divalent cations, pH, and temperature, 10 μg MAP3292c protein was incubated with different concentrations of casein (33, 16.7, 8.35, 4.2, 2.08, 1.04, 0.52, 0.26, 0.13, 0.065, and 0.033 mg/mL). The MAP3292c protein was replaced with 50 mM Tris-HCl buffer as a negative control. All of the assays were repeated three times.
2.4. Mass spectrometry analysis of MAP3292c
2.10. Site-directed mutagenesis
The purified MAP3292c protein was thoroughly decolorized by 4%–12% SDS PAGE, and the target band was cut out and sent to Beijing Protein Innovation (Beijing, China) for matrix-assisted laser desorption ion time-of-flight mass spectrometry analysis. The protein was identified by searching the SwissProt database.
Mutagenic oligonucleotide pairs (Supplementary Materials Table S3) were used to introduce S238A substitutions into the nucleotide sequence, and PCR amplification was conducted using the PrimerSTAR Max DNA Polymerase (TaKaRa Bio, Dalian, China). The PCR products were digested with Dpn I to damage the methylated parental template 2
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DNA, and the mutated plasmids were transformed into DH5α E. coli. All of the substitutions were confirmed by DNA sequencing. 2.11. Localization of MAP3292c in rMS and MAP Preparation of subcellular fractions from recombinant MAP3292c/ MS and MAP K-10 was performed as described in previous reports from our laboratory [22]. The components of whole cell lysate proteins (CL), culture filtrate (CF), cell wall (CW), cytoplasm (CP), and cell membrane (CM) were separated by 12% SDS-PAGE and then electrotransferred to a nitrocellulose membrane. Western blotting was performed using an anti-MAP3292c polyclonal antibody (BALB/c mice was immunized with purified MAP3292c protein) as the primary antibody and Dylight680-labeled goat anti-mouse IgG antibody (KPL, Gaithersburg, MD, USA) as the secondary antibody. Finally, the membrane was detected and visualized using the Li-Cor Odyssey imaging system (Li-COR Biosciences, Lincoln, NE, USA).
Fig. 2. Autocleavage activity of MAP3292c serine protease. (A) MAP3292c protein in pH 6.0. (B) MAP3292c protein in pH 8.0. (C) MAP3292c protein with autocleavage sites determined from N-terminal sequence spectrometry.
protein was expressed in inclusion body form and purified by Ni2+ affinity chromatography (Fig. 1A). Protein identification by mass spectrometry and peptide mass fingerprinting revealed that the purified protein was the expected MAP3292c protein (GeneBank Accession No. AAS05842) from MAP K-10 (Fig. 1B).
3.2. MAP3292c has protease activity and is localized in the extracellular MAP domain
2.12. Mice infection BALB/c mice (n = 3 per group) were infected intraperitoneally with 1 × 108 CFU recombinant MAP3292c/MS and S238A/MS in 100 μL PBS with 0.05% Tween 20 (PBST), and the mice were treated with PBST and recombinant pAIC/MS as the negative control. At 7 d post-infection, serum was separated from blood collected from the eyeballs of mice. TNF-α, IL-1β, and IL-6 levels in the serum were measured with a commercially available mouse TNF-α, IL-1β, and IL-6 ELISA Kit (NEOBIOSCINCE, Beijing, China) according to the manufacturer's instructions. The left lobe of the right liver, the entire left lobe of the lung, and the upper half of the spleen were removed and homogenized with aseptic PBS and spread onto 7H10 plates (50 μg/mL kanamycin) for counting bacteria. The small intestine, lobe of the liver, about one-third of the right lobe of the lung, and lower half of the spleen were fixed in 10% neutral buffered formalin for histopathological analysis.
National Center for Biotechnology Information conserved domain analysis revealed that MAP3292c is a trypsin-like serine protease, and MTB Rv3671c is a trypsin-like serine protease with autoproteolytic activity [23]. To determine if MAP3292c has autoproteolytic activity, purified MAP3292c protein in 20 mM PB (pH 6.0) was substituted with 20 mM Tris-HCl-pH 8.0, and showed autoproteolytic activity (Fig. 2B). N-terminal sequencing suggested that it is cleaved between serine (S)86 and asparagine (D)-87 (Fig. 2C). To understand the subcellular location of MAP3292c, the components (CF, CW, CP, and CM) were separated from recombinant MAP3292c/MS and MAP K-10. Western blot analysis showed that MAP3292c protein was localized to the CW and CF fractions of recombinant MAP3292c/MS and MAP K-10, and the secreted MAP3292c in CF had autoproteolysis activity (Fig. 3). Next, purified MAP3292c protein was incubated with casein. SDS-PAGE analysis revealed that MAP3292c could hydrolyze casein (Fig. 4A), showing that MAP3292c is an extracellular protease.
2.13. Statistical analysis Statistical analysis was performed using GraphPad Prism Version 8.0 software. All of the data are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance was used to evaluate statistical significance followed by Bonferroni's multiple comparison test. A P-value < 0.05 was considered to be statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).
3.3. Effects of divalent cations, temperature, and pH on MAP3292c activity To optimize the reaction conditions of MAP3292c protease, the effects of different divalent salts on the protease activity of MAP3292c were assessed. The protease activity was significantly improved in the presence of 5 mM CaCl2, while MgCl2, ZnCl2, BaCl2, NiCl2, CuCl2, and MnCl2 inhibited its activity to different degrees (Table 1). The results for different pH and different temperatures are shown in Fig. 4B and 4C. The optimum activity and pH of MAP3292c protease were 41 °C and 9.0, respectively.
3. Results 3.1. Purification and identification of MAP3292c protein To understand the function of MAP3292c serine protease, the
Fig. 1. Purification and Mass Spectrometry Analysis of MAP3292c protein. (A) Affinity chromatography purification of MAP3292c protein. 1: renaturation supernatant; 2: precipitation after renaturation; 3: traversing solution; 4–5: pre-washing medium; 6: eluent solution. (B) Peptide mass fingerprinting of MAP3292c protein by matrix-assisted laser desorption ion time-of-flight mass spectrometry. 3
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Table 1 Effect of divalent cations on MAP3292c activity. Divalent cation (5 mM)
Relative activity (%)
None MgCl2 ZnCl2 CaCl2 BaCl2 NiCl2 CuCl2 MnCl2
100.00 ± 5.90 75.45 ± 4.47 31.83 ± 5.93 167.95 ± 4.30 24.05 ± 2.31 33.74 ± 4.49 57.33 ± 6.02 34.05 ± 2.56
The errors are given as standard deviation.
3.4. Kinetic studies of MAP3292c activity The kinetic parameters of MAP3292c protease were measured at 41 °C (pH 9.0) with 5 mM CaCl2 and different concentrations of casein. Michaelis constant assays suggested that the Km and Vmax values were 0.151 ± 0.04 mg/mL and 14.75 U/mg/min, respectively (Fig. 4D). 3.5. MAP3292c is a serine protease To determine the active site on MAP3292c protease, multiple sequence alignment of MAP3292c from reported serine protease conserved domains was conducted (Fig. 5A). A total of 238 serine residues were replaced by site-directed mutagenesis, and the S238A mutant protein was produced by affinity chromatography (Fig. 5B). The S238A mutant caused a 95% loss of activity compared to wild-type MAP3292c (Fig. 5C). These results showed that MAP3292c is a serine protease.
Fig. 3. Cell fractionation experiments were performed to determine the subcellular localization of MAP3292c protein. (A) Subcellular localization of MAP3292c in MS, CL: whole bacterial protein; CF: culture supernatant; CW: cell wall; CP: cytoplasm; CM: cell membrane. (B) Subcellular localization of MAP3292c in MAP.
3.6. MAP3292c promotes the MS persistence in mice To determine the pathogenicity of MAP3292c serine protease in mycobacteria, we constructed recombinant pAIC/MS, MAP3292c/MS, and S238A/MS. The lung, liver, and spleen were removed from mice infected with rMS (pAIC/MS, MAP329c/MS, and S238A/MS) and PBST at 7 d. Bacterial colony counting results showed that bacterial load of the MAP3292c/MS group was greatly enhanced compared with the Fig. 4. Determination of MAP3292c protease activity. (A) Digestion of casein by purified MAP3292c protein. (B) Effects of temperature on MAP3292c activity. The reaction was performed in 50 mM Tris-HCl, pH 9.0, and 5 mM CaCl2 at 41 °C for 1 h. (C) Effects of pH on MAP3292c activity. (D) Michaelis-Menton kinetic assays of MAP3292c protease activity. The errors are given as the SEM of three independent experiments.
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Fig. 5. The substitution of S-238 by alanine reduces serine protease activity in MAP3292c protein. (A) Multiple sequence alignment of MAP3292c from reported serine protease conserved domains: MTB Rv3671c (NP_218188), MTB Rv0983 (NP_215498), E. coli Deg S (AAC44006), and T. maritima Htr A (AGL49494). Alignment was performed using MEGA5 and BoxShade. The GxSxG motif is underlined. (B) The activity of site-directed mutagenesis of MAP3292c.
epithelial cells in the lung, large necrotic foci and infiltration of inflammatory cells in hepatic parenchyma in the liver, microgranuloma around the splenic white pulp, and neutrophil infiltration in the spleen in the MAP329c/MS group (Fig. 8). There was mild proliferation of alveolar epithelial cells in the lung, small infiltration of inflammation cells in the liver, and no pathological changes in the spleen of the S238A/MS group (Fig. 8). There were no pathological changes in the lung, liver, and spleen in the PBST and pAIC/MS groups (Fig. 8). There were no pathological changes in the small intestine in the PBST, pAIC/ MS, S238A/MS, and MAP3292c/MS groups (Supplementary Materials Fig. 1). These results showed that MAP3292c serine protease promotes the release of inflammatory cytokines and induces pathological injury in mice.
pAIC/MS group in the lung, liver, and spleen (Fig. 6A–C). However, the bacterial load of the pAIC/MS group was significantly decreased compared with the MAP3292c/MS group (Fig. 6A–C). These results indicated that MAP3292c serine protease promotes MS persistence in mice.
3.7. MAP3292c induces the release of inflammatory cytokines and pathological injury in mice To measure the inflammatory response, the levels of cytokines TNFα, IL-1β, and IL-6 were determined in the serum separated from mice infected with rMS (pAIC/MS, MAP329c/MS, and S238A/MS) and PBST (Fig. 7A–C). TNF-α, IL-1β, and IL-6 levels in MAP329c/MS-infected mice were significantly greater than those in control mice (PBST and pAIC/MS). However, TNF-α, IL-1β, and IL-6 levels in mice challenged with S238A/MS were markedly reduced compared with mice treated with MAP3292c/MS. Histopathological determination showed pulmonary vasodilation and congestion, mild proliferation of alveolar
4. Discussion We discovered that MAP3292c is proteolytically active for bovine casein at pH ranging from 3.0 to 12.0, with an optimal pH of 9.0. This Fig. 6. Presence of persistent recombinant MS in mouse lung (A), liver (B), and spleen (C). Bacterial loads in infected lung, liver, and spleen tissues from BALB/c mice, as transmitted by intraperitoneal infection with recombinant pAIC/MS, MAP3292c/MS and S238A/MS were determined at 7 d after infection (3 mice/group/time point). (*P < 0.05, **P < 0.01,***P < 0.001).
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Fig. 7. The release of inflammatory indicators in BALB/c mice infected with recombinant MS. (A) Analysis of TNF-α. (B) Analysis of IL-1β. (C) Analysis of IL-6. (*P < 0.05, **P < 0.01,***P < 0.001.) Fig. 8. Histopathological analysis of BALB/c mice lung, liver, and spleen. BALB/c mice were intraperitoneally infected with rMS pAIC/MS, S238A/ MS, and MAP3292c/MS at 7 d, using PBST as the negative control. (Lung) PBST and pAIC/MS: no pathological changes; S238A/MS: mild proliferation of alveolar epithelial cells (white arrow); MAP3292c/ MS: pulmonary vasodilation and congestion (blue arrow), mild proliferation of alveolar epithelial cells (white arrow). (Liver) PBST and pAIC/MS: no pathological changes; S238A/MS: small infiltration foci of myeloid cells (blue arrow) and giant cells (white arrow); MAP3292c/MS: there were large necrotic foci and infiltration of inflammatory cells in hepatic parenchyma (blue arrow). Fibroblast proliferation could be seen at the edge of necrotic foci (white arrow). (Spleen) PBS, pAIC/MS, and S238A/MS: no pathological changes; MAP3292c/MS: microgranuloma around splenic white pulp (white arrow) and neutrophil infiltration (blue arrow). Scale bars = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cytokines, such as IL-1β and IL-6, which are major early regulators of pro-inflammatory responses [28]. The expression levels of IL-1β, TNFα, and IL-6 significantly increased in the infected ovine intestinal tissues by MAP [29]. MAP and CW components induce the expression of TNF-α and IL-6 in bovine monocytes [30]. TNF-α activates naive macrophages and is essential for the formation of granulomas around the sites of mycobacterial infection [31]. Our results showed that MAP3292c serine protease activity promoted the release of IL-1β, IL-6, and TNF-α in a mouse model, suggesting that MAP3292c is involved in the pathogenesis of mycobacterium. In S. aureus, clpP or clpX protease mutant strains render the bacterium avirulent in a mouse skin abscess model, which points to a significant role for the ClpXP protease in the virulence of S. aureus [32]. In Helicobacter pylori, HtrA protease is secreted into the extracellular surroundings [33] and degrades E-cadherin to efficiently destroy adherence junctions in polarized epithelial cells [34]. In MTB, Rv2869c protease regulates MTB intramembrane proteolysis to control membrane composition to affect the virulence of MTB [35]. Our data showed that MAP3292c promoted the persistent of the MS and induced injury to the lung, liver, and spleen in mice, indicating that MAP3292c is an important mycobacterium virulence factor. MS growth is quick and its infection model is often used to study the protein function and pathogenesis of slow growing mycobacteria, such as MTB [36–38]. However, the MS infection model is not a perfect in vivo model for MAP, as a multitude of virulence factors are missing in MS that are in present in MAP. MAP is the primary pathogen of enteric regions; however, our results showed that there were no pathological
pH range is consistent with the MAP of gastrointestinal pathogenic microorganism, which can tolerate the acid environment of the stomach and alkaline environment of the intestine, indicating that MAP3292c may be associated with MAP virulence. Divalent metal ions of Ca2+ increased the activity of MAP3292c protease, indicating that they play important roles in stabilizing the structure of protease. However, Mg2+, Zn2+, Ba2+, Ni2+, Cu2+, and Mn2+ inhibited its activity, which caused small changes in protein conformation. These phenotypes also appear in the serine protease of Agkistrodon blomhoffii ussurensis snake venom [24] and insulin-like growth factor binding proteins serine protease [25]. The subcellular localization results showed that MAP3292c protein was localized in the CW, CM, and CF. Studies have shown that proteins on the CM and CW are the main components of pathogen-infected host cells, and these proteins are more accessible to the host's immune system [26,27]. This localization of the MAP3292c protein indicates that it may play an important role in the pathogenesis of MAP. Rv3671c, a serine protease of MTB that can cleave β-casein and cause autocleavage, is required for MTB resistance in macrophages [23]. However, there have been no reports on serine protease autocleavage in MAP. We found that MAP3292c is the autoproteolytic active, and the cleavage site is between S-86 and D-87 to produce a ~28 kDa protein, consistent with the size of MAP3292c protein secreted by MAP. These results showed that MAP3292c protein obtained in vitro has the same function as that in wild-type MAP K-10. Studies have shown that cytokine signaling plays a major role in host defense MAP, characterized by the release of pro-inflammatory 6
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changes in the small intestine in the MAP3292c/MS groups. Thus, the MAP3292c mutant of MAP needs to be constructed to study the function of MAP3292c protein. In summary, this study characterized MAP3292c serine protease, an extracellular protein of MAP, which was found to degrade casein and have autoproteolytic activity. In addition, MAP3292c serine protease activity was shown to promote the colonization of MS in mice and induce pathological damage and the release of inflammatory cytokines in mice. These results provide the basis for future studies on the pathogenic mechanism of MAP.
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CRediT authorship contribution statement Hongxiu Liu: Formal analysis. Guanghui Dang: Formal analysis. Xinxin Zang: Formal analysis. Zhuming Cai: Formal analysis. Ziyin Cui: Formal analysis. Ningning Song: Formal analysis. Siguo Liu: Formal analysis. Declaration of competing interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by grants from the National Key Research and Development Program of China (2016YFD0500905), Key scientific and technological projects of Xinjiang Production and Construction Corps (XPCC) (2019AB029), and National Natural Science Foundation of China (No. 31772767). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2020.104055. References [1] O.D. Ekici, M. Paetzel, R.E. Dalbey, Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration, Protein Sci. 17 (2008) 2023–2037. [2] M.M. Krem, E. Di Cera, Molecular markers of serine protease evolution, EMBO J. 20 (2001) 3036–3045. [3] S.F. de Stoppelaar, H.J. Bootsma, A. Zomer, J.J. Roelofs, P.W. Hermans, C. van 't Veer, T. van der Poll, Streptococcus pneumoniae serine protease HtrA, but not SFP or PrtA, is a major virulence factor in pneumonia, PloS One 8 (2013) e80062. [4] A. Karlsson, P. Saravia-Otten, K. Tegmark, E. Morfeldt, S. Arvidson, Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases, Infect. Immun. 69 (2001) 4742–4748. [5] F.M. McAleese, E.J. Walsh, M. Sieprawska, J. Potempa, T.J. Foster, Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease, J. Biol. Chem. 276 (2001) 29969–29978. [6] Q.J. Zhao, J.P. Xie, Mycobacterium tuberculosis proteases and implications for new antibiotics against tuberculosis, Crit. Rev. Eukaryot. Gene Expr. 21 (2011) 347–361. [7] M.J. White, H. He, R.M. Penoske, S.S. Twining, T.C. Zahrt, PepD participates in the mycobacterial stress response mediated through MprAB and SigE, J. Bacteriol. 192 (2010) 1498–1510. [8] Q. Zhao, W. Li, T. Chen, Y. He, W. Deng, H. Luo, J. Xie, Mycobacterium tuberculosis serine protease Rv3668c can manipulate the host-pathogen interaction via Erk-NFκB axis-mediated cytokine differential expression, J. Interferon Cytokine Res. 34 (2014) 686–698. [9] I. Olsen, G. Sigurğardóttir, B. Djønne, Paratuberculosis with special reference to cattle. A review, Vet. Q. 24 (2002) 12–28. [10] R.J. Whittington, D.J. Begg, K. de Silva, A.C. Purdie, N.K. Dhand, K.M. Plain, Case definition terminology for paratuberculosis (Johne's disease), BMC Vet. Res. 13 (2017) 328. [11] P. Mercier, S. Freret, K. Laroucau, M.P. Gautier, I. Brémaud, C. Bertin, C. Rossignol, A. Souriau, L.A. Guilloteau, A longitudinal study of the Mycobacterium avium subspecies paratuberculosis infection status in young goats and their mothers, Vet. Microbiol. 195 (2016) 9–16. [12] R.J. Whittington, E.S. Sergeant, Progress towards understanding the spread, detection and control of Mycobacterium avium subsp. paratuberculosis in animal populations, Aust. Vet. J. 79 (2001) 267–278.
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Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp. paratuberculosis
T
Hongxiu Liu1, Guanghui Dang1, Xinxin Zang, Zhuming Cai, Ziyin Cui, Ningning Song, Siguo Liu∗ State Key Laboratory of Veterinary Biotechnology, Division of Bacterial Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 678 Haping Street, Harbin, 150069, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Mycobacterium avium subsp. paratuberculosis MAP3292c Serine protease Mycobacterium smegmatis Pathogenicity
Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.
1. Introduction Serine proteases, the best-known class of proteases, are involved in blood coagulation, cell signaling, inflammation, protein processing, and protein digestion [1,2]. In addition, they are reportedly found in many pathogenic microorganisms as a virulence factor of pathogens. Streptococcus pneumoniae serine protease HtrA promotes the dissemination of S. pneumoniae and lung inflammation in mice [3]. Staphylococcus aureus serine glutamyal endopeptidase SspA degrades cell surface protein A and clumping factor B to promote the release of S. aureus from colonization sites and spread infection [4,5]. Mycobacterium tuberculosis (MTB) serine proteases MycP1 and Rv3610c are important virulence factors and have important functions in the pathogenesis of MTB [6]. PepD, an HtrA-like serine protease of MTB, plays an important role in the stress response network by altering the expression of stress-responsive determinants [7]. The Rv3668c serine protease in MTB can promote the interaction between bacteria and host cells by activating the extracellular signal-regulated kinase-nuclear factor kappa B pathway, which mediates the secretion of inflammatory factors, such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) [8]. Paratuberculosis, also known as Johne's disease (JD), is a chronic irreversible consumptive disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), that is characterized by chronic
diarrhea, progressive extenuation, and hyperplastic enteritis [9,10]]. MAP is mainly infected with ruminants such as cattle and sheep [11], but it has also been found in other animals, including deer, hare, wild boar, and camel [12]. JD is dominated by sporadic herds, and largescale cattle farms are dominated by regional epidemics [13]. JD is widespread worldwide and causes significant economic losses to animal husbandry and severely threatens the healthy development of dairy farming [14,15]. Some reports have shown that MAP can be isolated from patients with Crohn's disease and type 1 diabetes [16,17]. In addition, MAP has a potential connection with rheumatoid arthritis, Hashimoto's thyroiditis, and Sardinian [18–20]. Kugadas [21] found that MAP0403 serine protease is upregulated in infected macrophages and bovine mammary alveolar cells associated with phagocytic acidification, suggesting that it may regulate bacterial survival in acidic environments via an acid stress response network. However, there have been few studies on the pathogenic mechanisms of proteases in MAP. MAP3292c belongs to the SdrC superfamily, 234–338 amino acid residues are predicted archaeal serine protease, S18 family domain. In this work, we identified and characterized MAP3292c, a serine protease from MAP. We showed that this protease was highly active in degrading casein at pH 9.0 and 41 °C and had autoproteolytic activity. In addition, MAP3292c serine protease activity promoted the colonization of Mycobacterium smegmatis (MS) in mice and induced pathological injury
∗
Corresponding author. E-mail address: [email protected] (S. Liu). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.micpath.2020.104055 Received 6 November 2019; Received in revised form 8 January 2020; Accepted 10 February 2020 Available online 11 February 2020 0882-4010/ © 2020 Elsevier Ltd. All rights reserved.
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2.5. MAP3292c autocleavage activity and N-terminal sequencing
and inflammation.
The purified MAP3292c protein dissolved in 20 mM PB (pH 6.0) was incubated with 20 mM Tris-HCl (pH 8.0) overnight at 37 °C. Then the MAP3292c protein was resolved by 4–12% SDS-PAGE and transferred to a PVDF membrane. The target protein was stained with Ponceau red, and the excess liquid was removed by filter paper. The membrane was stored in a dry ziplock bag at 4 °C and sent to Shanghai Applied Protein Technology Co. Ltd. (Shanghai, China) for N-end sequencing using the Edman degradation method to determine the autocleavage site of the MAP3292c protein.
2. Materials and methods 2.1. Animals, bacterial strains, and plasmids The BALB/c mice (6–8 weeks old, female) used in this study were purchased from Vital River (Beijing, China). All of the experiments involving animals were conducted according to the instructions by the Heilongjiang Animal Ethics Committee at the Heilongjiang Science and Technology government agency (Harbin, China). The study was approved and supervised by the Commissioner for Animal Welfare at the Harbin Veterinary Research Institute, representing the Institutional Animal Care and Use Committee. The MAP K-10 reference strain, Mycobacterium smegmatis mc2 155, pAIC shuttle vector (acetamide-inducible expression vector constructed based on pMV261 in this experiment, the nucleotide sequence of pAIC is in Supplementary Materials), and pET-28a vector were stored in our laboratory. The Escherichia coli DH5α host strain was used for plasmid preparation, and the E. coli BL21 host strain was used for protein expression.
2.6. Determination of MAP3292c serine protease activity To measure MAP3292c serine protease activity, 15 μg casein (C7078; Sigma-Aldrich, St. Louis, MO, USA) was incubated with 10 μg purified MAP3292c protein in 20 μL reaction buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 8.0) at 37 °C. The casein in buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 8.0) served as a negative control. After 1 h, 20 μL reaction solution was mixed with 5 μL of 5 × SDS loading buffer and analyzed by 12% SDS-PAGE. All of the assays were conducted in triplicate.
2.2. Cloning and expression 2.7. Effects of divalent cations on MAP3292c serine protease activity The amino acid sequence of MAP3292c was predicted by the online server SignalP 4.0. The results showed that 1–23 was its signal peptide sequence. According to the MAP3292c gene sequence published in GeneBank, the signal peptide sequence was removed, and primers (Supplementary Materials Tables S1 and S2) were designed to amplify the corresponding fragments from the MAP K-10 reference strain genomic DNA, followed by cloning into the pET28a and pAIC vectors. Then pET28a-MAP3292c was transformed into BL21(DE3) E. coli, and MAP3292c protein was expressed as an inclusion body. The recombinant plasmid MAP3292c-pAIC was transformed into the MS strain. Recombinant MS (rMS) was grown in 7H9 medium (0.05% Tween 80, 0.2% glycerol, 100 μg/mL kanamycin) at 37 °C until the optical density at 600 nm was 0.6–1.0, and then was induced with 0.4% acetamide inducer at 37 °C for 12 h. Protein expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
To test whether the proteolytic activity of MAP3292c was divalent cation-dependent, an enzymatic assay using casein as a substrate (https://www.sigmaaldrich.com/catalog/product/sigma/c7078? lang=zh®ion=CN) was used to quantitatively determine MAP3292c serine protease activity. With casein as the reactant, 10 μg purified MAP3292c protein was incubated with different divalent salts (5 mM MgCl2, ZnCl2, CaCl2, BaCl2, NiCl2, CuCl2, and MnCl2) at 37 °C for 30 min. The results were measured at 660 nm using a multimode microplate reader (EnSpire; PerkinElmer, Waltham, MA, USA). 2.8. Effects of temperature and pH on MAP3292c serine protease activity The influence of pH on MAP3292c activity was detected with optimal divalent cations in 40 mM Britton-Robinson buffer solution (pH 3.0–12.0, interval of 1.0) at 37 °C for 1 h. The impact of temperature on MAP3292c activity was identified with optimal divalent cations and optimal pH at temperatures of 31 °C to 51 °C (interval of 2 °C) for 1 h. All of the results were measured using a multimode microplate reader at 660 nm, and all of the experiments were repeated three times. One enzymatic activity unit was defined as the quantity of enzyme hydrolyze casein to produce color equivalent to 1.0 μM tyrosine per min at pH 9.0 at 41 °C.
2.3. Purification of MAP3292c Bacterial culture (1 L) was collected, and the cell pellet was resuspended in 50 mL lysis buffer A (20 mM PB, 150 mM NaCl, 10% glycerol, pH 6.0) and sonicated in an ice bath. The precipitate was collected by centrifugation at 15,000 rpm for 30 min at 4 °C, and the pellet was re-suspended in 50 mL buffer B (20 mM PB, 150 mM NaCl, 6 M urea, 10% glycerol, pH 6.0). The dissolved supernatant was dialyzed in 1 L phosphate-buffered saline (PBS) (pH 6.0). After 12 h, the dialyzed protein was centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatant was loaded onto a Ni Sepharose 6 Fast Flow resin (GE Healthcare, Chicago, IL, USA). Unbound proteins were washed with 40 mL buffer C (20 mM PB, 150 mM NaCl, 0.6% OB-2, 10% glycerol, pH 6.0) and 40 mL buffer D (20 mM PB, 150 mM NaCl, 20 mM imidazole, 10% glycerol, pH 6.0), and the target protein was sequentially eluted with 5 mL buffer E (20 mM PB, 150 mM NaCl, 10% glycerol, 500 mM imidazole, pH 6.0). The collected proteins were analyzed by SDS-PAGE.
2.9. Kinetic studies of MAP3292c serine protease activity Casein was used as a substrate to determine the kinetic parameters of MAP3292c serine protease by an enzymatic assay using casein as a substrate. Under conditions of optimal divalent cations, pH, and temperature, 10 μg MAP3292c protein was incubated with different concentrations of casein (33, 16.7, 8.35, 4.2, 2.08, 1.04, 0.52, 0.26, 0.13, 0.065, and 0.033 mg/mL). The MAP3292c protein was replaced with 50 mM Tris-HCl buffer as a negative control. All of the assays were repeated three times.
2.4. Mass spectrometry analysis of MAP3292c
2.10. Site-directed mutagenesis
The purified MAP3292c protein was thoroughly decolorized by 4%–12% SDS PAGE, and the target band was cut out and sent to Beijing Protein Innovation (Beijing, China) for matrix-assisted laser desorption ion time-of-flight mass spectrometry analysis. The protein was identified by searching the SwissProt database.
Mutagenic oligonucleotide pairs (Supplementary Materials Table S3) were used to introduce S238A substitutions into the nucleotide sequence, and PCR amplification was conducted using the PrimerSTAR Max DNA Polymerase (TaKaRa Bio, Dalian, China). The PCR products were digested with Dpn I to damage the methylated parental template 2
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DNA, and the mutated plasmids were transformed into DH5α E. coli. All of the substitutions were confirmed by DNA sequencing. 2.11. Localization of MAP3292c in rMS and MAP Preparation of subcellular fractions from recombinant MAP3292c/ MS and MAP K-10 was performed as described in previous reports from our laboratory [22]. The components of whole cell lysate proteins (CL), culture filtrate (CF), cell wall (CW), cytoplasm (CP), and cell membrane (CM) were separated by 12% SDS-PAGE and then electrotransferred to a nitrocellulose membrane. Western blotting was performed using an anti-MAP3292c polyclonal antibody (BALB/c mice was immunized with purified MAP3292c protein) as the primary antibody and Dylight680-labeled goat anti-mouse IgG antibody (KPL, Gaithersburg, MD, USA) as the secondary antibody. Finally, the membrane was detected and visualized using the Li-Cor Odyssey imaging system (Li-COR Biosciences, Lincoln, NE, USA).
Fig. 2. Autocleavage activity of MAP3292c serine protease. (A) MAP3292c protein in pH 6.0. (B) MAP3292c protein in pH 8.0. (C) MAP3292c protein with autocleavage sites determined from N-terminal sequence spectrometry.
protein was expressed in inclusion body form and purified by Ni2+ affinity chromatography (Fig. 1A). Protein identification by mass spectrometry and peptide mass fingerprinting revealed that the purified protein was the expected MAP3292c protein (GeneBank Accession No. AAS05842) from MAP K-10 (Fig. 1B).
3.2. MAP3292c has protease activity and is localized in the extracellular MAP domain
2.12. Mice infection BALB/c mice (n = 3 per group) were infected intraperitoneally with 1 × 108 CFU recombinant MAP3292c/MS and S238A/MS in 100 μL PBS with 0.05% Tween 20 (PBST), and the mice were treated with PBST and recombinant pAIC/MS as the negative control. At 7 d post-infection, serum was separated from blood collected from the eyeballs of mice. TNF-α, IL-1β, and IL-6 levels in the serum were measured with a commercially available mouse TNF-α, IL-1β, and IL-6 ELISA Kit (NEOBIOSCINCE, Beijing, China) according to the manufacturer's instructions. The left lobe of the right liver, the entire left lobe of the lung, and the upper half of the spleen were removed and homogenized with aseptic PBS and spread onto 7H10 plates (50 μg/mL kanamycin) for counting bacteria. The small intestine, lobe of the liver, about one-third of the right lobe of the lung, and lower half of the spleen were fixed in 10% neutral buffered formalin for histopathological analysis.
National Center for Biotechnology Information conserved domain analysis revealed that MAP3292c is a trypsin-like serine protease, and MTB Rv3671c is a trypsin-like serine protease with autoproteolytic activity [23]. To determine if MAP3292c has autoproteolytic activity, purified MAP3292c protein in 20 mM PB (pH 6.0) was substituted with 20 mM Tris-HCl-pH 8.0, and showed autoproteolytic activity (Fig. 2B). N-terminal sequencing suggested that it is cleaved between serine (S)86 and asparagine (D)-87 (Fig. 2C). To understand the subcellular location of MAP3292c, the components (CF, CW, CP, and CM) were separated from recombinant MAP3292c/MS and MAP K-10. Western blot analysis showed that MAP3292c protein was localized to the CW and CF fractions of recombinant MAP3292c/MS and MAP K-10, and the secreted MAP3292c in CF had autoproteolysis activity (Fig. 3). Next, purified MAP3292c protein was incubated with casein. SDS-PAGE analysis revealed that MAP3292c could hydrolyze casein (Fig. 4A), showing that MAP3292c is an extracellular protease.
2.13. Statistical analysis Statistical analysis was performed using GraphPad Prism Version 8.0 software. All of the data are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance was used to evaluate statistical significance followed by Bonferroni's multiple comparison test. A P-value < 0.05 was considered to be statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).
3.3. Effects of divalent cations, temperature, and pH on MAP3292c activity To optimize the reaction conditions of MAP3292c protease, the effects of different divalent salts on the protease activity of MAP3292c were assessed. The protease activity was significantly improved in the presence of 5 mM CaCl2, while MgCl2, ZnCl2, BaCl2, NiCl2, CuCl2, and MnCl2 inhibited its activity to different degrees (Table 1). The results for different pH and different temperatures are shown in Fig. 4B and 4C. The optimum activity and pH of MAP3292c protease were 41 °C and 9.0, respectively.
3. Results 3.1. Purification and identification of MAP3292c protein To understand the function of MAP3292c serine protease, the
Fig. 1. Purification and Mass Spectrometry Analysis of MAP3292c protein. (A) Affinity chromatography purification of MAP3292c protein. 1: renaturation supernatant; 2: precipitation after renaturation; 3: traversing solution; 4–5: pre-washing medium; 6: eluent solution. (B) Peptide mass fingerprinting of MAP3292c protein by matrix-assisted laser desorption ion time-of-flight mass spectrometry. 3
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Table 1 Effect of divalent cations on MAP3292c activity. Divalent cation (5 mM)
Relative activity (%)
None MgCl2 ZnCl2 CaCl2 BaCl2 NiCl2 CuCl2 MnCl2
100.00 ± 5.90 75.45 ± 4.47 31.83 ± 5.93 167.95 ± 4.30 24.05 ± 2.31 33.74 ± 4.49 57.33 ± 6.02 34.05 ± 2.56
The errors are given as standard deviation.
3.4. Kinetic studies of MAP3292c activity The kinetic parameters of MAP3292c protease were measured at 41 °C (pH 9.0) with 5 mM CaCl2 and different concentrations of casein. Michaelis constant assays suggested that the Km and Vmax values were 0.151 ± 0.04 mg/mL and 14.75 U/mg/min, respectively (Fig. 4D). 3.5. MAP3292c is a serine protease To determine the active site on MAP3292c protease, multiple sequence alignment of MAP3292c from reported serine protease conserved domains was conducted (Fig. 5A). A total of 238 serine residues were replaced by site-directed mutagenesis, and the S238A mutant protein was produced by affinity chromatography (Fig. 5B). The S238A mutant caused a 95% loss of activity compared to wild-type MAP3292c (Fig. 5C). These results showed that MAP3292c is a serine protease.
Fig. 3. Cell fractionation experiments were performed to determine the subcellular localization of MAP3292c protein. (A) Subcellular localization of MAP3292c in MS, CL: whole bacterial protein; CF: culture supernatant; CW: cell wall; CP: cytoplasm; CM: cell membrane. (B) Subcellular localization of MAP3292c in MAP.
3.6. MAP3292c promotes the MS persistence in mice To determine the pathogenicity of MAP3292c serine protease in mycobacteria, we constructed recombinant pAIC/MS, MAP3292c/MS, and S238A/MS. The lung, liver, and spleen were removed from mice infected with rMS (pAIC/MS, MAP329c/MS, and S238A/MS) and PBST at 7 d. Bacterial colony counting results showed that bacterial load of the MAP3292c/MS group was greatly enhanced compared with the Fig. 4. Determination of MAP3292c protease activity. (A) Digestion of casein by purified MAP3292c protein. (B) Effects of temperature on MAP3292c activity. The reaction was performed in 50 mM Tris-HCl, pH 9.0, and 5 mM CaCl2 at 41 °C for 1 h. (C) Effects of pH on MAP3292c activity. (D) Michaelis-Menton kinetic assays of MAP3292c protease activity. The errors are given as the SEM of three independent experiments.
4
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Fig. 5. The substitution of S-238 by alanine reduces serine protease activity in MAP3292c protein. (A) Multiple sequence alignment of MAP3292c from reported serine protease conserved domains: MTB Rv3671c (NP_218188), MTB Rv0983 (NP_215498), E. coli Deg S (AAC44006), and T. maritima Htr A (AGL49494). Alignment was performed using MEGA5 and BoxShade. The GxSxG motif is underlined. (B) The activity of site-directed mutagenesis of MAP3292c.
epithelial cells in the lung, large necrotic foci and infiltration of inflammatory cells in hepatic parenchyma in the liver, microgranuloma around the splenic white pulp, and neutrophil infiltration in the spleen in the MAP329c/MS group (Fig. 8). There was mild proliferation of alveolar epithelial cells in the lung, small infiltration of inflammation cells in the liver, and no pathological changes in the spleen of the S238A/MS group (Fig. 8). There were no pathological changes in the lung, liver, and spleen in the PBST and pAIC/MS groups (Fig. 8). There were no pathological changes in the small intestine in the PBST, pAIC/ MS, S238A/MS, and MAP3292c/MS groups (Supplementary Materials Fig. 1). These results showed that MAP3292c serine protease promotes the release of inflammatory cytokines and induces pathological injury in mice.
pAIC/MS group in the lung, liver, and spleen (Fig. 6A–C). However, the bacterial load of the pAIC/MS group was significantly decreased compared with the MAP3292c/MS group (Fig. 6A–C). These results indicated that MAP3292c serine protease promotes MS persistence in mice.
3.7. MAP3292c induces the release of inflammatory cytokines and pathological injury in mice To measure the inflammatory response, the levels of cytokines TNFα, IL-1β, and IL-6 were determined in the serum separated from mice infected with rMS (pAIC/MS, MAP329c/MS, and S238A/MS) and PBST (Fig. 7A–C). TNF-α, IL-1β, and IL-6 levels in MAP329c/MS-infected mice were significantly greater than those in control mice (PBST and pAIC/MS). However, TNF-α, IL-1β, and IL-6 levels in mice challenged with S238A/MS were markedly reduced compared with mice treated with MAP3292c/MS. Histopathological determination showed pulmonary vasodilation and congestion, mild proliferation of alveolar
4. Discussion We discovered that MAP3292c is proteolytically active for bovine casein at pH ranging from 3.0 to 12.0, with an optimal pH of 9.0. This Fig. 6. Presence of persistent recombinant MS in mouse lung (A), liver (B), and spleen (C). Bacterial loads in infected lung, liver, and spleen tissues from BALB/c mice, as transmitted by intraperitoneal infection with recombinant pAIC/MS, MAP3292c/MS and S238A/MS were determined at 7 d after infection (3 mice/group/time point). (*P < 0.05, **P < 0.01,***P < 0.001).
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Fig. 7. The release of inflammatory indicators in BALB/c mice infected with recombinant MS. (A) Analysis of TNF-α. (B) Analysis of IL-1β. (C) Analysis of IL-6. (*P < 0.05, **P < 0.01,***P < 0.001.) Fig. 8. Histopathological analysis of BALB/c mice lung, liver, and spleen. BALB/c mice were intraperitoneally infected with rMS pAIC/MS, S238A/ MS, and MAP3292c/MS at 7 d, using PBST as the negative control. (Lung) PBST and pAIC/MS: no pathological changes; S238A/MS: mild proliferation of alveolar epithelial cells (white arrow); MAP3292c/ MS: pulmonary vasodilation and congestion (blue arrow), mild proliferation of alveolar epithelial cells (white arrow). (Liver) PBST and pAIC/MS: no pathological changes; S238A/MS: small infiltration foci of myeloid cells (blue arrow) and giant cells (white arrow); MAP3292c/MS: there were large necrotic foci and infiltration of inflammatory cells in hepatic parenchyma (blue arrow). Fibroblast proliferation could be seen at the edge of necrotic foci (white arrow). (Spleen) PBS, pAIC/MS, and S238A/MS: no pathological changes; MAP3292c/MS: microgranuloma around splenic white pulp (white arrow) and neutrophil infiltration (blue arrow). Scale bars = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
cytokines, such as IL-1β and IL-6, which are major early regulators of pro-inflammatory responses [28]. The expression levels of IL-1β, TNFα, and IL-6 significantly increased in the infected ovine intestinal tissues by MAP [29]. MAP and CW components induce the expression of TNF-α and IL-6 in bovine monocytes [30]. TNF-α activates naive macrophages and is essential for the formation of granulomas around the sites of mycobacterial infection [31]. Our results showed that MAP3292c serine protease activity promoted the release of IL-1β, IL-6, and TNF-α in a mouse model, suggesting that MAP3292c is involved in the pathogenesis of mycobacterium. In S. aureus, clpP or clpX protease mutant strains render the bacterium avirulent in a mouse skin abscess model, which points to a significant role for the ClpXP protease in the virulence of S. aureus [32]. In Helicobacter pylori, HtrA protease is secreted into the extracellular surroundings [33] and degrades E-cadherin to efficiently destroy adherence junctions in polarized epithelial cells [34]. In MTB, Rv2869c protease regulates MTB intramembrane proteolysis to control membrane composition to affect the virulence of MTB [35]. Our data showed that MAP3292c promoted the persistent of the MS and induced injury to the lung, liver, and spleen in mice, indicating that MAP3292c is an important mycobacterium virulence factor. MS growth is quick and its infection model is often used to study the protein function and pathogenesis of slow growing mycobacteria, such as MTB [36–38]. However, the MS infection model is not a perfect in vivo model for MAP, as a multitude of virulence factors are missing in MS that are in present in MAP. MAP is the primary pathogen of enteric regions; however, our results showed that there were no pathological
pH range is consistent with the MAP of gastrointestinal pathogenic microorganism, which can tolerate the acid environment of the stomach and alkaline environment of the intestine, indicating that MAP3292c may be associated with MAP virulence. Divalent metal ions of Ca2+ increased the activity of MAP3292c protease, indicating that they play important roles in stabilizing the structure of protease. However, Mg2+, Zn2+, Ba2+, Ni2+, Cu2+, and Mn2+ inhibited its activity, which caused small changes in protein conformation. These phenotypes also appear in the serine protease of Agkistrodon blomhoffii ussurensis snake venom [24] and insulin-like growth factor binding proteins serine protease [25]. The subcellular localization results showed that MAP3292c protein was localized in the CW, CM, and CF. Studies have shown that proteins on the CM and CW are the main components of pathogen-infected host cells, and these proteins are more accessible to the host's immune system [26,27]. This localization of the MAP3292c protein indicates that it may play an important role in the pathogenesis of MAP. Rv3671c, a serine protease of MTB that can cleave β-casein and cause autocleavage, is required for MTB resistance in macrophages [23]. However, there have been no reports on serine protease autocleavage in MAP. We found that MAP3292c is the autoproteolytic active, and the cleavage site is between S-86 and D-87 to produce a ~28 kDa protein, consistent with the size of MAP3292c protein secreted by MAP. These results showed that MAP3292c protein obtained in vitro has the same function as that in wild-type MAP K-10. Studies have shown that cytokine signaling plays a major role in host defense MAP, characterized by the release of pro-inflammatory 6
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changes in the small intestine in the MAP3292c/MS groups. Thus, the MAP3292c mutant of MAP needs to be constructed to study the function of MAP3292c protein. In summary, this study characterized MAP3292c serine protease, an extracellular protein of MAP, which was found to degrade casein and have autoproteolytic activity. In addition, MAP3292c serine protease activity was shown to promote the colonization of MS in mice and induce pathological damage and the release of inflammatory cytokines in mice. These results provide the basis for future studies on the pathogenic mechanism of MAP.
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CRediT authorship contribution statement Hongxiu Liu: Formal analysis. Guanghui Dang: Formal analysis. Xinxin Zang: Formal analysis. Zhuming Cai: Formal analysis. Ziyin Cui: Formal analysis. Ningning Song: Formal analysis. Siguo Liu: Formal analysis. Declaration of competing interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by grants from the National Key Research and Development Program of China (2016YFD0500905), Key scientific and technological projects of Xinjiang Production and Construction Corps (XPCC) (2019AB029), and National Natural Science Foundation of China (No. 31772767). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micpath.2020.104055. References [1] O.D. Ekici, M. Paetzel, R.E. Dalbey, Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration, Protein Sci. 17 (2008) 2023–2037. [2] M.M. Krem, E. Di Cera, Molecular markers of serine protease evolution, EMBO J. 20 (2001) 3036–3045. [3] S.F. de Stoppelaar, H.J. Bootsma, A. Zomer, J.J. Roelofs, P.W. Hermans, C. van 't Veer, T. van der Poll, Streptococcus pneumoniae serine protease HtrA, but not SFP or PrtA, is a major virulence factor in pneumonia, PloS One 8 (2013) e80062. [4] A. Karlsson, P. Saravia-Otten, K. Tegmark, E. Morfeldt, S. Arvidson, Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases, Infect. Immun. 69 (2001) 4742–4748. [5] F.M. McAleese, E.J. Walsh, M. Sieprawska, J. Potempa, T.J. Foster, Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease, J. Biol. Chem. 276 (2001) 29969–29978. [6] Q.J. Zhao, J.P. Xie, Mycobacterium tuberculosis proteases and implications for new antibiotics against tuberculosis, Crit. Rev. Eukaryot. Gene Expr. 21 (2011) 347–361. [7] M.J. White, H. He, R.M. Penoske, S.S. Twining, T.C. Zahrt, PepD participates in the mycobacterial stress response mediated through MprAB and SigE, J. Bacteriol. 192 (2010) 1498–1510. [8] Q. Zhao, W. Li, T. Chen, Y. He, W. Deng, H. Luo, J. Xie, Mycobacterium tuberculosis serine protease Rv3668c can manipulate the host-pathogen interaction via Erk-NFκB axis-mediated cytokine differential expression, J. Interferon Cytokine Res. 34 (2014) 686–698. [9] I. Olsen, G. Sigurğardóttir, B. Djønne, Paratuberculosis with special reference to cattle. A review, Vet. Q. 24 (2002) 12–28. [10] R.J. Whittington, D.J. Begg, K. de Silva, A.C. Purdie, N.K. Dhand, K.M. Plain, Case definition terminology for paratuberculosis (Johne's disease), BMC Vet. Res. 13 (2017) 328. [11] P. Mercier, S. Freret, K. Laroucau, M.P. Gautier, I. Brémaud, C. Bertin, C. Rossignol, A. Souriau, L.A. Guilloteau, A longitudinal study of the Mycobacterium avium subspecies paratuberculosis infection status in young goats and their mothers, Vet. Microbiol. 195 (2016) 9–16. [12] R.J. Whittington, E.S. Sergeant, Progress towards understanding the spread, detection and control of Mycobacterium avium subsp. paratuberculosis in animal populations, Aust. Vet. J. 79 (2001) 267–278.
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