Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in Pseudomonas aeruginosa

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PMID: 29296265 (PubMed) - PMCID: PMC5742652 - View online: PubReader
Volume 10, Issue 1, January-March 2018, Page 34 to 40
Wednesday, September 14, 2016 :Received , Wednesday, February 15, 2017 :Accepted

• Faezi, Sobhan
  - Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
  - Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
  - Mycobacteriology Research Center (MRC), Tehran, Iran

• Nikokar, Iraj Central Research Laboratory of Modern Technologies of Medical Sciences, Faculty of Paramedicine, Guilan, Guilan University of Medical Sciences, Iran , Tel: +98 1342537171, Fax: +98 1342537171, E-mail: Nikokariraj@yahoo.com, Nikokariraj@gums.ac.ir
  - Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran
  - Laboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

• Elmi, Ali
  - Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

• Ghasemi, Yusuf
  - Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

• Farahbakhsh, Mojtaba
  - Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran

• Salimi Chirani, Alireza
  - Department of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science, Tehran, Iran

• Mahdavi, Mehdi
  - Department of Immunology, Pasteur Institute of Iran, Tehran, Iran

Pseudomonas aeruginosa (P. aeruginosa) as an opportunistic human pathogen has a remarkable capacity to cause disease in susceptible hosts. It is the major colonizing microbial pathogen for Cystic Fibrosis (CF) patients ¹ and a common infectious agent in nosocomial infections, such as patients with a severe burn, cancer, transplantation, AIDS, and other immunocompromising conditions. Despite improvements in antibiotic therapy, P. aeruginosa shows inherent and acquired resistance to many antimicrobial agents ². The pathogenesis of P. aeruginosa infections is multifactorial and is being affected by a complex of virulence factors; hence, it has made vaccine development difficult. Bacterial attachment is an initial and a critical step for the establishment of infection that involves bacterial adhesins and host receptors. One of the most important adhesins in P. aeruginosa is pili ³. 
Type IV pili (T4P) are the most common type of bacterial pili and are thin, long, flexible, and retractable protein filaments. T4P are polarly localized, filamentous surface appendages present at the cell surface of a broad range of pathogenic and environmental bacterial species ⁴. This adhesive cell surface structure is the prominent virulence factor that essential for initiation of infection by mediating attachment to host cells, where non-piliated strains were reported to exhibit a 90% decrease in their ability to bind human alveolar cells ⁵, and also mutant strains that are unable to produce T4P are attenuated in virulence ^6,7. Furthermore, another study revealed that piliated strains caused 28%-96% more cases of P. aeruginosa pneumonia as compared to non-piliated strains in a mouse model of infection ⁸. T4P play an important role in many processes including bacterial locomotion known as twitching motility, aggregation, infection by pilus-specific bacteriophage, DNA uptake, attachment to biotic and abiotic surfaces, host cell invasion and biofilm maturation ⁹. The pilus fiber is composed of hundred copies of PilA (or pilin, the major structural subunit) that are encoded by an operon that positively control by the algR regulator ¹⁰. The pilin monomer can be divided into three domains: a highly conserved hydrophobic N-terminal α-helix region; a hypervariable central region; and a semi-conserved C-terminal region containing β-strands. The C-terminal Receptor Binding Domain (RBD) of P. aeruginosa pilin is a suitable candidate for a peptide vaccine ¹¹. The RBD contains a disulphide-bonded loop (DSL) that structurally is highly conserved among T4P of all species of P. aeruginosa, although the size of the DSL (from 12-31 amino acids) and its sequence is varied among pilin alleles. 
The monoclonal antibody studies revealed that the C-terminal DSL of the pilin subunit mediates attachment to epithelium receptors, this finding suggests that PilA itself acts as both a major structural subunit and an adhesion ^7,12. Finally, Type IV pili has a common receptor among all strains of P. aeruginosa; however, the sequence diversity presents a considerable obstacle to the development of a protective RBD-based vaccine targeting the T4P ¹¹. 
Pili are rapidly extended and retracted via a most powerful molecular machine that organized with four subcomplexes: the cytoplasmic motor subcomplex (consisting of PilBTUCD), the inner membrane alignment subcomplex (PilMNOP), the outer membrane secretin pore subcomplex (PilQ and PilF), and the pilus itself (or PilA) ¹³. There are significant structural and functional similarities between this pilus assembly apparatus and type II secretion system ¹⁴. T4P passes the outer membrane through a large oligomeric channel and makes a single protein. The PilQ (77 kDa; ORF PA5040) that encoded by the highly conserved pilMNOPQ operon ¹⁵ is a member of the so-called "secretin" family required for configuration of the outer membrane pore through which the pilus is extruded ⁹. The PilQ protein that is essential for T4P biogenesis consists of five conserved domains; Secretin_N (380-449), Secretin (549-705), STN (306-354), HofQ (1-707) and AMIN (63-123). The secretin domains (Secretin_N and Secretin) of the PilQ are more highly conserved at their C-termini. This region facilitates the passage of folded proteins, filamentous phage particles, DNA, and other macromolecules across the outer membrane ¹⁶. The secretin domain of PilQ (that's mean PilQ_380-706) was chosen as a new antigen and designed into expression vector pET28a.
In the present study, we designed a chimeric plasmid contains the pilQ_1138-2118 gene, which codes the immunologic domains of PilQ secretin (the C-terminal domain of the PilQ). To the best of our knowledge, for the first time, we report the purification and characterization of a novel recombinant PilQ (r-PilQ380-706) from P. aeruginosa. Furthermore, our data suggest that the protein has biological activities in both in vivo and in vitro conditions.

Bacterial strains, plasmids, and media: Escherichia coli (E. coli) strains Top10F and BL21 (DE3) were used as preservation and expression hosts. The P. aeruginosa laboratory strain PAO1 (that kindly provided by Dr. Abdi from Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran) were performed. The recombinant plasmid pET28a/pilQ_1138-2118 synthesized by Biomatik Corporation (Cambridge, Ont., Canada). All enzymes for DNA manipulations were obtained from NEB (USA). The Ni²⁺-NTA agarose was purchased from Qiagen (USA). The HRP-conjugated goat anti-rabbit IgG and Protein A/G agarose were obtained from ThermoFisher (formerly Invitrogen, USA). The strains were cultured in LB broth or on agar (HIMEDIA, India) at 37°C with or without 30 µg kanamycin/ml (Bioscience, Canada). 
Construction of the expression vector: The pilQ_1138-2118 gene was inserted into the E. coli expression vector pET28a, in frame with a T7 promoter, kanamycin-resistant gene and the C and N-terminal six-His-tagged sequences. The gene containing BamHI and HindIII sites at the 5′ and 3′ ends, respectively. In the designation of the construct, we have inserted a start codon ATG immediately after the BamHI site (ggatccATG) of the pET28a vector, resulting in the correct framing of the gene of the insert. After transformation of the recombinant vector (pET28a/pilQ_1138-2118) into E. coli Top10F competent cells, transformants were screened on LB plates supplemented with 30 µg kanamycin/ml. The recombinant vector was extracted from E. coli Top10F using plasmid extraction kit (Bioneer, Korea) according to manufacture instruction. 
Confirmation of the recombinant vector: The pET28a/pilQ_1138-2118 vector was verified by polymerase chain reaction (PCR) and restriction enzyme digestion. The vector was treated with the restriction endonucleases BamHI and HindIII (Jena Bioscience Kit, Germany) according to manufacture instruction. The specific primers were designed for the pilQ_1138-2118 sequence of the P. aeruginosa PAO1 strain from NCBI (Gene ID: 880962). The gene was amplified from pET28a/pilQ1138-2118 vector using the following specific primers: Forward: 5’-CCA GGT GAA CTA CGC CAA GG- 3’ and Reverse: 5’-CGG TGT CAG GAA AAC CAG AAG T-3’, which incorporated by BamHI and HindIII restriction sites, respectively. Amplifications were performed in 25 µl volumes containing 50 pM of each primers, 1X PCR master mix, and 25 ng of the vector. The PCR was performed in a master gradient thermocycler (Bio-Rad, USA). Template DNA was denatured at 94°C for 4 min prior to the start of PCR cycling, which consisted of denaturation at 94°C  for 46 s, primer annealing at 61°C for 1 min, extension at 72°C for 1 min, repeating for 30 cycles followed by a final extension at 72°C for 10 min. The digested fragments and PCR product were separated by 1.2% (w/v) agarose gel electrophoresis.
Expression and isolation of inclusion bodies: In order to overexpress the protein, the recombinant construct pET28a/pilQ_1138-2118 was transformed into BL21 (DE3) and platted on LB agar containing kanamycin (30 µg/ml). To optimize the induction conditions, the colonies carrying pET28a/pilQ_1138-2118 were grown in 5 ml of LB medium supplemented with kanamycin at 22ºC. At OD₆₀₀ nm of 0.8, expression of the r-PilQ_380-706 was induced by addition of IPTG (BIOSYNTH, Switzerland) to a final concentration of 1 mM. After 0, 1, 2, 3, 4 and 5 hr of induction, cells were harvested and the induced level of r-PilQ_380-706 was determined by 12% SDS-PAGE electrophoresis.
For isolation of inclusion bodies, an overnight culture of E. coli BL21 (DE3) cells harboring pET28a/pilQ_1138-2118 was diluted 100-fold in LB medium (1 liter) containing kanamycin and incubated at 22°C with shaking. When the OD₆₀₀ of the culture reached 0.8, the promoter of the recombinant vector was induced by the addition of IPTG to the final concentration of 1 mM. After 4 hr, the induced cells were harvested by centrifugation at 8500×g for 10 min at 4ºC and suspended in lysis buffer [20 mM sodium phosphate (pH=7.5), 10 mM EDTA, 1% (v/v) Triton X-100] to remove the contaminant proteins. Following freezing and thawing, the lysozyme (100 µg/ml) was added to lyse the cell wall and incubated for 30 min in room temperature. The sonication was carried out on ice in the presence of PMSF (1 mM) as a protease inhibitor. To chelate the EDTA and remove DNA, 10 mM MgSO4 and DNase (0.01 mg/ml) was added, respectively and followed to incubate on ice for 20 min. After centrifugation, the pellet was thoroughly washed with the same buffer without EDTA and resuspended again in the buffer without EDTA and Triton-X100. The Inclusion Bodies (IBs) were harvested by centrifugation and stored at 4^oC.
Solubilization, refolding and purification of r-PilQ380-706: The IBs were solubilized with Guanidinium Lysis Buffer [20 mM sodium phosphate, 500 mM NaCl, 6 M guanidine hydrochloride, pH=7.4]. The solubilized proteins were purified using Ni2+-NTA agarose (Qiagen, USA) according to the manufacturer’s instructions with modifications. Purifications were performed under denaturing and renaturing conditions (hybrid conditions). Briefly, after applying the sample to the column and washing with denaturing binding buffer [20 mM sodium phosphate, 500 mM NaCl, 8 M urea, pH=7.8], a linear gradient of urea from 7 M to 0 M of refolding buffer [20 mM sodium phosphate, 500 mM NaCl, 5% (v/v) glycerol, pH=6.0] was used at flow rate of 0.6 ml/min. The contaminant proteins were washed using native wash buffer [20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH=8.0]. Finally, bound proteins were eluted in native elution buffer [20 mM sodium phosphate, 500 mM NaCl, 250-500 mM imidazole, pH=8.0]. The purified r-PilQ_380-706 was dialyzed against phosphate buffered saline (PBS, pH=7.4) for imidazole removal and analyzed by 12% (w/v) SDS-PAGE followed by Coomassie brilliant blue G-250 staining. The protein concentration was quantitatively measured by using a NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) and Bradford protein assay using standard albumin (Sigma, USA).
Preparation and purification of anti r-PilQ_380-706 IgG: To determine the immunogenic nature of purified r-PilQ_380-706, the female New Zealand white rabbits (Razi Vaccine and Serum Research Institute, Karaj, Iran) were immunized with 400 µg of the r-PilQ_380-706 protein administered subcutaneously and boosted twice with 200 µg with 2 weeks intervals. The rabbits were anesthetized intramuscularly with an injection of a mixture of xylazine (10 mg/kg) and ketamine (50 mg/kg). The rabbits were bled prior to immunization and 2 weeks after the last immunization. Sera were collected from the retracted clot, clarified by centrifugation (2500×g) and then aliquoted and stored at -20°C. The rich fractions pooled and the specific IgGs (except IgG3) purified by using protein A/G agarose (Invitrogen, USA) according to the manufacturer's instructions and analyzed by SDS-PAGE. Protein concentration was quantitatively determined using NanoDrop (2000c spectrophotometer, Thermo Scientific, USA) and Bradford protein assay. Anti r-PilQ_380-706 IgG and non-immune IgG were aliquoted at a concentration of 1-2 mg/ml and finally stored at -20^oC until use. 
SDS-PAGE electrophoresis and Immunoblot analysis: The bacterial pellets and purified protein were separated by SDS-PAGE. The samples were directly resuspended at a 2:1 ratio with 3× SDS-PAGE sample buffer. in an appropriate volume of sample buffer. The discontinuous gel consisted of a 5% stacking gel and a 12% resolving gel which was run on a vertical electrophoresis unit (Mini PPROTEAN 3 cell, Bio-Rad). To determine the functional activity, the purified r-PilQ_380-706 protein was electrophoresed, and then transferred onto a PVDF membrane (Hi-bond Amersham Biosciences, USA) by a Bio-Rad apparatus at 25 V for overnight. The membrane was blocked with 5% non-fat skim milk in TBST buffer (Tris buffer saline contain 0.1% Tween-20) for 2 hr at RT. After incubated with rabbit anti r-PilQ380-706 IgG (1:5000 diluted in blocking buffer) for 1 hr at RT, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG for 1 hr at RT. The membrane was then washed 5 times with TBST for 5 min each. Finally, it was developed by adding 3, 3′-diaminobenzidine (DAB) solution (Sigma, USA) allowing it to incubate until bands were seen. The reaction was stopped by rising the membrane with water.
Twitching inhibition assay: To verify the functionality of the r-PilQ_380-706-specific polyclonal IgG, the twitching inhibition assay was carried out by Castric et al ¹⁷ as follows. Different concentrations (0.1, 0.2 and 0.3 µg) of specific rabbit anti r-PilQ3_80-706 IgG (filter-sterilized) were added to LB broth (containing 1% (w/v) agar), which was poured into a 15×90 mm plastic Petri dish. After solidification, the plate was dried for 6 hr at room temperature. A single colony of the P. aeruginosa PAO1 strain to be tested was stab-inoculated with a toothpick to the bottom of the plates. After omitting an 18 hr incubation at 37^oC, the diameter zone of growth of different strains obtained at the interstitial surface of the agar and the plate was measured. For each assay, triplicate plates were examined. 
Statistical analysis: The data were analyzed using one-way analysis of variance (ANOVA) and Student’s t-test (StatView). Statistical analysis was performed using the software GraphPad Prism version 6.0 for Windows, (GraphPad Software, San Diego, CA, USA). All data of this study are expressed as mean±SD. The p-values less than 0.05 was considered to be statistically significant.

Confirmation of the pET28a/pilQ_1138-2118 construct: The coding sequence of the secretin domain of pilQ (pilQ_1138-2118) was constructed in the pET28a expression vector. Transformants were characterized by enzymatic digestion. The recombinant plasmid, pET28a/pilQ_1138-2118, was extracted and its orientation confirmed by digestion with two restriction enzymes that mentioned above. The target fragments with the expected sizes are shown in figure 1. DNA gel electrophoresis of the PCR product resulted in single 981 bp band, which confirmed amplification of pilQ_1138-2118 gene (Figure 1). Sequence analysis of recombinant pET26b/pilQ_1138-2118 confirmed that there are no amplification errors and that construction was accurate.
Expression and purification of r-PilQ_380-706: To construct an expression system, the coding sequence of PilQ_380-706, whose theoretical molecular size is approximately 35 kDa, was constructed into expression vector pET28a to express a recombinant protein. The recombinant plasmid, pET28a/pilQ_1138-2118, was transformed into E. coli BL21 (DE3). The His-Tagged recombinant protein was purified from inclusion bodies by using Ni2+-affinity chromatography. Results from SDS-PAGE analysis of expression products showed that the PilQ_380-706 protein expressed 4 hr after induction with IPTG. The expression product of the protein was approximately 37 kDa in molecular size (Figure 2). The purified protein was confirmed by western blotting as showed in figure 3. The yield of the purified PilQ380-706 protein was about 2.18 mg per liter of culture media. In term of measured protein, no significant difference was observed between NanoDrop and Bradford protein assay.
Production of PilQ_380-706-specific IgG and specificity analysis: Polyclonal antibodies against r-PilQ_380-706 were produced in rabbit and finally the specific IgG was purified by using protein A/G agarose (Invitrogen, USA) according to manufacturer's instruction. As shown in figure 4, β-Mercaptoethanol as a reducing agent break the hinge-region disulfide bonds and thus antibodies will dissociate into the heavy (51.4 kDa) and light (24.9 kDa) chains, respectively. To determine the specificity of the antiserum raised against purified r-PilQ_380-706, western blot analysis was performed. The total cell extracts (induced and non-induced) and the purified PilQ_380-706 protein were immunoblotted and then hybridized with specific polyclonal IgG. Addition of HRP-conjugated goat anti-rabbit IgG showed that the PilQ_380-706 protein was substantially expressed by using the pET28a/pilQ_1138-2118 expression vector when IPTG was added at the early-exponential phase of growth and collecting the cells 4 hr after induction (Figure 3).  Overall, our results indicated that the rabbit produced antibodies are highly specific to detect the r-PilQ_380-706. 
Twitching inhibition assay: Immunized and non-immunized rabbit sera were evaluated in the twitching inhibition assay for their biofunctional activity to inhibit the motility of PAO1 strain of P. aeruginosa. In this assay, NRS was used as control group. As shown in figure 5 and table 1, the r-PilQ_380-706 IgG was able to inhibit the motility of the PAO1 strain, so that the motility zone was significantly decreased compared to control group. In the presence of NRS, no immobilization was observed.

In conclusion, the present study described a modified method for expression, purification and refolding of r-PilQ_380-706 from P. aeruginosa in E. coli. The recombinant protein was expressed in the form of inclusion bodies under the pET28a expression vector. Here, we developed a reproducible and simplified method to achieve significant yields of the protein. The purification of r-PilQ_380-706 was done under the modified hybrid condition. The procedure developed in this study may be useful in the efficient purification of other recombinant proteins expressed in E. coli as inclusion bodies. This recombinant protein was biologically active and recommended to be used as a vaccine or an adjuvant.

The authors are grateful for technical support provided by personnel of the Central Research Laboratory of Modern Technologies of Medical Sciences, Paramedicine Faculty, Langarud, Guilan, Iran. This work was supported by a grant from Guilan University of Medical Sciences, Guilan, Iran.

Larger Image High Resolution Figure 1. Agarose gel electrophoresis analysis of recombinant pET28a/pilQ1138-2118 with restriction enzyme digestion. Lane M; DNA marker (1 kb), Lane 1; mono-digestion of the pET28a/pilQ1138-2118 vector with BamHI. One expected fragment was observed on the gel (~6350 bp band). Lane 2 and 3; BamHI/HindIII double digested the recombinant vector with BamHI and HindIII buffer, respectively. Two expected fragments from double digestion were observed on the gel (~5369 and 981 bp bands). Lane 4; the optimized PCR product of the pilQ1138-2118 gene (~ 961 bp band).

Larger Image High Resolution Figure 2. SDS-PAGE analysis of the expression of r-PilQ380-706 protein in E. coli. The total proteins of the BL21harboring pET28a/ pilQ1138-2118 plasmid was harvested and loaded on 12% (v/v) SDS-PAGE after 4 hr induction with or without IPTG. (lane M) denote molecular weight marker proteins; (lanes 1) total cell lysate of non-induced bacteria; (lanes 2-7) 1-6 hr incubation after induction; (lane 8) purified r-PilQ380-706 after HisTrap Chelating and Ni2+-affinity chromatography (~37 kDa).

Larger Image High Resolution Figure 3. Western blot analysis of the expressed r-PilQ380-706 protein in E. coli BL21. After running the SDS-PAGE, the protein transferred onto PVDF membrane and detected with HRP-conjugated goat anti-rabbit IgG. (lane 1) total cell lysate of non-induced bacteria; (lane 2) total cell lysate of bacteria after 4 hr induction; (lane 3 and 4) purified r-PilQ380-706 by Ni2+-affinity chromatography.

Larger Image High Resolution Figure 4. Coomassie blue-stained SDS-PAGE gel of affinity-purified antibodies. (lane M) molecular mass markers, in kDa; (lane 1) 10 mM glycine eluant; (lane 2) flowthrough from 100 mM glycine. Light chain (LC) and Heavy chain (HC) polypeptides were predicted to be 24.9 and 51.4 kDa, respectively.

Larger Image High Resolution Figure 5. Twitching inhibition of P. aeruginosa PA01 strain in the presence of r-PilQ380-706 IgG on motility agar (LB broth with 1% w/v agar). The motility agar plates supplemented with either different concentrations of anti r-PilQ380-706 IgG or non-immune rabbit serum (NRS) were stab inoculated with P. aeruginosa and incubated for 18 hr at 37°C. Different concentrations (0.3, 0.2 and 0.1 µg) of antiserum raised against r-PilQ380-706 inhibited motility of P. aeruginosa PAO1 (b to d, respectively) compared with NRS (a).