Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum

Abstract

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms—rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01235-4.

Introduction

Ralstonia pseudosolanacearum (RPS) is a gram-negative soil-borne phytopathogen that causes bacterial wilt in many plant species including the economically important Solanaceae family crops [1, 2]. Previously, the pathogen was a member of four phylotype groups, the Ralstonia solanacearum species complex (RSSC). Recently, RSSC was rearranged into three different species [3, 4]: R. solanacearum (RS) (phylotype II), RPS (phylotype I and III), and R. syzygii (phylotype IV and blood disease bacterium) [5–7]. RPS shares most of the virulence genes and other characteristics with RS [8, 9].

The pathogen invades the susceptible host through open wounds thereby infecting the cortex and spreading to the xylem blocking its flow, leading to the occurrence of marked symptoms of wilting [10, 11]. The infection can be preliminarily diagnosed by an onsite ooze test. For confirmation, the etiological agent firstly needs to be isolated in Kelman’s tetrazolium medium or modified semi-selective medium South Africa (mSMSA), followed by subsequent confirmation using molecular or immune-detection tools. The challenge lies in precisely identifying colonies of the etiological agent among the colonies of saprophytic bacterial cells and other secondary pathogens [12]. In such circumstances, immune-detection tools can provide a rapid and handy method to screen colonies for later confirmatory laboratory assays.

To our knowledge, no rapid immune-based detection platforms for the bacterial wilt pathogen have been described in scientific literature. Most of the studies were based on ELISA using either polyclonal or monoclonal antibodies raised against the whole cell or LPS of the pathogen [13–15]. The polyclonal antibodies raised against whole cells and lipopolysaccharides (LPS) have specificity limitations [13, 16–18]. Conversely, several immune-based detection systems targeting specific virulent proteins were described for other gram-negative bacteria [19, 20].

In the genus Ralstonia, several virulence factors including type III secretion system and effectors [21, 22], extracellular polysaccharide I [23, 24], flagella [25, 26], type II secretion system [27, 28], and cell wall degrading enzymes such as exo-and endo-polygalacturonase (PGs) [29, 30] contribute to the pathogenicity [31]. Among these, flagella-mediated swimming motility plays a crucial role in the ecological fitness and virulence of the pathogen [25, 26]. Flagella is a composite organelle comprising of flagellar filament, basal body, etc. [32]. The flagellar filament is a polymer of flagellin protein (FliC), encoded by the flagellin gene (fliC). In many gram-negative pathogens including human pathogens, the flagellar protein, FliC, has been successfully used in immune-based detection procedures [33–35]. Intended to explore the possibility of using flagellar protein FliC as an immune target for rapid and specific identification of RP colonies and cells in general, polyclonal antibodies were generated against the recombinant form of flagellin. The antibodies were then evaluated using ELISA and Western Blot to confirm the presence of flagellin protein in RPS cells. Thereafter, a latex agglutination reagent (LAT) was prepared and evaluated for screening colonies of RPS. Figure 1 embodies the schematic representation of the proposed detection pipeline involved in the present study.

Fig. 1.

Schematic representation of the immune-based detection scheme for the Ralstonia pseudosolanacearum induced bacterial wilt

Materials and methods

Bacterial strains, vector, and host strains used in the study

R. pseudosolanaceaerum and non-R. pseudosolanaceaerum strains

Six bacterial wilt strains including five RPS strains, i.e., DIBER115 (MG266193), DIBER116 (MG266202), DIBER117 (MG266199), DIBER118 (MG266203), and DIBER119 (MG266201), and one RS strain NAIMCC-B-00418 (National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau Nath Bhanjan, India) were used in this study. The non-bacterial wilt strains used were Ralstonia pickettii (R. pickettii) (MTCC 648), Ralstonia insidiosa (R. insidiosa) (ATCC 49129), Pseudomonas spp., Ochroboctrum spp., Cupriavidus paculus (C. paculus) (All DIBER culture collection), and Escherichia coli (E. coli) (ATCC 10536).

Vector and host strains used

Commercially available Qiagen’s pQE-30 vector, containing an ampicillin resistance gene, was utilized for both gene cloning and expression purposes. This vector is specifically designed for the expression of the cloned gene, accompanied by an N-terminal 6x histidine (His)-tag. For host strains, E. coli strain M15 [pREP4] was chosen, serving as both a cloning and expression host. This strain is known for its capability of high-level expression and harbors a native plasmid pREP4 rendering resistance to antibiotic kanamycin. Therefore, the growth of M15 cells was conducted in Luria-Bertani broth medium supplemented with 25 μg/mL kanamycin, maintained at 37°C for 12 h.

Molecular procedure for screening bacterial wilt isolates

Six bacterial wilt strains including five RPS strains, i.e., DIBER115 (MG266193), DIBER116 (MG266202), DIBER117 (MG266199), DIBER118 (MG266203), and DIBER119 (MG266201), and one RS strain NAIMCC-B-00418 (National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau Nath Bhanjan, India) were used in this study. Isolation of the pathogen, RPS, was carried out from the infected plants of tomato, pepper, and eggplants from diverse regions of the state Uttarakhand, India. Plants manifesting bacterial wilt symptoms were collected, as well as onsite screening by the stem streaming test (Ooze test) was performed [12]. Tissues from suspected plants were transported to the laboratory and crushed in 1× phosphate-buffered saline (PBS) (pH 7.4) to isolate the etiological agent in modified semi-selective medium South Africa (mSMSA) agar plates [36]. Colonies that resemble RPS and RS were selected and sub-cultured in casamino acid-peptone-glucose (CPG) broth [37–39]. Thereafter, the isolates were screened by commercially available DAS-ELISA (Adgia Co.) and PCR (Q-cycler 96 Hain Life sciences, UK, Ltd.). In-house designed primer pairs Pbh2F (5′-AAT GCC AGC AAG TGG AGC ACC-3′) and Pbh1/2R (5′-TAC GAC AAC GTG AGG ATG AAC G-3′) (ThermoFisher Scientific) targeting the 683bp region spanning nucleotides 1075 and 1757 of the exo-polygalacturonase (pehB) gene in RS strain GMI1000 (accession number NC_003295 available at National library of medicine, NCBI). Qiagen’s HotStarTaq DNA polymerase kit with Cat No. 203205 was used for conducting PCR. The thermal cycling encompasses initial denaturation at 95°C for 3 min, and 35 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 20 s, and extension at 72°C for 45 s. Consequently, the isolates were confirmed by 16S rDNA sequencing outsourced to professional sequencing services (AgriGenome Labs Pvt. Ltd., Kochi). The strains were preserved (30% glycerol stock) in ultra-low-temperature freezer at −80°C to prevent spontaneous phenotypic conversion from pathogenic fluidal to non-pathogenic non-fluidal colony morphology [40, 41].

Cloning of flagellin gene in E. coli and its expression

Nucleotides encoding the amino acids 4 to 266 of the fliC gene were amplified from the genomic DNA of NAIMCC-B-00418 strain. A standardized PCR protocol was employed with the temperature profile as shown in Table 1, involving an initial denaturation step at 95°C for 3 min, followed by 35 cycles of amplification at 95°C for 30 s, 60°C for 20 s, and 72°C for 1 min (Qiagen’s HotStarTaq DNA polymerase Kit; Cat No. 203205). This PCR amplified the 10th to 798th bp region of the fliC gene using specific cloning primers namely FliC forward and reverse primer, FliC-FP and FliC-RP. Table 2 shows sequence of the primer sets with incorporated restriction enzyme sites underlined and bold. The resulting amplicon was purified and subsequently cloned into the pQE-30 expression vector (Qiagen, Germany), known for its ability to co-express a poly-histidine tag. After transformation into chemically competent E. coli M15 cells using standard techniques, antibiotic selection employing ampicillin and kanamycin was used to isolate transformed cells [42]. Confirmation of successful clones was achieved through PCR screening using FliC-FP and FliC-FP. Thereafter, selected clones were induced to produce recombinant FliC protein (r-FliC) using 1.5 mM IPTG. This involved inoculating 5 mL of prewarmed Luria-Bertani broth with 100 μL of overnight cultures of the selected clones and incubating them at 300 rpm until reaching an OD600 of 0.6, typically taking around 3 h under our experimental conditions. Subsequently, induction with IPTG at a final concentration of 1 mM was performed. Verification of induced cultures expressing r-FliC was conducted via Western blot using anti-His-HRP conjugated antibodies [42]. These antibodies specifically detect the poly-histidine tag co-expressed at the N-terminal of the r-FliC protein. Clones exhibiting relatively higher expression levels of r-FliC were chosen for further experimentation.

Table 1.

Primer pair sequence with nucleotide targeted and encoded amino acids

Target gene	Primer pair	Primer sequence	Nucleotide targeted	Amino acids encoded
FliC	FliC F	5´AAGATGGATCCAGCCTCAATACCAACATCTCG-3´	10th to 798th	4th to 266th
	FliC R	5’CAGCAAAGCTTAGGTTCAGGATGCTGTTGGG-3´

Inserted restriction enzymes sites are marked with bold and underlined

Table 2.

Temperature profile used in PCR for the amplification of fliC gene with primer pair FliC-FP and RP

Step	Temperature (°C)	Time (minutes)	No. of cycles
Initial denaturation	95	3	1
Denaturation	95	0.5	35
Annealing	60	0.5
Extension	72	0.7
Final extension	72	3	1

Purification of r-flagellin protein

Determining protein solubility

Recombinant proteins expressed in E. coli are in vivo stored either in soluble form or as insoluble inclusion bodies [43]. Therefore, prior to bulk purification, the solubility of recombinant proteins was determined. One milliliter of IPTG-induced culture of the selected clone was harvested by centrifugation at 6500 rpm for 10 min and sonicated at 6× 10s with a 10-s pause at 40 amplitudes in a sonicator (Vibra-Cell^TM Sonics, USA). The cell lysate was kept on ice throughout the procedure. Sonicated cell lysate was then centrifuged at 10,000 rpm for 10 min at 4 °C; supernatant and pelleted cells were then individually analyzed by SDS–PAGE and Western blot [42]. Recombinant proteins expressed in E. coli, M15 were purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity purification column (Qiagen, Germany).

Preparation of cleared E. coli lysates under denaturing conditions

One liter culture of each of the selected clones was induced with IPTG. Induced cells were harvested by centrifugation at 5000 rpm for 20 min in 25-mL batches. Then the cell pellet was resuspended in lysis buffer pH 8.0 (100mM NaH₂PO₄, 10mM Tris-Cl, 8M Urea) at 5 mL per gram wet weight of the harvested pellet. Resuspended cells were stirred for 60 min (till the solution becomes translucent) at room temperature in a tube rotor (Tarsons Products Pvt. Ltd.). Then the lysate was centrifuged at 10,000 rpm for 30 min at room temperature to pellet the cellular debris. Supernatant or cleared lysate was further processed for purification of recombinant protein.

Purification of 6xHis-tagged proteins from E. coli under denaturing conditions

The denatured lysate was used for the purification of 6xHis-tagged proteins as per the manufacturer’s instruction (Qiagen Ni-NTA Spin Kit). Collected fractions of eluates were analyzed by SDS-PAGE [42].

Recombinant protein concentration and quantification

Eluates giving a single band corresponding to the molecular weight of the protein were pooled in respective 15-mL eppendorf tubes. Thereafter, the protein was concentrated using Amicon® Ultra centrifugal (EMD Millipore, Germany) of appropriate size according to the molecular weight of the recombinant protein. Pooled r-FliC protein was concentrated using an Amicon® Ultra 15K device with a molecular weight cut-off (MWCO) of 15,000 Da. Thereafter, the concentration of each of the proteins was determined spectrophotometrically using Total Protein Kit, Micro Lowry, Peterson’s Modification (Sigma-Aldrich, USA).

Production of polyclonal antibodies

Polyclonal antibodies against the recombinant proteins and whole cell of strain NAIMCC-B-00418 were produced in model animals, rabbits, and mice, through outsourcing to Abgenex Private Limited, Bhubaneswar, India. For the preparation of inactivated whole cells, 20 mL of 48 h CPG broth culture of Rs0418 was pelleted. Then the harvested cells were washed twice with 10 mL of sterile 1× PBS. Finally, the washed cells were resuspended in 5mL of sterile 1× PBS. The cell count of the washed cell suspension was done using the serial dilution technique by the direct viable counting (DVC) method. The plates were incubated at 28°C for 48 h and the resultant colonies were counted and the respective cell suspensions were standardized to 1×10⁶ CFU/mL using 1× PBS. Thereafter, formalin inactivation was done by adding formalin to the cell suspension at a final concentration of 0.5% (v/v).

Evaluation of antibody titer

Evaluation of antibody titer for anti-rFliC was done by indirect ELISA. Briefly, 100 μL of purified protein at a concentration of 20 ng/100 μL in coating buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) were coated onto the 96-well ELISA plates (Nunc™ PolySorp™), followed by incubation at 37°C for 1 h. Then, the plates were rinsed three times with 1× PBS-Tween 20 (PBS-T) washing solution for 3 min, followed by blocking the unbound sites in the wells by adding 200 μL of 1% BSA solution (in 1× PBS) and incubation overnight at 4°C. Doubling dilutions of polyclonal antisera (each of rabbit and mice) ranging from 1:1500 to 1:286,000 dilutions in 1× PBS were made, and 100 μL from each dilution was added in respective wells and the plates were incubated at 37°C for 1 h and washed. Then, either Goat anti-rabbit IgG or Goat anti-mice IgG conjugated to horse radish peroxidase (HRP) diluted to 1:5000 in 1× PBS was prepared, and 100 μL of the diluted conjugate was added onto the respective wells and incubated at 37°C for 1 h and washed. 100 μL of the development buffer [5 mg o-phenylenediamine (OPD) and 20 μL H₂O₂ in 20 mL of sodium phosphate buffer] was added to each well and incubated in dark at room temperature for 10 min. Then 50 μL of 5M H₂SO₄ was added to each well to stop enzyme activity. Thereafter, absorbance was measured at 405 nm using a microplate reader (Synergy HT Multi-Detection Microplate Reader, Bio Tek Instruments, Inc., USA). Further, cut-off value was calculated as described by Classen et al. [44] using the following formula:

$$\mathrm{Cut}-\mathrm{off}\mathrm{value}=\mathrm{mean}\mathrm{of}\mathrm{negatives}+5\mathrm{time}\mathrm{the}\mathrm{standard}\mathrm{deviation}$$

ELISA optimization

Before performing further experiments for specificity evaluation, the optimal concentrations of capture antibody and detection antibody for each of the raised antibodies were determined by checkerboard titration. For this, Indirect ELISA in checkerboard titration format was performed in 96-well ELISA plates (Nunc™ PolySorp™) as described previously. The highest dilution which gives OD values greater than the cut-off values and can detect minimum antigen is the optimum antigen and antibody concentration.

Briefly, wells were coated with an array of doubly diluted rFliC protein. Diluted protein in the range 5–320 ng/well in coating buffer was coated while anti-FliC antibodies were titrated in doubling dilutions in 1× PBS from 1:2500 to 1:80000. For anti-WC antibodies optimization, the coating was done with tenfold dilutions of RS strain NAIMCC-B-00418 cells in the range 1×10⁶ to 1 CFU/100 μL in coating buffer and antisera in the range of 1:2500 to 1:80000 in 1× PBS were used.

Evaluation of raised polyclonal antibodies

Evaluation of anti-rFliC Ab, as well as anti-WC Ab, was done by Indirect Double Antibody Sandwich (Indirect DAS) ELISA and Western blot.

Preparation of washed cell suspensions

For using RPS and non-RPS cells in Western blot and DAS-ELISA, washed cell suspensions of each of the strains were prepared. For this, respective RPS and non-RPS strains were grown for 48 h and 24 h respectively in 10 mL of CPG broth medium. Cells were then harvested by centrifugation at 10,000 rpm for 5 min, and harvested cells pellet were washed twice with 1× PBS. Finally, the washed cells were resuspended in 1 mL of 1× PBS. The cell counts were performed using the serial dilution technique by the DVC method. The plates were incubated at 28°C for 48 h and the resultant colonies were counted. And finally, the respective cell suspensions were standardized to 1×10⁶ CFU/ 100 μL using 1× PBS.

Specificity evaluation by Western blot

Western blot was performed to reveal the presence of target proteins in test microcosms. 1 mL of each of the prepared washed cells were pelleted and used for sample preparation for SDS-PAGE (10% polyacrylamide, 0.1% SDS). Proteins expressed in the respective pellets were resolved in the gel and electro-transferred onto the PVDF (polyvinylidene difluoride) membrane following standard protocol with slight modifications [45]. Briefly, the membrane was washed three times by soaking it in 1× PBS-T for 5 min each. Thereafter, unbound sites in the membrane were blocked by soaking the membrane in 1×-BSA in 1×-PBS and incubating overnight at 4°C. After consecutive incubation with the Rabbit anti-FliC polyclonal serum (1:10000) and anti-rabbit secondary antibody (1:5000) (Sigma-Aldrich, USA), water-soluble chromogenic substrate 3,3′-Diaminobenzidine (DAB) and hydrogen peroxide (H₂O₂) were used to visualize the immuno-labeled bands. Washing was performed after every incubation as explained above.

Specificity evaluation by Indirect DAS-ELISA

Washed cell suspension of RPS and non-RPS cells (1×10⁶ CFU/100 μL) were coated onto the 96-well ELISA (Nunc™ PolySorp™) plates. In DAS-ELISA, as performed previously, mice anti-WC polyclonal serum (1:10000) was used as capture antibodies, and Rabbit anti-FliC polyclonal serum (1:10000) as revealing antibodies to detect flagellin protein in the cells. Goat anti-rab IgG HRP conjugate (1:5000) was used as a secondary antibody-enzyme conjugate.

Latex agglutination tests

Preparation of latex agglutinating reagent

The preparation of polystyrene latex beads (Sigma-Aldrich, USA) for agglutination was done according to the manufacturer’s protocol with slight modifications. Briefly, the latex beads were preliminary washed in 1 mL of MES (2-morpholino ethane sulfonic acid) buffer, and 500 μg of anti-FliC antibodies was coupled to latex beads using 0.2 mM EDAC [1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide]. After 15 min incubation in a rocker and subsequent washing with wash buffer (50 mM MES, 0.1% Triton x-100; pH 6.1), the antibody coupled beads pellet was resuspended in 1 mL of storage buffer (50 mM glycine in MES buffer, 0.03% Triton x-100 and 0.1% sodium azide, pH 8.1).

Test procedure

Latex agglutination tests were performed in two sets of bacterial cells. In the first set, washed cell suspensions of bacterial wilt and non-bacterial wilt strains were tested for agglutination. Briefly, 1 mL of 24 h grown culture was pelleted and resuspended in 100 μL of PBS. 10 μL of each of this prepared cell suspension was mixed with an equal volume of coated particles on a clean grease-free slide and hand tilted gently for 2 min. Results were evaluated macroscopically and recorded qualitatively as positive (+) and negative (−). In the second set of experiments, a single colony from CPG-grown microcosms was washed twice, resuspended in 100 μL of saline, and tested for agglutination. The purified recombinant protein was used as the positive control. All the latex agglutination tests were performed in triplicate with cells from different batches of microcosms.

Results

Bacterial strains, vector, and host strains

Colonies namely DIBER115-DIBER119 showed the characteristic feature of RPS colonies such as white colonies with pink centers generating reddish-brown pigmentation (Fig. 2). These colonies were further confirmed by PCR using the primer pairs Pbh2F/Pbh1/2R targeting the gene pehB region respectively of RS/RPS [38]. Figure 3 shows the agarose gel electrophoresis of the PCR products for the primer sets Pbh2F/Pbh1/2R and 760F/759R. NAIMCC-B-00418 strain was used as the positive control, and PCR reaction with no template was used as the negative control. PCR product of all the stains showed a 683bp band in electrophoresis which corresponds to the exo-polygalacturonase (pehB) gene targeted by Pbh2F/Pbh1/2R. DIBER120 strain gave weak amplification with primer set Pbh2F/Pbh1/2R.

Fig. 2.

Colony morphology of Ralstonia pseudosolanacearum strains. a DIBER115; b DIBER116; c DIBER117; d DIBER118; e DIBER119

Fig. 3.

PCR for confirmation of the selected colonies. Lane 1–lane 5: DIBER115-DIBER119; +: positive control (NAIMCC-B-00418); −ve: non-template control. PCR results reveal that all the isolates were Ralstonia pseudosolanacearum

Further 16SrDNA sequencing confirms that all the positives were identified as Phylotype-I of RSSC which is taxonomically reclassified as RPS [46]. Hence, the strains were rechristened RPS DIBER115–DIBER120. The background colonies were identified as strains belonging to Pseudomonas, Ochroboctrum, Cupriavidus, and Burkholderia genera by 16S rDNA sequencing. Isolates were submitted to National Center for Biotechnology Information (NCBI) and received the NCBI gene bank accession numbers MG266193 (DIBER115), MG266202 (DIBER116), MG266199 (DIBER117), MG266203 (DIBER118), MG266201 (DIBER119), and MG266200 (DIBER120) respectively. Refer to supplementary files S1 for16S rRNA sequences of each strain and supplementary material for BLAST results. Information regarding RPS and non-RPS as well as host strains used in the study along with the culture media and growth conditions required to cultivate them is provided in supplementary file S1 (Table S1).

Cloning of flagellin gene in E. coli and its expression

Growth of recombinant clones of the fliC genes is observed in the form of colonies (>300) on the Luria-Bertani agar plates supplemented with ampicillin. Upon PCR of the extracted plasmid from the broth cultures of the selected clones, 7 clones showed a band corresponding to the 748 bp of the fliC gene. These PCR-positive clones were further sub-cultured for IPTG induction to express the recombinant protein and confirmed by Western blot [42]. In Western blot, it was observed that the 2 clones gave a band corresponding to the molecular weight of the protein, i.e., 28.7 kDa. Figure 4 shows transformation and screening results for recombinant FliC protein. The cloned sequence of the fliC gene was submitted to NCBI and received the accession number MW759461. Partial coding sequence of cloned fliC gene is provided in supplementary file S1 and refer to supplementary material for BLAST analysis.

Fig. 4.

Confirmation of observed clones. a Growth of clones in Luria-Bertani agar + Amp + Kan Plates. b Confirmation of positive clones by agarose gel electrophoresis of the PCR product showing band corresponding to fliC gene. Lanes 1–8: PCR product of the selected clones. c SDS PAGE and d Western Blot, analysis of induced and non-induced clones of M15 cells giving r-FliC protein expression. Lanes 1, 2, 3: non-induced cells of selected clones; lanes 4, 5, 6, 7: 4 h induced cells of selected clones, showing band corresponding to the molecular weight of FliC protein, i.e., 28.7 kDa; lane 8: positive control (purified r-FliC Protein); M—clearly stained Protein Ladder (Takara Bio USA, Inc. Code No. 3454A, 10–210 kDa)

Purification of r-flagellin protein

Western blot analysis of sonicated M15 cell lysate transformed with the fliC gene showed that the r-FliC protein was present in the sonicated pellet, indicating that the protein was expressed in an insoluble form inside the inclusion bodies (Fig. 5). So, further purification of the proteins was done under denaturing conditions. Figure 4 shows the result of SDS-PAGE for solubility determination.

Fig. 5.

a SDS-PAGE for solubility determination of the expressed r-FliC protein. Lane 1: pelleted suspension of clone; lane 2: non-induced clone; lane 3: induced clone; lane 4: sonicated supernatant; lane 5: sonicated pellet; M: stained protein ladder (Takara Bio USA, Inc. Code No. 3454A, 10-210 kDa). b SDS-PAGE of collected fractions of r-FliC purification. Lane 1: non-induced clone; lane 2: induced clone; lane 3: resuspended pellet; lane 4: flow through; lanes 5 and 6: wash fraction 1 and 2; lanes 7 to 23: eluates 1 to 17; M: stained protein ladder (Takara Bio USA, Inc. Code No. 3454A, 10–210 kDa)

During affinity purification, recombinant protein was successfully purified as observed by band corresponding to the molecular weight of the protein. Eluates 12–17 gave single band of 28.7 kDa, so these were pooled (Fig. 5). Purification was repeated five times, and all the eluates giving a single band corresponding to the molecular weight of r-FliC protein were pooled and concentrated using an amicon filter (Sigma-Aldrich, USA) of 15 kDa. The purified and concentrated recombinant protein was quantified using Total Protein Kit, Micro Lowry, Peterson’s Modification by Sigma-Aldrich, USA, and the concentration of protein was determined to be 3.4 mg/ mL.

Production of polyclonal antibodies

Outsourced polyclonal antibodies against concentrated r-FliC and formalin-inactivated whole cells of Rs0418 raised in rabbits and mice were received and used in further experiments.

Antibody titer

The optimum antibody titer for the produced anti-WC and anti-r-FliC antibodies was determined by indirect ELISA. The highest dilution of the antisera giving an O.D. value greater than the cut-off values of the respective normal rabbit and mice antisera was considered the optimum antibody titer for the respective antibodies produced against the whole cell as well as the recombinant protein. Cut-off value and optimum antibody titer for the respective antibody are summarized in Table 3.

Table 3.

Antibody titer for the synthesized anti-whole cell and anti-FliC antibodies

Protein/whole cell	Antibody	Cut-off value	Ab titer
Whole cell	Anti-WCrabbit	0.230	1:62000
	Anti-WCmice	0.215	1:32000
r-FliC	Anti- r-FliC rabbit	0.230	1:128000
	Anti- r-FliCmice	0.208	1:128000

ELISA optimization

Absorbance greater than the cut-off value was selected and antiserum dilution corresponding to that absorbance was further used in the experiments. The mean absorbance of the checkerboard titration involving serially diluted WC and rFliC protein with their corresponding rabbit/mice antiserum suspension is provided in the supplementary file S1 (Tables S2 and Table S3).

For rabbit/mice anti-r-WC antisera:

$$\mathit{Cut}\text{-}\mathit{off}\mathrm{value}\left(\mathrm{for},,\mathrm{rabbit},,\mathrm{antisera}\right)=0.317+5(0.061)=0.622$$

$$\mathit{Cut}\text{-}\mathit{off}\left(\mathrm{for},,\mathrm{mice},,\mathrm{antisera}\right)=0.3205+5(0.038)=0.510$$

Antiserum dilution of 1:5000 and 1:10000 respectively for rabbit and mice antisera were further used for evaluating the anti-r-WC antibodies using DAS-ELISA.

For rabbit/mice anti-r-FliC antisera:

$$\mathit{Cut}\text{-}\mathit{off}\left(\mathrm{for},,\mathrm{rabbit},,\mathrm{antisera}\right)=0.248+5(0.032)=0.408$$

$$\mathit{Cut}\text{-}\mathit{off}\left(\mathrm{for},,\mathrm{mice},,\mathrm{antisera}\right)=0.26+5(0.025)=0.385$$

Antiserum dilution of 1:10000 for rabbit anti-r-FliC and 1:5000 for mice anti-r-FliC antibodies were used for evaluating the anti-r-FliC antibodies in further ELISA.

Evaluation of antibodies

Anti-r-WC

Cut-off value of the absorbance of non-immunized sera was calculated and strains giving absorbance greater than the cut-off value were marked as positive for the reactivity with the anti-WC antibodies. Cut-off value of the negatives is:

$$\mathit{Cut}\text{-}\mathit{off}\mathrm{value}=0.18+5(0.010)=0.230$$

High degree of nonspecific reactions with non-bacterial wilt strains was observed in DAS-ELISA using anti-WC antibodies. Therefore, anti-WC antibodies were not chosen for agglutination studies.

Anti-r-FliC

In Western blot, all the RS strains reacted with the antibodies as bands corresponding to the molecular weight of flagellin (FliC) were observed, while no reactivity was seen for non-RS strains except for R. pickettii strain (Fig. 6 and Table 4). For indirect DAS-ELISA, cut-off value of the absorbance of non-immunized sera was calculated and strains giving absorbance greater than the cut-off value were marked as positive for the reactivity with the anti-FliC antibodies. Cut-off value of the negatives is:

$$\mathit{Cut}\text{-}\mathit{off}\mathrm{value}=0.19+5(0.009)=0.235$$

Fig. 6.

Western Blot results of washed cell suspensions (10⁶ CFU/ mL) of RPS and non RSSC strains. Lane 1: NAIMCC-B-00418; lane 2: DIBER115; lane 3: DIBER116; lane 4: DIBER117; lane 5: DIBER118; lane 6: DIBER119; lane 7: negative control-PBS; lane 8: positive control (recombinant protein rFliC); lane 9: R. pickettii; lane 10: Pseudomonas sp.; lane 11: E. coli; lane 12: Ochrobactrum sp.; lane M: low-range protein ladder

Table 4.

Evaluation results of synthesized anti-WC and anti-FliC antibodies

Culture	Source of the strain	DAS-ELISA using anti WC antibodies	Immunological tests with anti-flagellin antibodies
			Western Blotting	DAS-ELISA	Latex agglutination
					Washed cell suspension	Colony
DIBER 115	Tomato	+/+/+	+/+/+	+/+/+	+/+/+	+/+/+
DIBER 116	Tomato	+/+/+	+/+/+	+/−/−	+/−/+	+/+/+
DIBER 117	Tomato	+/+/+	+/+/+	+/+/+	+/+/+	+/+/+
DIBER 118	Tomato	+/+/+	+/+/+	+/+/+	+/+/+	+/+/+
DIBER 119	Tomato	+/+/+	+/+/+	+/+/+	+/+/+	+/+/+
NAIMCC-B-00418	NBAIMCC	+/+/+	+/+/+	+/−/+	+/−/−	+/+/+
R. picketti	ATCC 648	+/+/+	+/+/+	+/+/−	+/+/+	+/+/+
Pseudomonas sp.	DIBER114	+/+/+	−/−/−	−/−/−	−/−/−	−/−/−
E. coli	ATCC10536	+/+/+	−/−/−	−/−/−	−/−/−	−/−/−
Ochrabactrum sp.	DIBER102	+/+/+	−/−/−	−/−/−	−/−/−	−/−/−

Similar results were observed in indirect DAS-ELISA.

Latex agglutination test

Latex beads coated with anti-FliC antibodies agglutinated RPS, RS, and R. pickettii colony suspensions. No visible agglutination was observed with Pseudomonas spp., Ochrabactrum spp., and E. coli, which were routinely identified as background colonies (Fig. 7), whereas agglutination reactions with washed cell suspensions derived from liquid broth showed variable results similar to DAS-ELISA. Table 4 summarizes the evaluation results for anti-WC and anti-FliC antibodies.

Fig. 7.

Representative picture of agglutination using anti-r-FliC polyclonal antibody-coated latex particles. a NAIMCC-B-00418; b DIBER115; c DIBER116; d DIBER117; e DIBER118; f DIBER119; g R. pickettii; h Pseudomonas sp.; i E. coli; j Ochroboctrum sp.; k, l Cupriavidus paculus; m positive control (purified recombinant protein); n negative control (PBS)

Discussion

Various media such as 2,3,5-triphenyl tetrazolium chloride medium (TZC) and mSMSA are being employed for isolation of the bacterial wilt pathogens, RPS and RS [37, 47–49]. Along with pathogens, several saprophytes and secondary pathogens grow in the media. These nonspecific colonies account for more than 95% of colonies [50]. Most of them belong to the genus Pseudomonas, Ochroboctrum, Cupriavidus, and Burkholderia. In this work, the suspected colonies were identified by PCR targeting the exo-polygalacturonase gene (PehB) and the results were compared with commercially available DAS-ELISA. The PCR primer pairs Pbh2F (5′-AAT GCC AGC AAG TGG AGC ACC-3′) and Pbh1/2R (5′-TAC GAC AAC GTG AGG ATG AAC G-3′) were found theoretically specific and practically successful in identifying colonies of bacterial wilt pathogen.

During the identification of the bacterial wilt pathogen from the suspected plant samples, screening every colony by PCR or DAS-ELISA constitutes a complex tedious process. It involves colonies being subcultured in liquid media, a process requiring at least 24–48 h. This long procedure can be overcome by the introduction of a rapid immuno-based screening system. To our knowledge, such a rapid system has not been described for members of bacterial wilt pathogens in scholarly publications. In most of the immunological systems described, polyclonal antibodies (anti-WC) were directed toward the whole cell of the pathogen. This often results in non-specific antigen-antibody interactions with background organisms, which were also evident in this study [18, 51]. Henceforth, an effort was undertaken to determine the potential of the FliC protein encoded by the fliC gene as an immuno-target.

Flagella plays an important role in the virulence of bacterial wilt strains, RS and RPS. They mediate swimming motility required for host infection and plant colonization. Cells lacking flagella or deformed flagella either fail to infect the host or have reduced virulence [25, 52]. Recent experiments have proved that both twitching and swimming motility, respectively, mediated by type IV pili (TFP) and flagella are required for full virulence [26, 53]. The component proteins of these motility mediators can be excellent targets for antibodies as their in situ position is exocellular. Type IV pili component protein is conserved across many bacterial genera, while the amino acid sequences of flagellin are found to be specific for phylotypes I, II, and III of RSSC with 98–100% sequence similarity. Hence, considering the possibility of using the flagellin protein as an immuno-target, the sequence of the nucleotides encoding the 4th amino acid to 266th amino acid was cloned and expressed in recombinant form (r-FliC) and antibodies (anti-FliC) were raised against the purified recombinant protein.

The antibody evaluation was performed in two stages. In the first stage, the presence/absence of flagellin was evaluated in lysed cells by SDS-Western blotting. In the second stage, the whole cells of the strains were probed with anti-FliC antibodies by DAS-ELISA, to evaluate the presence of in-situ flagellin, and the results were compared with anti-WC mediated DAS-ELISA. In the third stage, latex beads coated with anti-FliC antibodies were evaluated for rapid identification of bacterial colonies and cells grown in broth culture.

Motility in RSSC is predominant when the concentration of cells in liquid broth reaches 10⁶–10⁸ CFU/mL [54]. Hence, all microcosms were harvested once the concentration reached 10⁷ and their counts were normalized to 5×10⁶ CFU/ mL for uniform analysis. In Western blot, flagellin protein was detected in all RPS strains and RS strain NAIMCC-B-00418. Among the non-bacterial wilt strains, the protein was detected in R. pickettii. In the genus Ralstonia, the flagellin protein was shared across RPS, RS, R. pickettii, R. mannitolilytica, and R. insidiosa as revealed by NCBI-BLAST analysis. This explains the positive reaction of antibodies with R. pickettii.

Results of DAS-ELISA using anti-fliC polyclonal antibodies were inconclusive about flagellin’s in situ presence on some batches of strains DIBER116 and NAIMCC-B-00418 (Table 4). In this species, motility is a quantitative trait; only 60% of cells exhibit flagella during the maximum motility phase [54]. Further, the minimum detection sensitivity of anti-fliC-mediated DAS-ELISA was 10⁵ CFU/mL. Colorimetric signals from cell counts below this were masked by background signals emerging from antibodies reacting with non-flagellin epitopes. Hence, the inconsistency in results may be due to the synergistic effect of the low sensitivity of DAS-ELISA and fewer cells with flagellin in some batches of broth culture (Table 4). This assertion can be true for inconclusive results observed in agglutination reactions of cells from broth cultures. However, cells derived from colonies were consistently agglutinated with the latex reagent (Table 4). Colony-derived cells are considered non-motile until exposed to the liquid broth for a few hours [52, 55]. However, this nonmotility is not due to the absence of flagella, but owing to deformed straight flagella [52]. Our tests with agglutination reagent endorse that flagellin is indeed present in cells affiliated with colonies of genus Ralstonia. The repeatable and reproducible agglutination results observed are due to the high cell numbers in colonies (>10⁸ CFU). Our results affirm the reliability of anti-FliC coated latex agglutinating reagent in identifying bacterial wilt strain.

The non-specific agglutination observed with R. pickettii and R. insidiosa will not influence the outcome of the final results, as this agglutination test will be used only for preliminary screening of colonies for subsequent confirmation by other molecular and immune tools. Further to note, R. pickettii, Ralstonia mannitolilytica (R. mannitolilytica) and Ralstonia insidiosa (R. insidiosa) are emerging nosocomial pathogens, which cause life-threatening bacteremia/septicemia in immunocompromised individuals and children [56–58]. Hence, the flagellin can be evaluated as a suitable immune target for detecting these pathogens.

The main aim of this study is to develop a proof of concept for immunodetection using flagellin as a target and to devise a prototype latex agglutinating reagent based on anti-flagellin antibodies for screening colonies. In a nutshell, two conclusions can be drawn from our results: (1) The flagellin protein can be used as a potential immune target for the identification of bacterial wilt pathogen; (2) anti-FliC polyclonal-coated latex beads can be used for preliminary screening of suspected RPS and RS colonies.

In conclusion, the present study illustrates the possible use of flagellin protein as an immune target for detecting RPS and RS cells. Antibodies raised against the flagellin protein coated over latex particles can be utilized for preliminary screening of the colonies obtained during the isolation of the bacterial wilt strains from the infected plant samples. Further, this proof of concept and the prototype need to be evaluated with bacterial wilt strains isolated from different hosts across various geographical regions.

Abbreviations

BSA

Bovine serum albumin

CFU

Colony-forming unit

CPG

Casamino acid-peptone-glucose

DAB

3,3′-Diaminobenzidine

DAS-ELISA

Double-antibody sandwich enzyme-linked immunosorbent assay

DVC

Direct viable count

EDAC

1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide

ELISA

Enzyme-linked immunosorbent assay

FliC

Flagellin

FP

Forward primer

His

Histidine

HRP

Horseradish peroxide

H202

Hydrogen peroxide

IgG

Immunoglobulin G

IPTG

Isopropyl β-d-1-thiogalactopyranoside

LAT

Latex agglutination test

MES

2-morpholino ethane sulfonic acid

mSMSA

Modified semi-selective medium South Africa

Ni-NTA

Nickel-nitrilotriacetic acid

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

PVDF

Ppolyvinylidene difluoride

RP

Reverse primer

RPS

Ralstonia pseudosolanacearum

RS

Ralstonia solanacearum

RSSC

Ralstonia solanacearum species complex

RPM

Revolutions per minute

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

WC

Whole cell

Author contribution

Study conceptualization: Shalini Bhatt, S. Merwyn P. Raj; methodology: Shalini Bhatt, S. Merwyn P. Raj, Neha Faridi; formal analysis and investigation: Shalini Bhatt, S. Merwyn P. Raj, Shraddha Mishra; writing—original draft preparation: Shalini Bhatt; writing—review and editing: Shalini Bhatt; supervision: Ankur Agarwal, S. Merwyn.

Declarations

Polyclonal antibodies used in the study were outsourced by Abgenex Private Limited, Bhubaneswar, India.

Competing interests

The authors declare no competing interests.