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Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism

Objectives To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. Results In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. Conclusions Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism. Supplementary Information The online version contains supplementary material available at 10.1007/s10529-021-03091-4.


Introduction
Clostridium botulinum is a Gram positive, obligate anaerobic and endospore forming bacterium that produces botulinum neurotoxins (Maikanov et al. 2019). It is the most potent neurotoxin so far known to humans hence it is categorized as a bio-warfare category 'A' agent. It has been divided into seven serotypes designated (A-G) which have similar structure but differ antigenically. Generally, serotypes A, B, E are associated with human clinical cases but rarely serotype F. Among the seven serotypes, A and B are the most poisonous serotypes. It causes neuroparalytic disease in humans and in animals called botulism (Lanci et al. 2019).There are three types of botulism; infant botulism, wound botulism and food borne botulism. Infant botulism is caused by swallowing of botulinum spores present in the environment. These spores are converted into vegetative cells followed by germination which then multiply and colonize in the large intestine leading to secretion of botulinum neurotoxin (BoNT) in situ (Galvis 2019). Similarly when the wounds are exposed to the environment, the C. botulinum spores enter into the wound followed by multiplication of the bacteria and produce the toxin. The toxin gets absorbed into the bloodstream and causes the wound botulism (Kuehn 2019). Both infant and wound botulism are caused by primary infection followed by secondary intoxication. But food borne botulism is caused by the intake of preformed BoNT containing food (Forss et al. 2012;Palma et al. 2019). The neurotoxin is absorbed and carried by the bloodstream to neuromuscular junctions where it blocks the release of acetylcholine and causes flaccid paralysis. So far there is no licensed vaccine commercially available for botulism. Similarly there is no rapid detection system available to diagnose botulism. The Centre for Disease Control and Prevention (CDC) used to provide the pentavalent toxoid as an investigational vaccine for select risk groups but it has been discontinued since 2011 due to the decrease in the antibody titer of certain serotypes as well as increasing side effects after the booster (Notice of CDC's discontinuation of investigational pentavalent (ABCDE) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins 2011). Several studies have been attempted for the development of recombinant protein based subunit vaccine using different domains of the heavy chain of different serotype (Przedpelski et al. 2018). Few studies have been directed towards the use of DNA based vaccines for different serotypes (Kim et al. 2019). Both the studies have revealed that, the binding domains are immunogenic and provide neutralization against the toxins but the risk of using the full domain of the toxin, reactivity of the recombinant proteins with other human proteins and other ethical concerns using the active toxin part for immunization. Immuno proteomic approach is one of the best tools available to study the host-pathogen interactions (McClean 2012). Researchers used combination of 2DE and immuno blotting with sera from infected animals and mass spectrometry to find out the immunogenic candidates (Pedersen et al. 2005). Similar approach has been widely used for the discovery of new biomarkers for vaccine development in cancer as well as infectious diseases (Forgber 2009;Martinez-Lopez 2008;Mendum 2009;Pitarch et al. 2006;Pitarch and Gil 2011). The secretory proteins play important roles in the pathogenesis of bacterial infection, represent the interphase of the bacterium-host interaction (Tsai-Tien Tseng 2009). In any pathogens the secretory proteins are exposed to the host immune system and are therefore the primary antigen targets of host immune response (Fulton and Twine 2013). Numerous novel secretory proteins produced by different bacteria such as Helicobacter pylori (Bernardini et al. 2007), Pseudomonas aeruginosa (Nouwens 2002) and Staphylococcus aureus (Ziebandt 2001) have been identified in secretome by using two-dimensional gel electrophoresis and mass spectrometry approach to develop the vaccine candidates as well as diagnostic system. Moreover the secretory immunogenic molecules are important for the development of diagnostics and passive immunotherapy (Vanitha Mariappan 2009). Identification of the secretory immunogenic proteins would be further step towards understanding the humoral immune response during C. botulinum infection. The aim of the present study was to identify and characterize immunogenic proteins from C. botulinum type B secretome by a combination of 2DE and immunoblotting assay using antisera raised in mice against the secretory proteins. Some of these immunogenic proteins have potential to be used as novel vaccine candidates or may be useful for enhancing the protective efficacy of a protectiveantigen-based vaccine. However, information regarding the secretome profile as well as immunogenic proteins of C. botulinum is not available in literature. The novel immunogenic proteins may be developed as alternative antigens for further study of botulism vaccine and diagnostics.

Materials and methods
Bacterial strains, growth and culture condition Indian isolates of C. botulinum type A SP08 (DRDE Accession No. DB120CLBO8), type B isolate SP11 (DB123CLB11), type E isolate SP01 (DB113CLBO1) and type F isolate SP04 (DB116CLBO4) were retrieved from the DRDE repository and further confirmed by polymerase chain reaction (PCR) using standard primers which were specific for BoNT/A, B, E and F (Lindstrom et al. 2001). Pure culture colonies were inoculated in serum vials containing 40 ml presterilized, de-aerated TPGY and CMM media (Hi Media, India). TPGY media is being used for enrichment and CMM media for maintenance the C. botulinum type B culture. These cultures were incubated at 37°C in an incubator shaker at 200 rpm for 5 days.

Preparation of extracellular proteins
Cultures were centrifuged at 80009g for 30 min at 4°C and collected the supernatant then it was filtered through 0.22 lm filters (Millipore, USA) to remove the suspended vegetative bacterial cells. Culture filtrates were concentrated using 10 kDa cutoff membranes (Millipore, USA) and precipitated using 10% Ice-cold trichloroacetic acid (Sigma USA) after that incubated on ice for 3 h. Precipitate were centrifuged at 80009g for 30 min at 4°C and washed the pellet three times with cold acetone and left the room for air dried (Hirose et al. 2000). Further, the pellets were resuspended in protein solubilization buffer (8 M urea, 2% CHAPS, and 2% carrier ampholytes pH 4-7) (Deatherage Kaiser et al. 2018). The resultant proteins were cleaned using protein clean up kit (Bio-Rad, USA). Protein concentration was estimated by Bradford method (SIGMA, USA) using Bovine serum albumin as a standard protein. Proteins was stored at -80°C for further use.

MALDI TOF-TOF analysis
Protein spots were excised from 2D gels and washed thrice with proteomic-grade deionized water, destained followed by reduction/alkylation and finally the proteins were digested with trypsin using the Montage In-Gel digestion kit (Millipore, USA) as described by the manufacturer's. The digested protein (0.8 ll) was mixed with 0.8 ll of the matrix solution (5 mg a-cyano-4-hydroxycinnamic acid in 80% acetonitrile and 0.1% trifluoroacetic acid [TFA]) thoroughly by pipetting for spectral scanning. This mixture (1 ll) was deposited onto the MALDI plate and proteins were identified by mass spectrometry (MS) using a matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF-TOF) mass spectrometer (Ultraflex III; Bruker Daltonics, Germany). MS mass spectra had been noted in the reflector positive mode using a laser (wavelength 355 nm) operated at a 200 Hz recurrence rate and at 2 kV accelerated voltage. The MS/MS mass spectra had been developed using data dependent acquisition method. In this method, 20 strongest precursors selected between 850 and 4000 Da and filtered through a signal-to-noise ratio more than 20 from one MS scan. Precursor ions had been selected by timed ion selector (TIS). Fragmentation had been done through collision induced dissociation (CID) method by air (collision gas) at 1 kV energy as well as recharge pressure threshold of 1.5e-006. MS and MS-MS spectra were obtained by accumulation of at least 1200 and 1600 laser shots, respectively. MS and MS/ MS data were analyzed and peak list were generated using the 4000 Series Explorer Software v. 3.5 (Applied Biosystem, USA). A peak intensity filter was used with no more than 50 peaks per 200 Da in the setting parameter of MASCOT search after acquisition. MS/MS peaks were selected based on a signal-tonoise ratio greater than 10 over a mass range of 60-20 Da below the precursor mass. MS and MS/MS data had been analyzed using Protein Pilot version 4.0 (Applied Biosystem) the MASCOT 2.0 search engine (Matrix Science, London). The peak list had been examined against 3,239,079 entries for Firmicutes at non-redundant protein sequence database of NCBI. Trypsin digestion with one missed cleavage, oxidation of methionine, carbamidomethylation of cystein and the peptide mass tolerance of 50 ppm for precursor ion and mass tolerance of ± 0.6 Da for fragment ion with ? 1 charge state were search parameters. MS/ MS was successfully identified proteins, MASCOT score more than 60 was accepted as significant (pvalue \ 0.05).

Immunization and generation of polyclonal antibody
The Animal experiments had been approved by the Institutional Animal Ethical Committee (IAEC) of the Defence Research & Development Establishment (DRDE), Gwalior, India. Polyclonal antibodies had been generated in BALB/c female mice via intra-peritoneal route against secretory proteins of C. botulinum type B, which were expressed in TPGY and CMM media. Active immunization (0, 14, 21, 28 days) schedule had been performed using 10 lg protein with complete Freund's adjuvant followed by three booster doses of 20, 30, 50 lg protein with incomplete Freund's adjuvant. Similarly polyclonal antibodies had been generated against C. botulinum type A/E and F.

Indirect ELISA
Indirect Enzyme-linked immunosorbent assay (ELISA) was performed to check the antibodies titre in mice against extracellular proteins of C. botulinum type B. Briefly, the 96 well ELISA plates were coated with 5 lg/ml extracellular proteins (CMM and TPGY medium) and incubated the plates overnight at 4°C. Then plate was washed three times with PBST (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4, 1.47 mM KH 2 PO 4 and 0.05% Tween-20, pH 7.4) followed by three times washing with phosphate buffer saline (PBS). Plates were blocked with 3% bovine serum albumin (BSA) at 37°C for 1 h. Plates were washed as mentioned previously followed by addition of 100 ll per well, two fold diluted primary antibody from 1:1000 to 20, 48,000 (mice sera against extracellular proteins expressed in TPGY and CMM media). Similarly the preimmunized serum incubated at 37°C for 1 h. Then plate was washed three times with PBST and three times with PBS. After washing, 100 ll per well of secondary antibody rabbit antimouse IgG-HRP (Dako, Denmark) 1:2000 dilution was added and incubated at 37°C for 1 h. Then the plate was washed as described previously. Finally the antigen and antibody interactions was developed using 2,2 0 -azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt solution (100 ll/well) containing H 2 O 2 and incubated at 37°C for 30 min. Absorbance was measured at 410 nm using an ELISA plate reader (Biotek, USA). Similarly ELISA assay was done for secretory proteins antiserum against C. botulinum type A/E and F.
Western blot analysis 2-DE gels were transferred onto PVDF membrane in semi-dry condition using transfer apparatus (Bio-Rad, USA) at 100 V for 1 h at 4°C in tris glycine transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol). The membranes were blocked with 5% skimmed milk and incubated at 4°C overnight. Further membranes were washed three times with PBST followed by PBS twice with 10 min interval. Membranes were probed with polyclonal antibodies (1:30,000 dilutions) generated in mice against the extracellular proteins expressed in TYGY and CMM media. Unimmunized mice serum (1:1000) was used as negative control for immunoblotting. The membranes were washed as washed previously and incubated for 1 h with 1:30,000 dilution Anti-Mouse IgG (Fc specific)-Peroxidase antibody. After washing with PBST, the membrane was developed by DAB substrate (SIGMA, USA). Similarly 2D-gel of type B secretory proteins were probed with other C. botulinum types A, E, F secretory proteins anti serum at dilutions of 1:30,000 and 1:1000.

Results and discussion
Identification of predominantly expressed protein spots in TPGY and CMM media Proteomics has been become a technique for identification of proteins involved during infection and invasion by the pathogens (Das et al. 2019). Bacterial secreted proteins perform key role in the interaction between the bacterial cell and the host environment (Siegrist et al. 2015). Thus secretome of a pathogen are hypothesized to comprise the components of effective vaccines. Secretome exploration facilitates identification of novel vaccine antigen and overall helps in progress of discovery of vaccine (McManus 2020). In the present work, predominantly expressed protein spots were successfully identified by mass spectrometry. Most of the proteins were over expressed in TPGY medium compared to CMM medium. The list of predominantly expressed proteins in both TPGY and CMM media are being shown in Fig. 1     Spot numbers refer to the numbers on the 2-DE gels shown in Fig. 1a, b Spot numbers refer to Fig. 1a and b Protein functions were predicted by UniProt Knowledgebase (Swiss-Prot and TrEMBL) Presence of signal peptide predicted by SignalP 3.0 Non-classical i.e. non-signal peptide triggered protein secretion by SecretomeP. For each input sequence the server predicts the possibility of non-classical secretion. For bacteria, four scores are generated by the SecretomeP server for each input sequence. The determining score is the 'SecP score', for which a value above 0.5 indicates possible secretion. Values in parenthesis are SecP scores Number of predicted transmembrane helices in proteins using TMHMM Server v. 2.0 Prediction of lipoproteins and signal peptides using LipoP 1.0 Grand average of hydropathy Prediction of protein sub cellular localization using PSORT version 2.0 hydrolase, family 18, ATP-dependent Clp protease proteolytic subunit, flagellin and M24 family metallopeptidase were predominant proteins. Elevated levels of these proteins had been also seen in C. tyrobutyricum secretome (Ma et al. 2015). Clostripain (Shimizu et al. 2002), ThiC (Martinez-Gomez and Downs 2008), peptidaseT (Strauch et al. 1983), Glycerol dehydrogenase (Monniot et al. 2012), glyceraldehyde-3-phosphate dehydrogenase and acetyl-CoA acetyltransferase (Prasad et al. 2013;Sengupta et al. 2010) predominant proteins had been also identified by other researchers in other microorganism using proteomic approach.

Bioinformatics analysis
Clostridium botulinum type B secretary proteins were grouped into different cellular functions and shown in Fig. 2. Identified secretary proteins are involved in carbohydrate 13.7%, amino acid and lipid 11.76% metabolism, protease activity (13.7%), metal binding, posttranslational modifications, flagellar synthesis and maintenance (5.88%), host cell interaction and transcription process (3.9%), protein translation, carbohydrate binding, endonuclease activity, stress response, DNA replication and recombination (1.9%) and unknown function (7.8%). Similar trends were also observed in B. anthracis secretome in which 17.8% proteins are involved in energy metabolism, 10.9% for protein synthesis, 8.7% cellular structure and 13% proteins functions are unknown (Walz et al. 2007). Hypothetical protein CLOSPO_00563, putative cell surface protein and cell surface protein were predicted as cell wall-associated protein, bacterial Iglike domain, secreted protease, flagellar hook-associated protein 2 and thermolysin metallopeptidase were identified as extracellular proteins, HNH endonuclease and M24 family metallopeptidase found as cytoplasmic membrane proteins and 35-cytoplasmic proteins and 7-proteins of unknown localization were predicted using PSORTb software. Utmost of the C. botulinum type B secretome proteins were situated at Fig. 2 Graphical representation of the cellular functions of secretome of C. botulinum type B Indian isolate SP11: the majority of the proteins were involved in transport and metabolism of carbohydrate, amino acid and lipid, protease activity, post-translational modification and chaperones the cytoplasmic region and did not contain signal sequence predicted by Signal P. Utmost of the enzymes involved in metabolism and energy production were present in the C. botulinum secretome. Similar results were also observed in Shigella flexneri (Liao et al. 2003). 15.68% of proteins were predicted by Signal P to be secreted in the classical Sec pathway, which is characterized by the presence of a signal peptide (Shah et al. 2009). But, three proteins were shown to possess the cleavage site for signal peptidase I (SpI) and one protein for signal peptide II (SpII) are being shown in Table 1. Four proteins have transmembrane helices as predicted by TMHMM, representing an extra cytoplasmic but membraneassociated location. Less than 50% of the proteins were predictably secreted by either non-classical pathways as indicated by the presence of a signal peptide (Signal P) or non-signal peptide triggered protein secretion (Dwivedi et al. 2015).
Immunogenic proteins of C. botulinum type B Immunoproteomics is an extension of proteomics, which permits specific elucidation of proteins based on immunoreactivity (Fulton et al. 2019). In procedure of immunoproteomics, 2-D gel blots are probed with antiserum collected from host post infection. This development has bypassed the extensive process of testing immunoreactivity and henceforth vastly enhanced the vaccine discovery by directly permitting the identification of those novel immunogenic proteins which evoke immune system. Secretary proteins of human disease causing agents are of special attention; in particular, these proteins play a role in the preliminary phase of pathogenesis when they arrive direct contact with host tissues (Kennedy 2018). If pathogenesis can be halted efficiently at the preliminary stage, infection can be paused. Secretary proteins can induce defensive immunity and also provoke an immune response which will be of specific attention for the development of vaccine/diagnostic markers. To determine the immunogenic proteins of C. botulinum secretome, antiserum of secretary proteins (1:30,000 dilution) were used as primary antibody and serum from unimmunized mice were used as control. Seventeen and ten immunogenic proteins were identified in TPGY and CMM media respectively, results are being shown in Figs. 3, 4 and tabulated in Tables 2, 3 Table 2. Three technical replicates were organized in independent experiments; these were very similar, and one of them was revealed Table 4. These proteins may be the best bioproducts for development of vaccine candidates or diagnostic markers against the infant and wound botulism. Cross reactive immunogenic proteins were also identified using secretary protein antisera of C. botulinum type A/E & F (supplement Fig. 1) Table 3. Three technical replicates were organized in independent experiments; these were very similar, and one of them was revealed perfringens endo-beta-N-acetylglucosaminidase, SagA protein, phospholipase C, translation elongation factor, acetyl-CoA acetyltransferase, fructose-bisphosphate aldolase and ornithine carbamoyl transferase were reported as immunogenic proteins and the recombinant ornithine carbamoyl transferase extended the death time of mice challenged with C. perfringens (Sengupta et al. 2010). Phosphoglycerate kinase was successfully expressed by prokaryotic expression system and the recombinant protein showed favorable immunogenicity in mice (He et al. 2013) Peptidase protein had been known as immunogenic protein in anopheles culicifacies or gambiae for epitope design using immuno-informatics models (Jakhar et al. 2019). Metallopeptidase was identified as immunogenic proteins in various cancer  including breast cancer (Chantada-Vázquez et al. 2020). Glycosyl hydrolase had been screened as immunogenic proteins in Clostridium chauvoei and identified as cell surface associated proteins by immunoproteomic method (Coral 2009). Phenyl alanyl-tRNA synthetase had been identified as immunogenic protein using immunoproteomic analysis of protective response obtained with subunit and commercial vaccine against glasser's disease caused by haemophilus parasuis (Li et al. 2017). Immunogenic proteins which identified in different microorganism involved in different disease had also been explore in C. botulinum type B secretome these proteins may be used as vaccine candidate as well as diagnostic marker against the botulism. In this work, there were several highly plentiful proteins in the 2-DE gels which did not obtain as immunoreactive.
Hence it is rational to recommend that the immunogenic proteins ones may be the noteworthy vaccine candidate as well diagnostic marker against botulism. The protective efficiencies of immunoreactive proteins identified either by alone or in altered combinations remain to be determined in further works.

Conclusions
The present study explored the secretome proteins of C. botulinum type B and identified the predominant immunogenic proteins using 2-DE immuno proteomic approach. To the best of our knowledge this is the first study to explore the secretome proteomics as well as identification of immuno dominant proteins of C. botulinum. In this study we have identified 17 immuno dominant in TPGY media and 10 proteins in CMM media. Five immunogenic proteins were common in both the media. Apart from that, cross reactive study of proteins had been also carried out to find out the cross reactive proteins in among C. botulinum types A, B, E and F. C. botulinum types A, B and E secretome showed the cross reactivity but C. botulinum type F did not show cross reactivity with the secretome of C. botulinum type B. Cross reactive proteins and common immunogenic proteins will be further validated in future study for their potential to be used as a vaccine/diagnostic candidates against infant and wound botulism. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.