Recombinant FimH, a fimbrial tip adhesin of Vibrio parahaemolyticus, elicits mixed T helper cell response and confers protection against Vibrio parahaemolyticus challenge in murine model
Introduction
Infectious diseases have remained a global threat to public health since the dawn of agriculture. The threat has become worse owing to climate change driven by global warming, development of antibiotic resistance, escalated import and export of foodstuff, migration, tourism and bioterrorism. Bacterial infections top the list of infectious diseases. Of these, emerging food borne gastrointestinal infections have raised grave concerns about food safety issues globally. The leading cause of foodborne “gastroenteritis” caused by Vibrios, also known as vibriosis, is V. parahaemolyticus, a halophilic, facultative-anaerobic, oxidase-positive, non-spore forming bacterium (Letchumanan et al., 2014; Farto et al., 2019). The bacterium is found as a free-living organism in marine and estuarine waters, sediments or associated with marine zooplanktons. These are one of the major contagions that infect humans as well as other organisms; as they are responsible for epizootic outbreak among marine fish population and infect human hosts that are sea-food consumers (Letchumanan et al., 2014). Approximately 45,000 of 80,000 vibriosis cases reported every year in the USA are caused due to V. parahaemolyticus infection (CDC report, 2021; https://www.cdc.gov/vibrio/index.html). Consumption of raw sea-food/water or contaminated food is primarily responsible for vibriosis as observed from most of the countries bordering oceans along coastal, estuarine, brackish and even fresh water worldwide (Esteves et al., 2015; Soto-Rodriguez et al., 2015; Ghenem et al., 2017). The hightened outbreak of V. parahaemolyticus mediated enteritis has been noticed during warm season, possibly due to the elevated sea surface temperature attributed to global warming, which is conducive to the bacterial proliferation (Vezzulli et al., 2016).
V. parahaemolyticus infection in broad range of edible crustaceans, mainly shrimp results in acute necrotizing hepatopancreatitis (AHPND) (Soto-Rodriguez et al., 2015; Taha and Mohamed, 2020). Consumption of contaminated seafood results in gastrointestinal discomfort in humans and the infected individuals suffer from diarrhoea, cramps, nausea, vomiting, high fever, and dysentery with bloody or mucoid stools (Shimohata and Takahashi, 2010). The exposure of open wound to contaminated seafood and sea water also contributes to wound infection and also causes life-threatening septicemia in immuno-compromised patients (Johnson et al., 1984; Guillod et al., 2019). Thus, the bacterium extensively impacts both marine and human population, and severely affects the farmers involved in aquaculture/mariculture industry.
Widespread use of antibiotics as feed additives or for immersion bath for marine fishes meant for human consumption to control infection, and for the treatment of vibriosis has led to accumulation of antibiotics in the food chain, and development of antibiotics resistant strains, ultimately resulting in failure to control V. parahaemolyticus infection with antibiotics (Lee et al., 2018). Thus, emergence of multiple antibiotic resistant V. parahaemolyticus strains poses significant global threat towards seafood safety and public health. Further, V. parahaemolyticus establishes infection in the host through colonization and biofilm formation, which are orchestrated by several bacterial proteins. The biofilm enclosed bacteria are generally antibiotic and phagocytosis resistance, thus even if the strains by itself are not resistant to antibiotics, the antibiotics are rendered ineffective (Sharma et al., 2019).
Vaccination against several bacterial and viral infections has reduced the disease burden and death caused due to these infections, and has been proven to be the most effective approach in controlling the infectious diseases. Use of vaccines have also resulted in reduction in antibiotics usage and thereby reducing antimicrobial resistance development. Heat-killed cultures or live attenuated cultures are being currently used as commercial vaccine against vibriosis in white shrimp (Powell et al., 2011). These vaccines are not very efficacious as these formulations generate non-focussed immune response due to the presence of large number of bacterial antigens, in addition to posing the risk to persons involved in culturing the live bacteria. Thus, a subunit vaccine targetting a virulence factor that would intercept the establishment of infection in the host is desirable.
V. parahaemolyticus causes virulence through quorum sensing mediated cell-cell communication to form biofilm using an array of proteins involved at various stages of infection. Several outer membrane proteins involved in adhesion are flagella with swimming and swarming motility, pili and the metalloenzymes like enolase secreted on the extracellular surface play critical role during the initial stages of establishment of the bacterium in the host cells (Park et al., 2004; Okada et al., 2009; Wu et al., 2019).
The surface exposed virulent fimbrial/afimbrial adhesive organelles such as long whipped flagella, thinner hair like fimbriae or pili, toxins and outer membrane lipoproteins are assembled and exported to the surface as either end products or their integral components through a number of secretion pathways (Park et al., 2004; Okada et al., 2009; Costa et al., 2015).
The components of secretion system including several pili or fimbriae have been found to be highly virulent and have been identified as potential protective antigen against pathogenic bacteria responsible for a numerous human diseases (Sharma et al., 2011; Luiz et al., 2015; Forsyth et al., 2020). Attempts are being made to utilize different fimbrial adhesins as potential human vaccines towards urinary tract infection caused by uropathogenic E. coli (Duan et al., 2020; Forsyth et al., 2020 and references therein).
Type 1 fimbriae have been thoroughly investigated in uropathogens (Connell et al., 1996; Langermann et al., 1997; Hung et al., 2002; Klemm and Schembri, 2004; Wright et al., 2007). Approximately 100–500 fimbriae are present on the extracellular surface as long tubular, non-flagellar, peritrichous hair like adhesive structures. The fimbria comprises of heteropolymeric assemblies of major subunit FimA (2000 copies) and minor subunits FimF, FimG, FimH encoded by the fimAICDFGHK operon (Schwan, 2011). Presence of chaperone FimC and the usher subunit FimD is essential for the export and assembly of fimbrial subunits into a fimbria. The FimH of chaperone-usher pathway is the minor type 1 fimbrial tip adhesin. Crystal strucutre of FimC-FimH chaperone-adhesin complex from uropathogenic E. coli has been elucidated (Choudhury et al., 1999). FimH contains N-terminal mannose binding lectin/carbohydrate domain (1–156 amino acid), short intrapeptide loop (157–160 amino acid) to connect C-terminal pilin domain (161–279 aa) for integrating the tipped adhesin into the fimbrial structural assembly (Schembri et al., 2000; Munera et al., 2008; Busch and Waksman, 2012). Adhesion of type 1 fimbriae to the host cell is mediated through binding of the terminal adhesin FimH lectin domain to D-mannosylated proteins present on the eukaryotic host cell surface (Bouckaert et al., 2005; Tomašić et al., 2014). Interaction of type 1 fimbriae with the host cell is blocked by mannose or mannose derived FimH antagonists (Sokurenko et al., 1997; Klemm and Schembri, 2004; Mydock-McGrane et al., 2016).
A number of fimbrial adhesins have beern reported to be involved in the pathogenesis of different bacteria as they play an essential role in adhesion, and colonization. Few of these include FimH, PapG, Lpf1, F4, F5, F6, F18, F41,K99,CFA/I, S pilli and Dra adhesin of E. coli (Ascón et al., 1998; Klemm and Schembri, 2004 and references therein; Yang et al., 2011; Pereira et al., 2015), MrpH of P. mirabilis (Asadi et al., 2018); Salmonella fimbrial component SafD, FimA, FimA-mC3d2, and FimA-mC3d3 (Musa et al., 2014); FimA of Porphyromonas gingivalis (Choi et al., 2016); Fim2, Fim3, FimD of Bordetella pertussis (Guevara et al., 2016); Bordetella bronchiseptica Fim (Scheller et al., 2015). Immunization with some of these fimbrial proteins have been found to confer protective immunity against different bacteria namely Yersinia pestis, uropathogenic E. coli, Salmonella enterica (Chalton et al., 2006; Yang et al., 2011; Musa et al., 2014), albeit to different degrees.
It was noted that the fimH null mutants of E. coli showed relatively poor survival and neutrophil influx in comparison to the wild type E. coli in mouse urinary tract infection model, clearly indicating the role of FimH in bacterial establishment in vivo (Connell et al., 1996; for review please see Klemm and Schembri, 2004). Further, immunization of mice with enteropathogenic E. coli FimH, its fragments or its recombinant fusion with other surface proteins inhibited (>99 %) bacterial colonization in bladder (Langermann et al., 1997; Luna-Pineda et al., 2016). Polyclonal antiserum generated against the E. coli FimH inhibited binding of the bacterium with human bladder cells in vitro (Langermann et al., 1997). Immunization with E. coli FimCH fusion protein also conferred protection (∼ 75 %) in monkeys against bacterial challenge (Langermann et al., 2000). Thus, vaccine potential of uropathogenic E. coli FimH is well established. FimH adhesin of type 1 fimbriae has also been found to be present in most of the virulent strains of V. parahaemolyticus, and is critical for bacterial adherence and invasion (Chao et al., 2010). The V. parahaemolyticus FimH can therefore effectively be used as vaccine candidates against the bacterium. The present study reports the immunogenic and protective potential of heterologously expressed recombinant FimH, a minor type 1 fimbrial tip adhesin against V. parahaemolyticus in mice model.
Section snippets
Bacterial strains, vectors, chemicals
For recombinant cloning and expression studies, Escherichia coli DH5α and BL21 (λDE3) pLysS cells, respectively obtained from GIBCO-URL, USA were used. V. parahaemolyticus (MTCC#451) was procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. All reagents used in the study were of molecular biology grade and were obtained from Sigma Aldrich Chemical Co., St. Louis, MO, USA, SD fine chemicals, Mumbai India or Sisco Research Laboratories,
V. parahaemolyticus fimH gene sequence
The sequence of type 1 fimbrial adhesin FimH sequence from V. parahaemolyticus (VpFimH) with protein id as KKY42922.1 (Geninfo identifier # GI: 82108904) was identified by BLASTX analysis of E. coli fimH gene sequence (Accession no. JX847135.1) against V. parahaemlyticus genome sequence (GenBank: LCUL01000008.1). The encoded protein of 301 residues gives rise to a mature VpFimH of 279 amino acid residues, after the removal of signal peptide of 22 amino acid residues.
Multiple sequence alignment
Discussion
Continued use of antibiotics has led to the development of multi-drug resistant V. parahaemolyticus, the major Vibrio species responsible for vibriosis in humans. Due to this, the mariculture industry suffers major losses as significant percentage of cargos are rejected for the fear of transmission of bacteria from the infected marine species to humans. Due to the development of drug resistance, prophylactic measures such as vaccination remains the method of choice to control V. parahaemolyticus
CRediT authorship contribution statement
Aparna Dixit, Lalit C. Garg, Devapriya Choudhury: Conception and design of the study.
Sweta Karan: Execution of experiment and acquisition of data.
Sweta Karan, Lalit C. Garg, Aparna Dixit: Analysis and/or interpretation of data.
Sweta Karan, Aparna Dixit: Preparation of the original manuscript draft.
Sweta Karan, Lalit C. Garg, Aparna Dixit, Devapriya Choudhury: review & editing of the manuscript and approval of the final version of the manuscript.
Aparna Dixit, Devapriya Choudhury, Lalit C. Garg:
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Ethical statement
The study does not involve human subjects. Animal studies were approved by the Institutional Animal Ethics Committee (IAEC) of the Jawaharlal Nehru University, New Delhi and all animal procedures were performed in compliance with the IAEC guidelines.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors acknowledge intramural support from the Jawaharlal Nehru University, New Delhi. SK thanks the Indian Council of Medical Research, New Delhi for research fellowship.
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