Review on Fusobacterium necrophorum Putative Candidates for Vaccine Development Strategies: Outer Membrane Proteins and Outer Membrane Vesicles

Mini Review

Fusobacterium necrophorum, a Gram-negative anaerobic bacillus, is an opportunistic pathogen isolated from oral cavities, gastrointestinal and genitourinary tracts of humans and animals [1,2]. The bacterium is associated with Lemierre’s syndrome affecting young and healthy individuals and necrotic infections in hepatic, abdominal, and respiratory organs in animals [3,4]. The bacteria are primary causative agents of liver abscess, foot rot, and calf diphtheria, sometimes in mixed infections with other bacteria such as Trueperella pyogenes and Porphyromonas species [5,6]. F. necrophorum has been classified into four biotypes: A, B, AB, and C [7]. Biotypes A and B are of veterinary importance and associated with cattle liver abscesses. Biotype A, subspecies necrophorum, and Biotype B, subspecies funduliforme, vary in cell morphology, colony characteristics, virulence capacity, virulence factors, 16s rRNA sequences, and DNA gyrase B subunit [7,8]. The subspecies necrophorum is more virulent and frequently isolated than the subspecies funduliforme, and is the primary causative agent in liver abscesses [9]. Annually, the rate of incidence of liver abscess in feedlot cattle is 10-20% [10].

Generally, the incidence is higher in cattle fed with high grainbased diets where the progression occurs from chronic acidosis and rumenitis to liver abscess [11]. The National Beef Quality Report 2016 has reported a liver abscess rate of 20.7%, causing liver condemnation [12]. Thus, this infection and search for a cure have been of economic importance in the feedlot industry. So far, the antibiotics such as tylosin-phosphate and virginiamycin have been approved as antimicrobial feed additives to control liver abscesses [13]. More generally, with the potential threat of antimicrobial resistance [14], new approaches and preventive strategies are needed, including vaccination. There is no successful vaccine against F. necrophorum, which has pointed out the need to investigate different vaccine candidates. This mini-review provides an overview of different vaccine candidates investigated in the past and other virulence factors that could be explored as a promising target for a vaccine.

Lipopolysaccharide (LPS) and Hemagglutinin

F. necrophorum has several virulence factors, including leukotoxin, lipopolysaccharides (LPS), hemolysin, hemagglutinin, outer membrane adhesins, extracellular proteases, and other enzymes. [1,9,15-17]. Currently, the focus has been looking into proteins as vaccine candidates, mainly surface proteins (LPS), membrane proteins (OMPs and OMVs), and secreted proteins (hemolysins).

F. necrophorum lipopolysaccharide and hemagglutinin plays a crucial role in disease pathogenesis. LPS can induce endothelial cell injury, toxic hepatitis and has anti-phagocytic property, thus indicating its role in eliciting an immune response [18,19]. Similarly, haemagglutinin of F. necrophorum has the ability to agglutinate chicken, human RBCs, and bovine platelets. Kanoe and Yamanaka reported that antisera specific for hemagglutinin reduced bacterial adherence and platelets aggregation, indicating the role of haemagglutinin in the bacterial attachment during the initial stages of abscess formation [20,21]. However, the protective functions of LPS and hemagglutinin have not been reported.

Exotoxins: Hemolysin and Leukotoxin

Leukotoxins are critical virulence factors involved in the pathogenesis of anaerobic infection. In F. necrophorum, leukotoxin plays a significant role in the pathogenesis of bovine liver abscesses. Its production is directly proportional to the severity of abscesses in cattle [22]. Leukotoxin induces cellular activation and apoptosis of bovine leukocytes for inflammation modulation [15]. Studies have demonstrated that recombinant leukotoxoid challenge in a mouse model induced good immune protection [1].

Similarly, another virulence factor that has a role in the pathogenesis of F. necrophorum is hemolysin. Iron acquisition is required for bacterial colonization and is critical for invasive infections such as liver abscess. Studies show that the production of hemolysin helps in successful colonization of F. necrophorum during infection by iron acquisition mechanism- a key role in pathogenesis [23].

The fact about the co-existence of F. necrophorum with T. pyogenes in liver abscesses in cattle is well documented in the literature. This symbiotic relation is mediated through pathogenic synergy between these two pathogens where T. pyogenes creates an anaerobic environment for the initial establishment of F. necrophorum. In turn, F. necrophorum produces leukotoxin to protect T. pyogenes from phagocytosis [24]. A study was conducted to examine the combination of leukotoxoids of F. necrophorum and bacterin of T. pyogenes [25]. However, the vaccine was only effective in low prevalence settings because of the biases related to the pen effect and antibiotics effect on recurrent infections in the studied group.

T. pyogenes is also found in mixed infections with other anaerobes such as Clostridium perfringens. A study conducted in a mouse model showed that pyolysin of T. pyogenes and phospholipase C of Clostridium perfringens, when used in combination, was effective in immuno-protection and reduced infections in mice challenged with T. pyogenes or Clostridium perfringens [26–28]. Based on the studies mentioned above, evaluating pyolysin and leukotoxin/ hemolysin combinations would be a possible combination to explore.

Outer Membrane Proteins (OMP)

OMPs of Gram-negative bacteria serve as a barrier for any toxic materials entering the bacterial cell. The OMPs are associated with host-bacteria interaction, adhesion, and induction of protective immunity [29]. Like other Gram-negative bacteria, the primary infection in F. necrophorum commences by attachment to the epithelial and endothelial cells of the liver and ruminal wall [17, 30] The attachment is facilitated by different adhesins and toxins, causing colonization and establishment in the liver parenchyma to cause an abscess [9,31]. Studies show that after the rumen entry, F. necrophorum enters through aggravated regions of the ruminal surface and enters portal circulation. Once trapped in the liver, it causes abscesses [11]. The OMPs of F. necrophorum facilitate direct interactions with the host and likely contain important constituents involved during infection, transmission, and survival, including putative vaccine candidates [17,32]. Therefore, a multivalent vaccine including OMPs and leukotoxin has been proposed in the past.

Previous studies identified adhesins that could have a potential role in the attachment of F. necrophorum to the host cells. Kumar et al., 2013 identified four adhesins (17kDa, 24kDa, 40kDa, and 74kDa) with high binding affinity to bovine adrenal gland endothelial cell line (EJG), in-vitro. Later, one of these adhesins was characterized as 42.4 kDa OMP FomA. [32]. FomA has been characterized in F. nucleatum and F. periodonticum as well. Based on the N-terminal sequences, FomA protein in F. necrophorum has 96% homology with FomA of F. nucleatum [32]. This protein is immunogenic and plays a role in the attachment of bacteria to the host cells [33]. The FomA protein in F. nucleatum is TLR2 and voltage-dependent porin [34]. FomA is involved in NF-kB, regulating genes responsible for host immune response. The activation of NF-kB is through TLR2 dependent fashion [35], thus indicating FomA could trigger host immune response. These studies suggest that FomA could be a potential vaccine candidate for controlling F. necrophorum infections. However, detailed research on the mechanism of action and receptors is necessary to understand the virulence mechanism of FomA in F. necrophorum.

FadA (13.6 kDa) is another membrane protein extensively studied for its role in the adhesion, invasion, and colonization of F. nucleatum in the host body [36,37]. FadA interacts with the vascular endothelial cadherin causing endothelial impermeability to allow the bacteria to cross through the tight junction of endothelium and proliferate to cause infections [38]. Moreover, many studies have suggested FadA adhesion is significant in inducing inflammation and suppressing host immunity by modulating the E-cadherin/ßcatenin pathway leading to colorectal cancer (CRC) [39].

OmpA and OmpH Family Protein

OmpA is studied for its membrane-associated pathogenicity and biofilm formation in Gram-negative bacteria [40,41]. OmpA family proteins are attached to peptidoglycan layer (via diaminopimelic acid) with the conserved domain at the C terminus. [42]. These proteins are known for their role at different stages during infections, such as interfering with the complement system, adhesion to the host cell, and mediating biofilm formation in several Gram-negative pathogens such as Pseudomonas, Escherichia coli, and Acinetobacter baumannii [43]. OmpA also helps in the intracellular survival of bacterial pathogens [44–47].

OmpH, a structural component of OMP in Gram-negative bacteria, is closely related to the family of porins [48]. Immune efficacy of OmpH based vaccines preparation has been studied in bacterial species such as Pasteurella multocida. The OmpH based vaccine has been used for protecting swamp buffaloes from hemorrhagic septicemia in South Asian countries. [49]. OmpH has other functions as well such as in Pseudomonas aeruginosa, it provides stability to the outer membrane through interaction with lipopolysaccharide [50].

Hence, exploring and identifying these different OMP family proteins in F. necrophorum and their role in adhesion and inducing protective immunity during liver abscesses in cattle could be exploited to study their protective function and vaccine potential.

Outer Membrane Vesicles (OMVs)

Outer membrane vesicles (OMVs) are spherical, membraneenclosed nanostructures released during bacterial growth. These nanostructures are composed of periplasmic proteins, toxins and sometimes genetic materials [51]. The OMVs play an important role in transporting toxins into the host cell and modulating the host immune reponse [51,52]. Therefore, OMVs, as efficient vaccine candidates, have received significant attention. In most cases, OMVs are shown to positively minimize infections in animal models [53,54]. The OMV based vaccine is successfully approved for Neisseria meningitidis and is currently the only licensed vaccine in humans [55,56]. OMV has also been studied as a targeted drug delivery vehicle and vaccine adjuvants [57,58]. OMVs are identified in Fusobacterium species, including F. nucleatum [59]. The OMVs of F. nucleatum have modulated the innate immune response by promoting inflammation [35,60]. Based on the proteomics analysis, OMPs serve as the significant components of OMVs.

Conclusion

OMPs and OMVs could be potential vaccine candidates to control F. necrophorum infections in cattle based on the virulence and immunomodulatory role observed in different bacterial species, including Fusobacterium species. Therefore, identifying and characterizing these OMPs and OMV components in F. necrophorum could widen the area to explore and develop an effective vaccine.

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