**4. Features of the twenty virulent proteins**

All the virulent proteins from different serovars of *Salmonella* are discussed here, with their characteristic features along with a note on their existing vaccine potential.

**SptP** is one of the most important SPI-1 Type III Secretion System (T3SS) effector proteins which facilitates the bacterial translocation and survival into the host non-phagocytic cells by inhibition of the extracellular-regulated kinase (ERK) mitogen-activated protein kinase (MAP) pathways [46]. It requires SicP as a chaperone protein for its secretion and stabilization [46]. Moreover, SptP is directly responsible for the reversal of the actin cytoskeletal changes in the host cells by acting as a GTPase-activating protein (GAP) for Rac-1 and Cdc42. In fact, the efficacy of *sptP* deletion mutation of *S.* Enteriditis has been shown to be effective for live attenuated vaccine (LAV) in chickens [47].

**SsaQ** is a member of FliN/YscQ/Spa33/HrcQ family of both T3SS and flagellum proteins [48]. The gene *ssaQ* is encoded in the *ssaMVNOPQ* operon within the SPI-2 and transcribes to two products namely, SsaQL of 322 residues and SsaQS of 106 residues. SsaQS acts as a chaperone-like protein for SsaQL and optimize its function. SsaQ interact with SsaK and SsaN to form the C-ring complex, which have a crucial role in secretion by acting as a cytoplasmic sorting platform at the base of T3SS as well as rotation and direction switching of the flagella [49].



*Computational Identification of the Plausible Molecular Vaccine Candidates… DOI: http://dx.doi.org/10.5772/intechopen.95856*

**Table 2.**

*Basic screening of plausible vaccine candidates.*

**49**

**SpaO** is a major invasion factor of *S. enterica spp.* and the core component of the sorting platform in *S.* Typhimurium. SpaO is comprised of 303 residues of two translated products with SpaOS (the shorter product) encompassing the last 101 amino acids of SpaOL (full length protein) [50]. It is a highly conserved element in T3SS that shares similarity with limited residues with flagellar C-ring substructure [51]. In fact, SpaO, along with H1a, has been suggested to be promising new vaccine candidates to prevent typhoid fever caused by *S.* Paratyphi A infection [52].

**PrgH** is a 55 kDa protein encoded within *prgHIJK* operon in the SPI-1. All the genes of *prg* operon are essential for the formation of T3SS needle complex (NC) and known to share sequence similarity with the flagellar protein, FliF [53]. PrgH inserts in the inner membrane by its hydrophobic domain where it forms the MS-ring of the flagellar basal body as well as provides the structural foundation required for *prgK* oligomerization for further assembly of the NC [53].

**SicA** is a wide acting chaperone protein (18 KDa) which aids in the secretion process of all T3SS proteins through the invasion of host cells. Accordingly, it is encoded upstream to the *Sip/SspABCD* operon in SPI-1. SipB and SipC proteins are responsible for the translocon formation in the host cell membrane to facilitate the injection of Type III effector proteins into the host cell to manipulate it [54]. Moreover, SicA is essential for the expression of the most virulence genes that encode T3SS effector proteins and is identified as a co-regulator with InvF for *SigDE* and *SptP* [55].

**HilA** is a member of the OmpR/ToxR regulator protein family and the central activator of SPI-1 genes, belonging to T3SS. The *hilA* gene is encoded within SPI-1 and is the key factor in SPI-1-T3SS regulation, starting from the expression of downstream genes *sicA* and *invF* to ultimate regulation of the effector genes *sipA* and *sipB* [56]. The upregulation of *hilA* results in the high expression of all genes encoded within the SPI-1 which are necessary for the invasion of epithelial cells. Moreover, the expression of *hilA* is controlled by many different activators and suppressors in response to specific environmental changes during invasion of the host cells, such as, temperature, bile, fatty acids, osmolarity, pH, oxygen concentrations and growth state [57]. Additionally, certain studies considered HilA as a promising drug target to inhibit the activity of T3SS without affecting the growth of *Salmonella* [58].

**SiiE** is the largest protein in *Salmonella* proteome*,* with the size of 595 kDa. It consists of 53 repetitive bacterial immunoglobulin domains, each containing several conserved residues [59]. The protein helps to contact the host cell membrane and positions the SPI T3SS, to initiate the translocation of effector proteins. A study states that *Salmonella* SiiE-mediated entry of enterocytes via the apical route requires transmembrane mucin MUC1 [60]. Moreover, it is shown that, *siiE* is required for the prevention of efficient humoral immune response against the pathogen and it induces the high tires of specific *Salmonella*-specific IgG [61].

**PrgK** is a component from the inner membrane of *Salmonella* SPI-1 T3SS basal body*,* in its N-terminus. It possess the canonical lipoproteins which acts as anchor for the hydrophilic proteins onto the surface of the bacterial cell membranes [62]. In addition, C-terminus of PrgK is found in the cytoplasm which confirms that the protein traverses the inner membrane. A study observed reduced fever in swine which were vaccinated with *prgK* gene attenuated *S.* Typhimurium in comparison with mock-vaccinated swine [63].

**SscA** is a chaperone protein of about 18 KDa size. It is an independent α-helical protein, that consists of eight α-helices and repeated large tetratricopeptide domain from 36 to 137 amino acids. SscA is a virulence factor which encodes the chaperonin of SseC and the translocon is involved during the adaptation and survival to

#### *Computational Identification of the Plausible Molecular Vaccine Candidates… DOI: http://dx.doi.org/10.5772/intechopen.95856*

desiccation [64]. A huge effect of the gene expression level of *sscA,* has been noted on treatment of the samples with ciprofloxacin [65].

**SsaJ** is a core encoding component of the T3SS. It is required for SpvB, in-order to induce the actin depolymerization, especially inside the human macrophages. *Salmonella* depends on SsaJ effector protein as it prevents the interaction of NADPH oxidase subunit Cytb558 with the *Salmonella* containing vesicle (SCV) thereby helping to avoid the oxidative burst [66]. An *in vivo* study, conducted with the peptide of SsaJ, however, showed its inability to provide antigen specific immunity when compared with the other chosen peptides [67].

**SctC** is a layer of outer membrane anchor forming two distinct outer rings namely, OR1 and OR2. It is homologous to a protein of Type II Secretion System (T2SS) which requires pilotin lipoprotein for its optimal assembly and localization [68]. SctC serves as a midline between the inner and outer membrane, with evidence showing that the translocation of foreign antigens can induce potent immune response against pathogens [69].

**SsrB** is responsible for the survival and replication of *Salmonella* in the host cell and plays an important role in the transcription of multiple genes of SPI-2. SsrB has been claimed as one of the most important factors for *Salmonella*'s virulence by the fact that, a mutated *ssrB*, resulted in reduced ability of colonization on comparing with the wild type [70]. Moreover, one alteration in the gene *ssrB*, preferentially silencing the acquired DNA, can have a high contribution towards low transcription in the virulence factors of *Salmonella* [71].

**BcfD** is a fimbrial protein and part of the operon Bcf [72]. BcfD is a surface molecule, which helps in the adherence through specific receptors on the host cell. This step of adhesion is considered to be an important course during infection as it allows bacteria to initiate the colonization [73]. A research shows that the knockout of this gene influenced in the low adhesion capacity of *Salmonella* to the host cell [74].

**InvE**, encoded within SPI-1, is a protein located in the cell membrane and said to be essential for the translocation of *Salmonella* proteins into the host cells by regulating the functions of the Sip protein translocases [75]. An investigation of finding the region of InvE, as the T3SS regulator protein, indicates that it may have two functional domains which are responsible for regulating the secretion of translocases as N-terminal secretion signal and C-terminal regulatory domain [76]. An *in-vivo* study conducted with the BALB/c mice, showed less pathogenicity when it is injected with the mutated *invE* gene *Salmonella* on comparing with the wild strain [77].

**SipB** is one of the effector proteins of SPI-1 T3SS which facilitates the entry of *Salmonella* into the host cell. It is also called as an invasion protein as it initiates the bacterial entry process. It forms a complex along with the SipC to assemble into plasma membrane-integral structure which mediates the effectors delivery [78]. It also affects the membrane fluidity and bacterial osmotolerance and hence a small alteration of this gene will pave a huge way to prevent *Salmonella* entry into the host cell [79]. In fact, a study evaluating the effect of *sipB* deleted mutants, showed significant decrease in the virulence of *sipB* mutants when compared with the wildtype strains [80].

**SsaD** is an important cellular component which is responsible for the virulence of *Salmonella.* It is found to be in the transmembrane of the bacteria. The gene *ssaD* encodes for the proteins related to the basal body, cytoplasmic rings and export apparatus and it is also involved in the ATPase complex, regulation and translocation of T3SS [81]. A study shows that there is an important defect in the intercellular survival with the mutant *ssaD* strains on comparing with the wild-type *Salmonella* [82].

**DD95\_23890** refers to the computationally predicted protein, mapping to the autotransporter adhesin BigA protein. The BigA protein in *Salmonella* has recently been identified via automated genome annotation in 2015. Thus, studies on this protein has been scarce. Inferring from its homolog in *Brucella*, the cell surface BigA protein promotes adhesion of bacteria on host epithelial cells [83, 84]. The adhesive properties of the BigA protein can be established by binding onto the cell adhesion molecules on the host epithelial cellular surface [85].

**DD95\_21695** maps to the RING-type E3 ubiquitin transferase (SspH2) protein. The SspH2 protein aids in *Salmonella* pathogenicity by conferring anti-inflammatory properties, hence delaying the host immune response in reaction to bacterial invasion [86]. Moreover, the ability of SspH2 to ubiquitinate host NOD1 protein, through an essential interaction with host SGT1 protein, can result in NOD1 mediated IL-8 secretion in host [87].

**DD95\_16310** maps to the *Salmonella* TorS histidine kinase sensor. The TorS protein comprises the two-component systems along with the TorT response regulator [88]. Upon stimulation by Trimethylamine-N-oxide, TorS, along with TorT, carry out osmoregulation and protect the cellular proteins against low-pH induced denaturation in urea [88].

**DD95\_14775** refers to the putative transcriptional regulator *marT\_1* in *Salmonella*. The MarT protein mainly regulates the expression of MisL autotransporter protein, which is a fibronectin-binding protein that is involved in the cell adhesive properties of *Salmonella* [89]. Moreover, MarT has also been reported to regulate the expression of genes related to bacterial biofilm formation [90].

## **5. Initial screening of the candidate proteins**

All the twenty proteins were screened to ascertain their potential for plausible candidatures as vaccines (**Table 2**). Proteins were localized in extracellular matrix (3), cytoplasm (7), cytoplasmic membrane (3) and outer membranes (2), besides some of them being predicted with unknown cellular location (5). Of these, surface/outer membrane proteins and vesicles have been deployed for prospective vaccinations against bacterial pathogens [91–94]. Again, extracellular proteins have been potentiated as drugs for prospects against disease management, *albeit,* in a different scenario [95, 96]. Our results predict the proteins namely, SptP, SipB, SsaD, PrgK and TorS to be potentially antigenic except InvE, SctC and SspH2. Notably, the five proteins of unknown location, namely, BcfD, SsaJ, BigA, SiiE, and MarT\_1 are all potentially antigenic. Of the two signal peptides BcfD and SctC, the latter was predicted to be non-antigenic while SsaJ and PrgK belongs to another category of signal peptides (lipoproteins) with good antigenic potential. Of these, SsaJ has been predicted with two transmembrane (TM) spanning helices and poses itself a good candidate for vaccines. Other candidates with more TM helices are BigA (5), SiiE (3) and TorS (3). Furthermore, a BLASTp alignment of these 20 proteins revealed SptP and SspH2 to have 40–50% similarity for 101 and 106 hits, respectively, against human counterparts, thereby completely ruling out their candidature as potential vaccines.

#### **6. Selection of potential vaccine candidates**

The 20 top ranked proteins were further screened for B cell epitopes. Therein, InvE, SsrB, SicA, and SscA were omitted from being considered as vaccine candidates due to the absence of predicted epitopes that fall within the normal range of peptide length (**Table 3**). Moreover, in allergenicity prediction, HilA,


*Computational Identification of the Plausible Molecular Vaccine Candidates… DOI: http://dx.doi.org/10.5772/intechopen.95856*


*Only predicted peptides of length between 15 to 25 amino acids were selected [97]. SiiE protein were omitted from prediction because of its overly huge sequence.*

#### **Table 3.**

*BepiPred v2.0 prediction of linear B-cell epitopes.*


#### **Table 4.**

*Allergenicity assessment through different predictive tools. Potential allergens are in bold case.*

BcfD, SicA, BigA, SiiE, and MarT\_1 were predicted to be potential allergens (**Table 4**), and thus, were excluded from consideration as well. Hence, we report SptP, SsaQ, SpaO, PrgH, SipB, SsaD, SctC, SsaJ, PrgK, SspH2, and TorS to be potentially utilized as B cell epitopes. Moreover, in discontinuous B cell epitope prediction, the localizations of the highly antigenic regions were illustrated in 3D (**Figure 2**). For successful vaccination, these regions should be prioritized and retained as much as possible due to their important roles in antigenicity.

*Computational Identification of the Plausible Molecular Vaccine Candidates… DOI: http://dx.doi.org/10.5772/intechopen.95856*

**Figure 2.**

*DiscoTope v2.0 prediction of discontinuous B-cell epitopes. The residues are colored according to their respective DiscoTope scores (red: high, white: threshold of −3.7 and blue: low).*

### **7. Potential druggable proteins**

Besides potential vaccine candidates, we have conducted predictions on the druggability and druggable sites of the 20 top ranked proteins which have their 3D crystallized structures available in PDB. Eventually, the localization of the top ranked druggable pockets of SptP, SipB, SctC, SpaO, SsrB, PrgK, PrgH, and SiiE were illustrated in 3D (**Figure 3**). This can help future research in structure-aided drug discovery, by designing drugs specific for the druggable pockets to suppress the virulence of *Salmonella*.

#### **8. Conclusions**

The study depicted here essentially delineates a schematic approach of shortlisting the most probable virulent proteins as potential vaccine and/or drug candidates from the proteome of *Salmonella* spp. It starts with the building of the theoretical PIN comprising the known and predicted virulent proteins followed by the graph theoretical parametric analyses for identifying a probable set of them. These were further screened through different essential tools enabling the prediction of cellular localisation, signal peptides, transmembrane helices, antigenicity, epitopes, allergenicity and molecular crevices besides comparing with any human homologs. A thorough analysis revealed SsaJ and PrgK to come to the forefront among those already known to be virulent. PrgK even has nice druggable pocket to be targeted through potential drugs. Our approach can pave the way for screening such effective molecular vaccines and/or drug targets for such pathogens. Newer candidates, however, could be unraveled through other effective methods.

#### **Figure 3.**

*P2Rank predicted druggable pockets colored in light green. For SpaO, chain A is in blue, while B is in red. Residues contributing to druggability are tabulated.*

#### **Acknowledgements**

The authors wish to acknowledge the support of Sunway University, Malaysia for the provision of computational facilities.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Computational Identification of the Plausible Molecular Vaccine Candidates… DOI: http://dx.doi.org/10.5772/intechopen.95856*
