Computer-aided genomic data analysis of drug-resistant Neisseria gonorrhoeae for the Identification of alternative therapeutic targets

Abstract

Neisseria gonorrhoeae is an emerging multidrug resistance pathogen that causes sexually transmitted infections in men and women. The N. gonorrhoeae has demonstrated an emerging antimicrobial resistance against reported antibiotics, hence fetching the attention of researchers to address this problem. The present in-silico study aimed to find putative novel drug and vaccine targets against N. gonorrhoeae infection by the application of bioinformatics approaches. Core genes set of 69 N. gonorrhoeae strains was acquired from complete genome sequences. The essential and non-homologous metabolic pathway proteins of N. gonorrhoeae were identified. Moreover, different bioinformatics databases were used for the downstream analysis. The DrugBank database scanning identified 12 novel drug targets in the prioritized list. They were preferred as drug targets against this bacterium. A viable vaccine is unavailable so far against N. gonorrhoeae infection. In the current study, two outer-membrane proteins were prioritized as vaccine candidates via reverse vaccinology approach. The top lead B and T-cells overlapped epitopes were utilized to generate a chimeric vaccine construct combined with immune-modulating adjuvants, linkers, and PADRE sequences. The top ranked prioritized vaccine construct (V7) showed stable molecular interaction with human immune cell receptors as inferred during the molecular docking and MD simulation analyses. Considerable response for immune cells was interpreted by in-silico immune studies. Additional tentative validation is required to ensure the effectiveness of the prioritized vaccine construct against N. gonorrhoeae infection. The identified proteins can be used for further rational drug and vaccine designing to develop potential therapeutic entities against the multi-drug resistant N. gonorrhoeae.

Keywords: Neisseria gonorrhoeae; chimaric vaccine; epitope; multi-drug resistant; novel drug target.

Figure 1

Flowchart of subtractive proteomics and reverse vaccinology approach followed for the potential drug and vaccine identifications.

Figure 2

Tertiary strucutre validation results for the designed construct (V7). (A) Ramachandran plot, (B) ProSA web graph.

Figure 3

MD simulation plots of V7-TLR4 docked complex. (A) Variations in temperature during the simulation. During 100 ps, the system temperature reached 300 K and showed little variations. (B) During simulation time, the radius of gyration vaccine construct is stable in its compact form. (C) Pressure variations during simulation. During 100 ps, the average pressure is 2.73710 bar, according to the pressure plot. (D) RMSD plot of backbone. The RMSD graph demonstrates that the RMSD of the protein backbone reaches ~0.4 nm and is generally maintained, indicating that the vaccine construct has few structural aberrations. (E) RMSF (Root Mean Square Fluctuation) plot. The RMSF plot of side chains illustrates the regions in peaks that have a lot of flexibility. (F) The expected density is 1017.19 kg/m3 on average.

Figure 4

Immune simulation profile of vaccine construct (V7) in two injections by C-immsim server. (A) High primary response of IgM and IgG antibodies by antigen exposure. (B) B-cells immune reaction where active B-cells are produced in constant amount after second injection. (C) Total B-cell antibodies immune response. (D) Cytotoxic T-cells immune reaction with high active T-cell production. (E) Helper T-cell response with significant increase at second injection. (F) Total Helper T-cells immune response. (G) Natural Killer immune cells profile. (H) Simpson Index D graph representing cytokines and Interleukin levels where “D” indicates the danger level.

Figure 5

In-silico restriction cloning of final vaccine construct (V7) into the E. coli pET28a (+) expression vector where red color shows the cloned vaccine construct.