Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to SalmoneUla typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cellsurface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cellsurface-exposed regions of porins tend to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria.

Background: Single nucleotide substitutions (SNS) in genetic codon are of prime importance due to their ability to alter an amino acid sequence as a result. Given the nature of genetic code, any SNS is expected to change the protein sequence randomly into any of the 64 possible codons. In this paper, we present a theoretical analysis of how single nucleotide substitutions in genetic codon may affect resulting amino acid residue and what is the most likely amino acid that will get selected as a result. Methods: A probability matrix was developed showing possible changes and routes likely being followed as a result of base substitution mutation causing changes at the translational level for the amino acid being encoded. Results: We observe that in event of single base pair substitution in a given amino acid; a chosen set of amino acids is theoretically more probable to be resulted suggesting a directional rather than a random change. This study also indicates that for a given amino acid coded by a number of synonymous codons, all synonymous codons will result into same list of amino acids in case of all possible SNS at three positions. Conclusion: The present work has resulted into development of a theoretical probability matrix which can be used to predict changes in amino acid residues in a protein sequence caused by single base substitutions.

The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs). We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively. In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing. A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S. typhimurium showed 88 and 98% identity with OmpC from E. coli and S. typhi, respectively. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane ␤ strands. The MAbs reacting with surface-exposed loops showed no cross-reaction with E. coli OmpC, whose sequence has diverged extensively from that of S. typhi and (probably) S. typhimurium OmpC only in regions of the externally exposed loops. In contrast, MAbs reacting with transmembrane ␤ strands, whose sequence is strongly conserved, showed strong cross-reaction with E. coli OmpC. These results show that comparison with the E. coli OmpF structure predicts the folding pattern of S. typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops. The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagnostic assays and vaccine development are discussed.

47The recent outbreak of severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 48 (SARS-CoV-2) causing coronavirus disease (covid19) has posed a great threat to human 49 health. Previous outbreaks of SARS-CoV and Middle East respiratory Syndrome CoV (MERS-50 CoV) from the same CoV family had posed similar threat to human health and economic 51 growth. To date, not even a single drug specific to any of these CoVs has been developed nor 52 any anti-viral vaccine is available for the treatment of diseases caused by CoVs. Subunits 53 present in spike glycoproteins of SARS-CoV and SARS-CoV-2 are involved in binding to 54 human ACE2 Receptor which is the primary method of viral invasion. As it has been observed 55 in the previous studies that there are very minor differences in the spike glycoproteins of 56 SARS-CoV and SARS-CoV-2. SARS-CoV-2 has an additional furin cleavage site that makes it 57 different from SARS-CoV (Walls et al., 2020). In this study, we have analyzed spike 58 glycoproteins of SARS-CoV-2 and SARS-CoV phylogenetically and subjected them to selection 59 pressure analysis. Selection pressure analysis has revealed some important sites in SARS-60 CoV-2 and SARS-CoV spike glycoproteins that might be involved in their pathogenicity. 61 Further, we have developed a potential multi-epitope vaccine candidate against SARS-CoV-62 2 by analyzing its interactions with HLA-B*15:03 subtype. This vaccine consists of multiple 63 T-helper (TH) cells, B-cells, and Cytotoxic T-cells (CTL) epitopes joined by linkers and an 64 adjuvant to increase its immunogenicity. Conservation of selected epitopes in SARS, MERS, 65 and human hosts, suggests that the designed vaccine could provide cross-protection. The 66 vaccine is designed in silico by following a reverse vaccinology method acknowledging its 67 antigenicity, immunogenicity, toxicity, and allergenicity. The vaccine candidate that we have 68 designed as a result of this work shows promising result indicating its potential capability of 69 simulating an immune response.70