Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/1574-6976.12063

S-layers: principles and applications

ATP-binding cassette (ABC) transporters are widespread among living organisms and comprise one of the largest protein families. For example, components of ABC transporters are encoded by approximately 5% of the Escherichia coli and Bacillus subtilis genomes ([47][1], [80][2]). These transporters are

https://iai.asm.org/content/iai/72/12/6757.full.pdf

ATP-binding cassette transporters are targets for the development of antibacterial vaccines and therapies.

Vaxign is the first web-based vaccine design system that predicts vaccine targets based on genome sequences using the strategy of reverse vaccinology. Predicted features in the Vaxign pipeline include protein subcellular location, transmembrane helices, adhesin probability, conservation to human and/or mouse proteins, sequence exclusion from genome(s) of nonpathogenic strain(s), and epitope binding to MHC class I and class II. The precomputed Vaxign database contains prediction of vaccine targets for <path id="x30" d="M241 635q53 0 94 -28.5t63.5 -76t33.5 -102.5t11 -116q0 -58 -11 -112.5t-34 -103.5t-63.5 -78.5t-94.5 -29.5t-95 28t-64.5 75t-34.5 102.5t-11 118.5q0 58 11.5 112.5t34.5 103t64.5 78t95.5 29.5zM238 602q-32 0 -55.5 -25t-35.5 -68t-17.5 -91t-5.5 -105
q0 -76 10 -138.5t37 -107.5t69 -45q32 0 55.5 25t35.5 68.5t17.5 91.5t5.5 105t-5.5 105.5t-18 92t-36 68t-56.5 24.5z" /> genomes. Vaxign also performs dynamic vaccine target prediction based on input sequences. To demonstrate the utility of this program, the vaccine candidates against uropathogenic Escherichia coli (UPEC) were predicted using Vaxign and compared with various experimental studies. Our results indicate that Vaxign is an accurate and efficient vaccine design program.

/pdf/vaxign-the-first-web-based-vaccine-design-program-for-4gl92no3jg.pdf

Vaxign: The First Web-Based Vaccine Design Program for Reverse Vaccinology and Applications for Vaccine Development

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown. The complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

/pdf/cereulide-synthetase-gene-cluster-from-emetic-bacillus-4dwdc1add6.pdf

Cereulide synthetase gene cluster from emetic Bacillus cereus : Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Vaccination has played a significant role in controlling and eliminating life-threatening infectious diseases throughout the world, and yet currently licensed vaccines represent only the tip of the iceberg in terms of controlling human pathogens. However, as we discuss in this Review, the arrival of the genome era has revolutionized vaccine development and catalyzed a shift from conventional culture-based approaches to genome-based vaccinology. The availability of complete bacterial genomes has led to the development and application of high-throughput analyses that enable rapid targeted identification of novel vaccine antigens. Furthermore, structural vaccinology is emerging as a powerful tool for the rational design or modification of vaccine antigens to improve their immunogenicity and safety.

/pdf/vaccinology-in-the-genome-era-3e1rrjc6hl.pdf

Vaccinology in the genome era

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from ∼30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

https://iai.asm.org/content/iai/70/12/6817.full.pdf

Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening.

Many members of the alphavirus family are important human or veterinary pathogens. These viruses are extremely similar in molecular architecture yet differ in host range and in the pathological consequences of infection. Despite marked structural conservation, the various species are inefficient in eliciting immunological cross protection. Previously(l) we described a systematic approach for selection of epitope-cassettes of the E2 envelope of the alphavirus family that induce virus species specific protective immunity. This approach was tested and proved successful(2–5) with two viruses - Sindbis (SIN) and Semliki Forest (SF) - which are very remote phylogenetically. Vaccination was performed with recombinant peptide cassettes fused to a bacterial protein carrier (β-galactosidase) formulated with different adjuvants. In order to create a vaccine compatible with human use, we designed several live vector vaccines for presentation of the protective epitope cassette. Attenuated salmonella vaccine vector which can potentially allow presentation of many different cassettes and thus generation of a pan-alphavirus vaccine, proved to be inefficient. On the other hand the live attenuated viral vaccine based on a benign alphavirus such as SIN vector, appears to be promising. The principle of such vaccines is substitution of E2 epitopes of SIN virus vector with parallel protective epitopes from a different alphavirus. This system should have the advantage of presentation of specific virus epitopes in the context of the SIN alphavirus backbone which in turn could provide cross protective immunity on the basis of multiple common antigen determinants.

Y. Inbar

Papers

Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening.

Alphavirus Hybrid Virion Vaccines