Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins

AbstractThe radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the ‘interstitial’ layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug

Environmentally Responsible Bioengineering for Spore Surface Expression of <em>Helicobacter pylori </em>Antigen

The development of genetic technologies and bioengineering are creating an increasing number of genetically engineered microorganisms with new traits for diverse industrial applications such as vaccines, drugs and pollutant degraders. However, the destiny of genetically engineered bacterial spores released into the environment as long-life organisms has remained a big environmental challenge. In this study, an environmentally responsible and sustainable gene technology solution based on the concept of thymine starvation is successfully applied for cloning and expression of a Helicobacter pylori antigen on Bacillus subtilis spore surface. As an example, a recombinant Bacillus subtilis strain A1.13 has been created from a gene fusion of the corresponding N-terminal fragment of spore coat protein CotB in B. subtilis and the entire urease subunit A (UreA) in H. pylori and the fusion showed a high stability of spore surface expression. The outcomes can open the door for developing highly safe spore vectored vaccines against this kind of pathogen and contributing to reduced potential risks of genetically engineered microorganisms released in the environment

Immunological Studies Of Dna (Pjwvacii) And Surface Display (R-Stvacii) Vaccine Candidates Expressing A Synthetic Multiepitope Gene Of Mycobacterium Tuberculosis In A Prime Boost Strategy Using A Mouse Model [QR82.M95 N839 2008 f rb].

Tuberculosis (TB) pada manusia adalah disebabkan oleh patogen bakteria Mycobacterium tuberculosis dan merupakan salah satu penyakit utama di dunia. Tuberculosis (TB) in humans is caused by the bacterial pathogen Mycobacterium tuberculosis and is still one of thea major health problems worldwide

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

<p>Abstract</p> <p>Background</p> <p>Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in <it>B. thuringiensis </it>using a LysM-containing peptidoglycan hydrolase, endo-<it>β</it>-<it>N</it>-acetylglucosaminidase (Mbg), as the anchor protein.</p> <p>Results</p> <p>Homology searching in the <it>B. thuringiensis </it>YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of <it>cry3Aa</it>. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM₁, or LysM₂were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM₁was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of <it>B. thuringiensis </it>target cells using the optimum (Mbgn)₂anchor, without radically altering its catalytic activity.</p> <p>Conclusion</p> <p>Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of <it>B. thuringiensis </it>cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system.</p

Oral vaccination of fish against vibriosis using spore-display technology

Publisher Copyright: Copyright © 2022 Gonçalves, Santos, Coutinho, Pedrosa, Curado, Machado, Costas, Bonneville, Serrano, Carvalho, Díaz-Rosales, Oliva-Teles, Couto and Serra.Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.publishersversionpublishe

Implementation of phenotypic methods to characterize guaiacol-producing strains of Alicyclobacillus’ genus

The food industry has recurrently been facing several quality-associated issues that harm brand reputation and consumer confidence. Bacteria of the genus Alicyclobacillus have been identified as one of the responsible microorganisms for spoilage events in fruit and vegetable-based products. Alicyclobacillus are endospore-forming bacteria that can produce off-flavours and unpleasant odours. These bacteria represent an economical threat to the food industry since their endospores can survive commercial pasteurization processes, as well as germinate and grow in acidic food environments (e.g., fruit juices and tomato pulps). Consumers usually describe the off-flavours and off-odours of the affected products as medicinal, disinfectant-like, antiseptic, smoky, and hammy. The phenolic compounds associated with these taste and smell alterations have been identified as guaiacol and two halophenols: 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). As guaiacol is considered the main offensive off-flavour, studying guaiacol-producing Alicyclobacillus strains remains relevant. Therefore, implementing a platform to phenotypically characterize these strains will set in place the tools to collect the necessary data to advance the knowledge on this genus. For this reason, in this work, a group of phenotypical methods were tested and optimized with the aim of implementing them in the laboratory where this research was performed. Thus providing a base protocol that can be applied to Alicyclobacillus strains that are isolated in the laboratory itself or from spoilage events pointed out by food companies. These methods allowed to elucidate microbial behaviours related to guaiacol production in different Alicyclobacillus strains and isolates from Portuguese food products.A indústria alimentar tem enfrentado, repetidamente, várias questões associadas à qualidade, que prejudicam a reputação da marca e a confiança dos consumidores. As bactérias do género Alicyclobacillus foram identificadas como um dos microrganismos responsáveis por eventos de deterioração em produtos à base de fruta e vegetais. Os Alicyclobacillus são bactérias formadoras de endósporos que podem produzir sabores e odores desagradáveis. Estas bactérias representam uma ameaça económica para a indústria alimentar, uma vez que os seus endósporos conseguem sobreviver a processos de pasteurização comercial, bem como germinar e crescer em ambientes alimentares acídicos (por exemplo, sumos de fruta e polpas de tomate). Os consumidores descrevem, geralmente, os sabores e odores estranhos dos produtos afetados como se cheirassem ou soubessem a medicamentos, desinfetantes, antisséticos, produtos fumados e afiambrados. Os compostos fenólicos associados a estas alterações de sabor e odor foram identificados como o guaiacol e dois halofenóis: 2,6-dibromofenol (2,6-DBP) e 2,6-diclorofenol (2,6-DCP). Como o guaiacol é considerado o principal composto desagradável, o estudo de estirpes de Alicyclobacillus que produzem guaiacol continua a ser relevante. Por conseguinte, a implementação de uma plataforma para caracterizar fenotipicamente estas estirpes permitirá criar as ferramentas para recolher os dados necessários para fazer avançar os conhecimentos sobre este género. Por esta razão, neste trabalho, foi testado e otimizado um grupo de métodos fenotípicos com o objetivo de os implementar no laboratório onde esta investigação foi realizada, fornecendo, assim, um protocolo base que pode ser aplicado às estirpes de Alicyclobacillus que são isoladas no próprio laboratório ou provenientes de eventos de deterioração apontados pelas empresas alimentares. Estes métodos permitiram elucidar o comportamento microbiano relacionado com a produção de guaiacol em diferentes estirpes de Alicyclobacillus e isolados de produtos alimentares portugueses

Immunological studies of dna (pjwvacll) and surface display (r-stvacll) vaccine candidates expressing a synthetic mul tiepitope gene of mycobacterium tuberculosis in a prime boost strategy using a mouse model

Tuberculosis (TB) in humans is caused by the bacterial pathogen Mycobacterium tuberculosis and is still a major health problem worldwide. The only TB vaccine currently available is an attenuated strain of M. bovis; Bacille Calmette Guerin (BCG). BCG demonstrated variable protective efficacies ranging from 0 to 80% in different field trials. BCG is effective at preventing childhood manifestation of TB but it does not prevent the most prevalent disease which is pulmonary TB in adults. DNA vaccination is an important new approach to the control of infectious agents and induces both humoral and cellular immune responses. Two previously constructed vaccine candidates, pJWVacll and r-STVacll were used in this study employing a prime-boost strategy. The naked DNA vaccine, pJWVacll was given intramuscularly to mice whilst the surface display vaccine, r-STVacll was given orally. Splenocytes from the vaccinated mice were tested for various immunological tests. The results showed that splenocytes from immunized mice were found to proliferate more aggressively when stimulated with the antigen (lnak-nVacll). Flow cytometric intracellular cytokine analysis of splenocytes from vaccinated mice also showed that both CD4+ and CD8+ T cells produce IL-2 and IFN-y following stimulation with the antigens. In the prime-boost approach, the study showed that mice primed with the naked DNA vaccine, pJWVacll and boosted with the surface display vaccine, r-STVacll is the best strategy to stimulate immune response in mice. As a conclusion, the data obtained from this study suggest that DNA vaccination in combination with surface display vaccination using prime-boost approach provides a new strategy for developing a candidate vaccine against TB

Environmentally Responsible Bioengineering for Spore Surface Expression of Helicobacter pylori Antigen

The development of genetic technologies and bioengineering are creating an increasing number of genetically engineered microorganisms with new traits for diverse industrial applications such as vaccines, drugs and pollutant degraders. However, the destiny of genetically engineered bacterial spores released into the environment as long-life organisms has remained a big environmental challenge. In this study, an environmentally responsible and sustainable gene technology solution based on the concept of thymine starvation is successfully applied for cloning and expression of a Helicobacter pylori antigen on Bacillus subtilis spore surface. As an example, a recombinant Bacillus subtilis strain A1.13 has been created from a gene fusion of the corresponding N-terminal fragment of spore coat protein CotB in B. subtilis and the entire urease subunit A (UreA) in H. pylori and the fusion showed a high stability of spore surface expression. The outcomes can open the door for developing highly safe spore vectored vaccines against this kind of pathogen and contributing to reduced potential risks of genetically engineered microorganisms released in the environment