Immunogenic Properties of Lactobacillus plantarum Producing Surface-Displayed Mycobacterium tuberculosis Antigens

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guerin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum. The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo. The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guerin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigenspecific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice

Omega-3 and omega-6 PUFAs induce the same GPR120-mediated signalling events, but with different kinetics and intensity in Caco-2 cells

Background Omega-3 PUFAs are known to have anti-inflammatory properties, and different mechanisms are involved. GPR120 is a G-protein coupled receptor that has recently received attention because of its anti-inflammatory signalling properties after binding omega-3 PUFAs. However, both omega-3 and omega-6 PUFAs are natural GPR120 ligands. The aim of this study was to study possible differences in GPR120-mediated signalling events after treatment with different long-chain PUFAs in intestinal epithelial cells. We also investigated possible GPR120-mediated anti-inflammatory effects of different long-chain PUFAs that may be relevant in the understanding of how dietary PUFAs influence inflammatory responses in inflammatory diseases such as IBD. Methods We used Caco-2 cells as a model system to study GPR120-mediated signalling events because we found this cell line to express GPR120, but not GPR40, another plasma membrane receptor for medium- and long chain fatty acids. Increase in cytosolic Ca2+concentration, activation of MAP kinase ERK1/2 and the inhibition of IL-1β induced NF-κB activity were studied to reveal potential differences in the activation of GPR120 by the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 PUFA arachidonic acid (AA). Results We found that EPA, DHA and AA enhanced the cytosolic concentration of the second messenger Ca2+ with the same efficiency, but with different kinetics. Both omega-3 and omega-6 PUFAs activated MAP kinase ERK1/2, but differences regarding kinetics and intensity were also observed in this pathway. ERK1/2 activation was shown to be dependent upon EGFR and Raf-1. We further investigated the ability of EPA, DHA and AA to inhibit NF-κB activity in Caco-2 cells. All PUFAs tested were able to inhibit IL-1β induced breakdown of IκBα after binding to GPR120, but with different potency. Conclusions Our results show that EPA, DHA and AA elicit the same signalling events, but with different kinetics and efficiency through GPR120 in Caco-2 cells. We show, for the first time, that both omega-3 and omega-6 PUFAs inhibit NF-κB activation in intestinal epithelial cells. Our results may be important for understanding how dietary PUFAs influence inflammatory processes relevant in delineating effects of PUFAs in the treatment of IBD

EP receptor expression in human intestinal epithelium and localization relative to the stem cell zone of the crypts.

There is substantial evidence for PGE2 affecting intestinal epithelial proliferation. PGE2 is also reported to be involved in the regulation of growth and differentiation in adult stem cells, both effects mediated by binding to EP-receptors. We have used the Lgr5 as a marker to scrutinize EP-receptor and COX expression in human intestinal epithelial cells with focus on the stem cell area of the crypts. Normal tissue from ileum and colon, but also duodenal biopsies from patients with untreated celiac disease, were investigated by immunohistochemistry and RT-PCR. The combination of fresh flash-frozen tissue and laser microdissection made it possible to isolate RNA from the epithelial cell layer, only. In the small intestine, Lgr5 labels cells are in the +4 position, while in the colon, Lgr5 positive cells are localized to the crypt bottoms. Epithelial crypt cells of normal small intestine expressed neither EP-receptor mRNA nor COX1/2. However, crypt cells in tissue from patients with untreated celiac disease expressed EP2/4 receptor and COX1 mRNA. In the colon, the situation was different. Epithelial crypt cells from normal colon were found to express EP2/4 receptor and COX1/2 transcripts. Thus, there are distinct differences between normal human small intestine and colon with regard to expression of EP2/4 receptors and COX1/2. In normal colon tissue, PGE2-mediated signaling through EP-receptors 2/4 could be involved in regulation of growth and differentiation of the epithelium, while the lack of EP-receptor expression in the small intestinal tissue exclude the possibility of a direct effect of PGE2 on the crypt epithelial cells

A representative silver stained SDS-PAGE gel showing proteins from cell free culture supernatant of <i>M. capsulatus</i> (Bath) culture supernatants from early-, mid, and late exponential growth.

<p>Precision Plus Protein Dual Color Standard; molecular weights are indicated in kDa. Proteins precipitated from 10 ml of culture supernatant were applied to the gel.</p

Computational and Experimental Analysis of the Secretome of <i>Methylococcus capsulatus</i> (Bath)

<div><p>The Gram-negative methanotroph <i>Methylococcus capsulatus</i> (Bath) was recently demonstrated to abrogate inflammation in a murine model of inflammatory bowel disease, suggesting interactions with cells involved in maintaining mucosal homeostasis and emphasizing the importance of understanding the many properties of <i>M. capsulatus</i>. Secreted proteins determine how bacteria may interact with their environment, and a comprehensive knowledge of such proteins is therefore vital to understand bacterial physiology and behavior. The aim of this study was to systematically analyze protein secretion in <i>M. capsulatus</i> (Bath) by identifying the secretion systems present and the respective secreted substrates. Computational analysis revealed that in addition to previously recognized type II secretion systems and a type VII secretion system, a type Vb (two-partner) secretion system and putative type I secretion systems are present in <i>M. capsulatus</i> (Bath). <i>In silico</i> analysis suggests that the diverse secretion systems in <i>M.capsulatus</i> transport proteins likely to be involved in adhesion, colonization, nutrient acquisition and homeostasis maintenance. Results of the computational analysis was verified and extended by an experimental approach showing that in addition an uncharacterized protein and putative moonlighting proteins are released to the medium during exponential growth of <i>M. capsulatus</i> (Bath).</p></div

Proteins identified in the culture supernatant of <i>M. capsulatus</i> (Bath).

<p>Proteins identified from <i>M. capsulatus</i> (Bath) culture supernatant from cultures in early, mid and late exponential growth phase. ^aColumn shows the cumulative number of exclusive unique peptides from three biological replicates. Proteins were considered significant if ≧2 peptides were identified with a probability of ≧95% and a total protein probability of ≧99% and the protein was represented in at least two of the three biological replicates. ^bTotal coverage was calculated as the percentage of all aas in a protein, after removing the signal sequence, that is identified from sample spectra. ^c-fThe predicted presence (Y) or absence (N) of: ^csignal peptide, ^dlipoprotein signal peptide, ^etwin-arginine signal peptide or ^fprepilin-like signal peptide as predicted by SignalP 4.1, LipoP, LIPO, TatFind, TatP and PilFind is indicated. ^gColumn shows the number of transmembrane helixes predicted by the TMHMM prediction program. An asterisk indicates that >10 of the aas found in helixes within the first 60 aas, indicating that TMH may be a signal sequence. ^hNumber of TMH and/or presence of signal peptide predicted by the Phobius prediction program. ⁱSubcellular location predicted <i>in silico</i>.</p><p>Proteins identified in the culture supernatant of <i>M. capsulatus</i> (Bath).</p

Proteins predicted to be secreted by <i>M. capsulatus</i> (Bath).

<p>Proteins predicted to be secreted by <i>M. capsulatus</i> (Bath) and the secretion systems responsible are shown. ^aSignificant hits obtained after searches against the Pfam database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114476#pone.0114476-Punta1" target="_blank">[34]</a>. ^bNo conserved domain was identified in MCA2160 and MCA0338 searching Pfam 27.0, but Polycystic Kidney Disease (PKD) domains were identified by CD-search <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114476#pone.0114476-MarchlerBauer1" target="_blank">[33]</a>. ^cAlthough annotated as a lipoprotein, no lipoprotein signal peptide was identified in MCA0303 by the lipoprotein signal peptide prediction tools used in this study.</p><p>Proteins predicted to be secreted by <i>M. capsulatus</i> (Bath).</p

Growth curve showing average OD440 nm for three cultures of <i>M. capsulatus</i> (Bath) during growth.

<p>Cultures were sampled at OD440 0.424, 0.601 and 0.701 ± 0.15, indicated by X.</p