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-Guérin (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-Guérin (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 antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice.

Activation of the bile acid receptor TGR5 enhances LPS-induced inflammatory responses in a human monocytic cell line.

INTRODUCTION: Bile acids are recognized as signaling molecules, mediating their effects both through the cell surface receptor TGR5 and the nuclear receptor FXR. After a meal, approximately 95% of the bile acids are transported from terminal ileum and back to the liver via the portal vein, resulting in postprandial elevations of bile acids in blood. During the digestion of fat, components from the microbiota, including LPS, are thought to reach the circulation where it may lead to inflammatory responses after binding TLR4 immune cells. Both LPS and bile acids are present in blood after a high-fat meal; we therefore wanted to study consequences of a possible interplay between TGR5 and TLR4 in human monocytes. METHODS: The monocytic cell line U937 stably transfected with the NF-κB reporter plasmid 3x-κB-luc was used as a model system to study the effects of TGR5 and TLR4. Activation of MAP kinases was studied to reveal functional consequences of triggering TGR5 in U937 cells. Effects of TGR5 and TLR4 activation were monitored using NF-κB luciferase assay and by quantification of the pro-inflammatory cytokines IL-6 and IL-8 using ELISA. RESULTS: In this study, results show that triggering TGR5 with the specific agonist betulinic acid (BA), and the bile acids CDCA or DCA, activated both the main MAP kinases ERK1/2, p38 and JNK, and the NF-κB signaling pathway. We further demonstrated that co-triggering of TLR4 and TGR5 enhanced the activation of NF-κB and the release of inflammatory cytokines in a synergistic manner compared to triggering of TLR4 alone. CONCLUSIONS: Thus, two different and simultaneous events associated with the digestive process coordinately affect the function of human monocytes and contribute to enhanced inflammation. Because elevated levels of circulatory LPS may contribute to the development of insulin resistance, the results from this study suggest that bile acids through the activation of TGR5 may have a role in the development of insulin resistance as well.

The noncommensal bacterium Methylococcus capsulatus (Bath) ameliorates dextran sulfate (Sodium Salt)-Induced Ulcerative Colitis by influencing mechanisms essential for maintenance of the colonic barrier function.

Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlantic salmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate disease development in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human inflammatory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weight BioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp., Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in the drinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet containing the BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage also showed significant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response to inflammation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced mucin 2 (Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcus capsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were similar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the noncommensal bacterium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, as judged by increased epithelial proliferation and increased Muc2 transcription.

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-1ß 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-1ß 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.

Draft genome sequence of the methane-oxidizing bacterium Methylococcus capsulatus (Texas).

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.

Surface display of N-terminally anchored invasin by Lactobacillus plantarum activates NF-κB in monocytes.

The probiotic lactic acid bacterium Lactobacillus plantarum is a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties of L. plantarum by expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogen Yersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs from L. plantarum were used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatory L. plantarum strains constructed here represent promising mucosal delivery vehicles for vaccine antigens.

In vitro comparison of commensal, probiotic and pathogenic strains of Enterococcus faecalis.

In vivo studies have provided evidence that micro-organisms have important roles in immunological, digestive and respiratory functions, conferring health benefits on the host. Several in vitro methods have been advised for the initial screening of microbes with potential health effects. The objective of the present study was to employ such in vitro methodology to characterise different strains of Enterococcus faecalis. The characteristics of a commercial product marketed as a probiotic, Symbioflor-1 (Symbiopharm), were compared with the characteristics of both pathogenic and commensal strains. Tolerance towards low pH and viability after exposure to human gastric and duodenal juices were assayed. Symbioflor-1 was the most susceptible strain to these treatments when compared with the other E. faecalis strains. Furthermore, Symbioflor-1 exhibited the lowest adhesion capacity to intestinal epithelial cells (IEC) and mucus. Competitive binding studies using heparin indicated that glycosaminoglycans might be involved in the adhesion to IEC, but also that differences in these putative bacteria-host interactions do not cause the relative low adhesion capacity of Symbioflor-1. Maturation of dendritic cells (DC) after exposure to bacteria was assayed as an indication of an immunomodulatory effect. All strains induced a moderate elevation of the DC maturation markers CD83 and CD86; however, no strain-specific differences were detected. Correlations between in vitro and in vivo studies are discussed. Although in vitro assaying is a rational starting point for the selection of microbes with a potential health benefit, it is emphasised that human clinical trials are the definite tool for establishing probiotic status.

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3+RORγt+IL-17+) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.

Ancestral Wheat Types Release Fewer Celiac Disease Related T Cell Epitopes than Common Wheat upon Ex Vivo Human Gastrointestinal Digestion.

Celiac disease (CeD) is an autoimmune enteropathy triggered by immunogenic gluten peptides released during the gastrointestinal digestion of wheat. Our aim was to identify T cell epitope-containing peptides after ex vivo digestion of ancestral (einkorn, spelt and emmer) and common (hexaploid) wheat (Fram, Bastian, Børsum and Mirakel) using human gastrointestinal juices. Wheat porridge was digested using a static ex vivo model. Peptides released after 240 min of digestion were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ESI MS/MS). Ex vivo digestion released fewer T cell epitope-containing peptides from the ancestral wheat varieties (einkorn (n = 38), spelt (n = 45) and emmer (n = 68)) compared to the common wheat varieties (Fram (n = 72), Børsum (n = 99), Bastian (n = 155) and Mirakel (n = 144)). Neither the immunodominant 33mer and 25mer α-gliadin peptides, nor the 26mer γ-gliadin peptide, were found in any of the digested wheat types. In conclusion, human digestive juice was able to digest the 33mer and 25mer α-gliadin, and the 26mer γ-gliadin derived peptides, while their fragments still contained naive T cell reactive epitopes. Although ancestral wheat released fewer immunogenic peptides after human digestion ex vivo, they are still highly toxic to celiac patients. More general use of these ancient wheat variants may, nevertheless, reduce CeD incidence.

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS.

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.

The Soil Bacterium Methylococcus capsulatus Bath Interacts with Human Dendritic Cells to Modulate Immune Function.

The prevalence of inflammatory bowel disease (IBD) has increased in Western countries during the course of the twentieth century, and is evolving to be a global disease. Recently we showed that a bacterial meal of a non-commensal, non-pathogenic methanotrophic soil bacterium, Methylococcus capsulatus Bath prevents experimentally induced colitis in a murine model of IBD. The mechanism behind the effect has this far not been identified. Here, for the first time we show that M. capsulatus, a soil bacterium adheres specifically to human dendritic cells, influencing DC maturation, cytokine production, and subsequent T cell activation, proliferation and differentiation. We characterize the immune modulatory properties of M. capsulatus and compare its immunological properties to those of another Gram-negative gammaproteobacterium, the commensal Escherichia coli K12, and the immune modulatory Gram-positive probiotic bacterium, Lactobacillus rhamnosus GG in vitro. M. capsulatus induces intermediate phenotypic and functional DC maturation. In a mixed lymphocyte reaction M. capsulatus-primed monocyte-derived dendritic cells (MoDCs) enhance T cell expression of CD25, the γ-chain of the high affinity IL-2 receptor, supports cell proliferation, and induce a T cell cytokine profile different from both E. coli K12 and Lactobacillus rhamnosus GG. M. capsulatus Bath thus interacts specifically with MoDC, affecting MoDC maturation, cytokine profile, and subsequent MoDC directed T cell polarization.

The relationship between pro-inflammatory cytokines and pain, appetite and fatigue in patients with advanced cancer.

BACKGROUND: Systemic inflammation is associated with reduced quality of life and increased symptoms in patients with advanced cancer. The aims of this study were to examine the relationships between inflammatory biomarkers and the Patient Reported Outcome Measures (PROMs) of pain, appetite and fatigue; and to explore whether levels of baseline biomarkers were associated with changes in these PROMs following treatment with corticosteroids. MATERIAL AND METHODS: An exploratory analysis was done on a trial examining the analgesic properties of corticosteroids in patients with advanced cancer. Inclusion criteria were: >18 years, taking opioids for moderate or severe cancer pain; pain ≥4 (numerical rating scale 0-10). Serum was extracted and levels of inflammatory biomarkers were assessed. PROMs of pain, appetite and fatigue were assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30). The relationships between PROMs and inflammatory biomarkers were examined using Spearman Rho-Rank and multiple regression analysis. RESULTS: Data were available on 49 patients. Levels of sTNF-r1, IL-6, IL-18, MIF, MCP-1, TGF-ß1, IL-1ra, and C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR) were elevated; IL-1ß, IL-2, IL-4, IL-8, IL-10, IL-12(p70), interferon-γ, MIP-1α, and TNF-α were below the level of detection. The following correlations were observed: appetite and IL-6 and CRP; fatigue and IL-1ra (rs: 0.38-0.41, p< .01). There was no association between pretreatment biomarkers and effect from corticosteroid treatment. CONCLUSION: In patients with advanced cancer and pain, some pro-inflammatory cytokines were related to appetite and fatigue. Inflammatory biomarkers were not associated with pain or with the efficacy of corticosteroid therapy. Further research examining the attenuation of the systemic inflammatory response and possible effects on symptoms would be of interest.

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7.

BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239-256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239-334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239-334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239-334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.

Transport of a Prolyl Endopeptidase Inhibitory Peptide across the Blood-Brain Barrier Demonstrated Using the hCMEC/D3 Cell Line Transcytosis Assay.

The blood-brain barrier (BBB) remains a significant hurdle for treatment of central nervous system (CNS) and mental health disorders. A prolyl endopeptidase (PEP) inhibitory peptide with the amino acid sequence proline-proline-leucine (PPL) was chemically synthesized labeled with 5-FAM and assessed using a transcytosis assay for its ability to cross the BBB. Transport of this peptide across the BBB was determined using an in vitro model of the human BBB, which utilizes the human cerebral microvascular endothelial cell line (hCMEC/D3). Uptake and transport of 5-FAM-PPL across the hCMEC/D3 cell model was determined using confocal microscopy and mass spectrometry. This is an important parameter in determining whether peptides may reach the target organ (i.e., the brain and central nervous system).This work assessed, for the first time, the ability of a food-derived PEP inhibitory peptide to cross the BBB without the use of animal models.

Effects of the non-commensal Methylococcus capsulatus Bath on mammalian immune cells.

Dietary inclusions of a bacterial meal consisting mainly of the non-commensal, methanotrophic bacteria Methylococcus capsulatus Bath have been shown to ameliorate symptoms of intestinal inflammation in different animal models. In order to investigate the molecular mechanisms causing these effects, we have studied the influence of this strain on different immune cells central for the regulation of inflammatory responses. Effects were compared to those induced by the closely related strain M. capsulatus Texas and the well-described probiotic strain Escherichia coli Nissle 1917. M. capsulatus Bath induced macrophage polarization toward a pro-inflammatory phenotype, but not to the extent observed after exposure to E. coli Nissle 1917. Likewise, dose-dependent abilities to activate NF-κB transcription in U937 cells were observed, with E. coli Nissle 1917 being most potent. High levels of CD141 on human primary monocyte-derived dendritic cells (moDCs) were only detected after exposure to E. coli Nissle 1917, which collectively indicate a superior capacity to induce Th1 cell responses for this strain. On the other hand, the M. capsulatus strains were more potent in increasing the expression of the maturation markers CD80, CD83 and CD86 than E. coli Nissle 1917. M. capsulatus Bath induced the highest levels of IL-6, IL-10 and IL-12 secretion from dendritic cells, suggesting that this strain generally the post potent inducer of cytokine secretion. These results show that M. capsulatus Bath exhibit immunogenic properties in mammalian in vitro systems which diverge from that of E. coli Nissle 1917. This may provide clues to how M. capsulatus Bath influence the adaptive immune system in vivo. However, further in vivo experiments are required for a complete understanding of how this strain ameliorates intestinal inflammation in animal models.

Enhanced plasma IL-6 and IL-1ra responses to repeated vs. single bouts of prolonged cycling in elite athletes.

The impact of repeated bouts of exercise on plasma levels of interleukin (IL)-6 and IL-1 receptor antagonist (IL-1ra) was examined. Nine well-trained men participated in four different 24-h trials: Long [two bouts of exercise, at 0800-0915 and afternoon exercise (Ex-A), separated by 6 h]; Short (two bouts, at 1100-1215 and Ex-A, separated by 3 h); One (single bout performed at the same Ex-A as second bout in prior trials); and Rest (no exercise). All exercise bouts were performed on a cycle ergometer at 75% of maximal O(2) uptake and lasted 75 min. Peak IL-6 observed at the end of Ex-A was significantly higher in Short (8.8 +/- 1.3 pg/ml) than One (5.2 +/- 0.7 pg/ml) but not compared with Long (5.9 +/- 1.2 pg/ml). Peak IL-1ra observed 1 h postexercise was significantly higher in Short (1,774 +/- 373 pg/ml) than One (302 +/- 53 pg/ml) but not compared with Long (1,276 +/- 451 pg/ml). We conclude that, when a second bout of endurance exercise is performed after only 3 h of recovery, IL-6 and IL-1ra responses are elevated. This may be linked to muscle glycogen depletion.

Computational and experimental analysis of the secretome of Methylococcus capsulatus (Bath).

The Gram-negative methanotroph Methylococcus capsulatus (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 M. capsulatus. 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 M. capsulatus (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 M. capsulatus (Bath). In silico analysis suggests that the diverse secretion systems in M.capsulatus 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 M. capsulatus (Bath).