Major Secretory Protein Research Articles

Mycobacterium tuberculosis antigen 85B modifies BCG-induced antituberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis has developed strategies not only to evade host immunity but also to manipulate it for its survival. We investigated whether Mycobacterium tuberculosis exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host antituberculosis immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin impaired the protection elicited by vaccination, causing a more severe infection when mice were challenged with Mycobacterium tuberculosis. Ag85B administration reduced Bacillus Calmette-Guérin-induced CD4 T-cell activation and IFN-γ, CCL-4, and IL-22 production in response to Mycobacterium tuberculosis-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses, and activation of regulatory and exhaustion pathways, including programmed death ligand 1 expression on macrophages. All this resulted in impaired intracellular Mycobacterium tuberculosis growth control by systemic immunity, both before and after the Mycobacterium tuberculosis challenge. Interestingly, Mycobacterium tuberculosis infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mycobacterium tuberculosis in both unvaccinated and Bacillus Calmette-Guérin-vaccinated mice. Our data suggest that Mycobacterium tuberculosis can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and is commonly found in the lungs of the Mycobacterium tuberculosis-infected host, our findings may advance the understanding on the mechanisms of Mycobacterium tuberculosis pathogenesis and immune evasion.

Legionella pneumophila and Free-Living Nematodes: Environmental Co-Occurrence and Trophic Link.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella's major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

Dietary Indole‐3‐Carbinol Supplementation Restores Epithelial Abnormalities in SAMP1/YitFc Mouse Model of Ileitis

SAMP1/YitFC (SAMP) mice exhibit spontaneous ileitis as they age with morphological and functional epithelial abnormalities mimicking human Crohn’s disease, a chronic disease characterized by ileal inflammation. Indole‐3‐carbinole (I3C), found in broccoli and Brussel sprouts, plays a crucial role in the anti‐inflammatory processes by activating the aryl hydrocarbon receptor (AhR), a transcription factor vital for epithelial and immune cell functions. The impact of I3C diet on disease progression and epithelial integrity in SAMP model of ileitis is not known.Methods6‐week‐old male and female (n=8 per group) SAMP and control AKR/J mice (Jackson) were fed for 14 weeks either a standard chow diet or a purified diet supplemented with 200 ppm with indole‐3‐carbinole (+I3C diet), or purified diet depleted of AhR ligands (‐I3C diet). Ileum samples were collected for qPCR and histological analyses. Statistical analysis was performed by Tukey’s multiple comparison test for all three diets. To detect changes between +I3C vs. ‐I3C, delta values (Δ +/‐I3C = SAMP‐AKR) were analyzed by unpaired T‐test.ResultsAs expected, H&E staining for the ileum of SAMP mice fed chow diet, showed thickening of the muscle layer, neutrophil infiltration, and epithelial tufting vs. AKR mice. Severity of inflammation in SAMP mice, as assessed by fecal calprotectin ELISA, mRNA expression of cytokines, and histological analysis was not significantly impacted by dietary depletion or supplementation of I3C. With respect to epithelial abnormalities, Periodic Acid Schiff staining showed increased number of goblet cells per villi (~47‐75%) in SAMP mice on all three diets. While mRNA levels of the major goblet cell secretory proteins, mucin2 (Muc2) and intestinal trefoil factor (ITF), were dramatically decreased in SAMP mice vs. AKR, supplementation of I3C attenuated this decrease in Muc2 and ITF by ~35% and 51% (p<.001), respectively, indicating defective synthesis or secretion. Similarly, I3C diet significantly (p<.01) attenuated the decrease in Paneth cell markers lysozyme C‐1 (LYZ1) alpha defensin 5 (Def5α) and regenerating islet‐derived protein 3 alpha (Reg3α) in SAMP mice by ~65%, 60%, and 24%, respectively. A decrease in fibroblast growth factor 15 (FGF15) and the epithelial ion transporters serotonin transporter (SERT) and organic solute transporter (OST) were observed in ileal mucosa of SAMP mice vs. AKR, and the supplementation of I3C mitigated these decreases by 30‐58% (p<.01). There were no significant changes in the expression of tryptophan hydroxylase 1 (tph‐1), C‐X‐C motif chemokine ligand 2 (CXCL2), or AhR by I3C diet in SAMP mice. In summary, I3C supplementation significantly attenuated the goblet cell, Paneth cell, and epithelial‐specific abnormalities seen in SAMP mice. These data imply that dietary supplementation of AhR ligands may be a viable therapeutic strategy for Crohn’s patients to restore epithelial integrity.

Serological immune response against major secretory proteins expressed by Pseudomonas aeruginosa upon intratracheal infection in mice.

Antimicrobial-resistant Pseudomonas aeruginosa is an opportunistic pathogen that severely affects human health. Immunotherapy has attracted substantial attention as an alternative treatment to decrease antimicrobial drug use. Considering previous studies on antibody and vaccine therapy, we focused on quantifying antibodies specific to the V antigen (PcrV) and type III secretory protein (ExoU) expressed by P. aeruginosa to evaluate the serological immune response. We intratracheally infected male ICR mice with several P. aeruginosa strains and quantified antigen-specific antibody titers across 8 weeks. Intratracheal infection of P. aeruginosa PA103 at a sublethal dose decreased the body temperature of mice. The IgG and IgA serum titers against PcrV and ExoU did not increase over 8 weeks, and the IgM titer initially increased for 4 weeks and then decreased. Specific antibodies against PcrV and ExoU may be difficult to produce naturally. Therefore, the IgM expression against major secretory proteins of P. aeruginosa is critical.

Identification of other Cellular Receptors for Edema Factor of Bacillus anthracis by Independent Inhibition of Protective Antigen Evidenced by Inhibition of Embryo Growth and Angiogenesis.

Edema factor (EF) is one of the major secretory proteins of anthrax bacteria along with protective antigen (PA) and lethal factor (LF). Edema factor is a calmodulin-and calcium-dependent adenylate cyclase that increases intracellular levels of cAMP. Intracellular trafficking of EF occurs through PA by binding to ATR/CMG2 receptors, which are also involved in other physiological functions of cells. cAMP is a secondary messenger which activates multiple signaling cascades involved in the cytokinetics of actin molecules and cell junction formation. The present study evaluated the effect of EF on growth and angiogenesis patterns in chicken embryos in the in ovo model. Angiogenesis in the chorioallantoic membrane (CAM) of an embryonated chicken egg was decreased and embryo growth was delayed by EF despite the absence of trafficking moiety PA, which is required for transferring the EF molecule inside the cell. Angiogenesis inhibition and embryo growth retardation indicate the use of an alternative receptor by EF to modulate these cellular functions. Additionally, docking was performed between EF as a ligand and hepatocyte growth factor receptor (cMET) and vascular endothelial growth factor (VEGF) receptors, which are mainly involved in growth and angiogenesis. The analysis revealed a very strong binding of EF to cMET receptor (in terms of the number of hydrogen bonds and energy) compared to its ligand hepatocyte growth factor (HGF), which indicates the use of cMET receptor by EF and induction of angiogenesis and embryo growth retardation possibly by competitive inhibition of HGF ligand or receptor-mediated endocytosis.

Embryo–uterineinteractions in pigs during week 2 of pregnancy

The second week of pregnancy is a particularly critical period for embryonic survival in pigs. Within that time, conceptus oestrogen synthesis is initiated, spacing and final placement of conceptuses is completed, and the signal for extending the functional lifespan of the corpora lutea is received by the mother. There is also a marked increase in blood flow to the uterus and the uterine endometrium produces and secretes nutrient histotrophe. Coneeptus-derived oestrogen has been implicated in many of these events. It is also during this period that the trophoblast elongates dramatically and the inner cell mass starts to differentiate into the embryo proper. Here, we critically review the evidence that oestrogen is the sole factor initiating long-term corpus luteum maintenance in pigs. We also review the functions and general properties of the major secretory proteins in histotrophe and the role of oestrogen in controlling their expression. It is now generally accepted that asynchrony within a litter underlies much of the losses of conceptuses that are otherwise genetically normal, but which are lagging in their development; however, the underlying mechanisms remain unclear. Here we hypothesize that oestrogenic compounds derived from more advanced conceptuses or provided prematurely, either by injection or in the diet, trigger a massive increase in uterine expression and secretion of retinol-binding protein laden with retinol. We propose that less developed, smaller conceptuses are least able to contend with the sudden exposure to this potential teratogen at a time when they are particularly susceptible to imbalance in retinol supply. Hence, even though their growth proceeds for a few days, their developmental potential is irrevocably compromised.

Isolation of a Novel Bacterial Strain Capable of Producing Abundant Extracellular Membrane Vesicles Carrying a Single Major Cargo Protein and Analysis of Its Transport Mechanism.

Extracellular membrane vesicles (EMVs) play an important role in various bacterial activities. EMVs have potential for use as vaccines, drug-delivery vehicles, platforms for extracellular production of recombinant proteins, and so on. In this study, we newly isolated a cold-adapted bacterium, Shewanella vesiculosa HM13, which abundantly produces EMVs, characterized them, and analyzed their cargo transport mechanism. S. vesiculosa HM13, isolated from the intestine of a horse mackerel as a prospective host for a low-temperature secretory protein expression system, produced a single major secretory protein, P49, of unknown function in the culture supernatant. Analysis using sucrose density gradient ultracentrifugation indicated that P49 is a cargo protein carried by EMVs. S. vesiculosa HM13 displayed extensive blebbing on the surface of the outer membrane, and the size of blebs was comparable to that of EMVs. These blebs are thought to be precursors of the EMVs. Disruption of the P49 gene resulted in only a marginal decrease in the EMV production, indicating that the EMVs are produced even in the absence of the major cargo protein. Whole genome sequencing of S. vesiculosa HM13 revealed that this bacterium has a gene cluster coding for a non-canonical type II protein secretion system (T2SS) homolog in addition to a gene cluster coding for canonical T2SS. The P49 gene was located downstream of the former gene cluster. To examine the role of the putative non-canonical T2SS-like translocon, we disrupted the gene coding for a putative outer membrane channel of the translocon, named GspD2. The gspD2 disruption lead to disappearance of P49 in the EMV fraction, whereas the production of EMVs was not significantly affected by this mutation. These results are indicative that the T2SS-like machinery functions as a novel type of protein translocon responsible for selective cargo loading to the EMVs. We also found that GFP fused to the C-terminus of P49 expressed in S. vesiculosa HM13 was transported to EMVs, indicating that P49 is useful as a carrier to deliver the fusion partner to EMVs. These findings deepen our understanding of the mechanism of biogenesis of EMVs and facilitate their applications.

Identification, recombinant protein production, and functional analysis of a M60-like metallopeptidase, secreted by the liver fluke Opisthorchis viverrini

The carcinogenic liver fluke Opisthorchis viverrini (O. viverrini) is endemic in Thailand and neighboring countries including Laos PDR, Vietnam and Cambodia. Infections with O. viverrini lead to hepatobiliary abnormalities including bile duct cancer–cholangiocarcinoma (CCA). Despite decades of extensive studies, the underlying mechanisms of how this parasite survives in the bile duct and causes disease are still unclear. Therefore, this study aims to identify and characterize the most abundant protein secreted by the parasite. Proteomics and bioinformatics analysis revealed that the most abundant secretory protein is a metallopeptidase, named Ov-M60-like-1. This protein contains an N-terminal carbohydrate-binding domain and a C-terminal M60-like domain with a zinc metallopeptidase HEXXH motif. Further analysis by mass spectrometry revealed that Ov-M60-like-1 is N-glycosylated. Recombinant Ov-M60-like-1 (rOv-M60-like-1) expressed in Escherichia coli (E. coli) was able to digest bovine submaxillary mucin (BSM). The mucinase activity was inhibited by the ion chelating agent EDTA, confirming its metallopeptidase identity. The enzyme was active at temperatures ranging 25–37 °C in a broad pH range (pH 2–10). The identification of Ov-M60-like-1 mucinase as the major secretory protein of O. viverrini worms warrants further research into the role of this glycoprotein in the pathology induced by this carcinogenic worm.

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp. CBMAI 1063, a laccase hyper-producer strain

The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.

Selection of DNA aptamers against Mycobacterium tuberculosis Ag85A, and its application in a graphene oxide-based fluorometric assay.

The Mycobacterium Ag85 complex is the major secretory protein of M. tuberculosis. It is a potential marker for early diagnosis of tuberculosis (TB). The authors have identified specific aptamers for Ag85A (FbpA) via protein SELEX using magnetic beads. After twelve rounds of selection, two aptamers (Apt8 and Apt22) were chosen from different groups, and their binding constants were determined by flow cytometry. Apt22 (labeled with Atto 647N) binds to FbpA with high affinity (Kd = 63nM) and specificity. A rapid, sensitive, and low-cost fluorescent assay was designed based on the use of Apt22 and graphene oxide, with a limit of detection of 1.5nM and an analytical range from 5 to 200nM of FbpA. Graphical abstract Schematic illustration of graphene oxide-based aptasensor for fluorometric determination of FbpA.

Early endosome motility mediates \u03b1-amylase production and cell differentiation in Aspergillus oryzae

Recent research in filamentous fungi has revealed that the motility of an endocytic organelle early endosome (EE) has a versatile role in many physiological functions. Here, to further examine the motility of EEs in the industrially important fungus Aspergillus oryzae, we visualized these organelles via the Rab5 homolog AoRab5 and identified AoHok1, a putative linker protein between an EE and a motor protein. The Aohok1 disruptant showed retarded mycelial growth and no EE motility, in addition to an apical accumulation of EEs and peroxisomes. We further demonstrated that the Aohok1 disruptant exhibited less sensitivity to osmotic and cell wall stresses. Analyses on the protein secretory pathway in ΔAohok1 cells showed that, although distribution of the endoplasmic reticulum and Golgi was not affected, formation of the apical secretory vesicle cluster Spitzenkörper was impaired, probably resulting in the observed reduction of the A. oryzae major secretory protein α-amylase. Moreover, we revealed that the transcript level of α-amylase-encoding gene amyB was significantly reduced in the Aohok1 disruptant. Furthermore, we observed perturbed conidial and sclerotial formations, indicating a defect in cell differentiation, in the Aohok1 disruptant. Collectively, our results suggest that EE motility is crucial for α-amylase production and cell differentiation in A. oryzae.

Listeria-Vectored Vaccine Expressing the Mycobacterium tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium bovis BCG Efficacy against Tuberculosis.

A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).

Phosphorylation of secreted proteins by a new family of kinases

Protein kinases are evolutionarily conserved enzymes that transfer a molecule of phosphate from ATP to protein substrates in a process known as phosphorylation. In 1883, the secreted milk protein casein was shown to contain phosphorous. In hindsight, this was the first indication for the existence of protein kinases that were ultimately discovered nearly one‐century later using casein as the substrate. We now know that protein phosphorylation is a universal mechanism that regulates nearly every aspect of cellular life. Despite the fact that casein was identified as the first phosphoprotein, the responsible kinase had remained obscure. The enzymes classically referred to as “casein kinases” do not mediate the physiological phosphorylation of casein because they are nuclear and cytosolic proteins and do not encounter casein within the secretory pathway. Numerous other secreted proteins and peptide hormones are phosphorylated; however, the molecular identities of the kinases responsible for these modifications were unknown as well. As a consequence, research on extracellular protein phosphorylation has been largely undeveloped.We discovered a novel family of atypical secretory pathway kinases that phosphorylate secreted proteins. This new kinase family is so different from canonical kinases that it was not included as a branch on the human “kinome” tree. One member of this new family, Fam20C, is the Golgi casein kinase, an enzyme that escaped identification for many years. Fam20C contains a signal peptide that directs it to the lumen of the secretory pathway where it phosphorylates proteins on S‐x‐E/pS motifs. The importance of this discovery is highlighted by the fact that ~75% of human serum, plasma and cerebrospinal fluid phosphoproteins are phosphorylated within this motif.Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing and mass spectrometry, we identify more than 100 phosphoproteins as Fam20C substrates. Functional annotations of Fam20C substrates suggest roles for the kinase in a broad spectrum of biological processes, including cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

A Single Kinase Generates the Majority of the Secreted Phosphoproteome

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

PA-824 is as effective as isoniazid against latent tuberculosis infection in C3HeB/FeJ mice

The bicyclic nitroimidazole-like molecule PA-824 has activity both against replicating and hypoxic non-replicating Mycobacterium tuberculosis, raising the possibility that it may have a role in the treatment of latent tuberculosis infection (LTBI). This study aimed to examine the bactericidal and sterilising activities of PA-824 against LTBI in C3HeB/FeJ mice, which develop hypoxic, necrotic granulomas histologically resembling their human counterparts. Female 5–6-week-old C3HeB/FeJ mice were immunised via the aerosol route with a recombinant BCG strain overexpressing the 30-kDa major secretory protein (rBCG30) and were aerosol-infected 6 weeks later with virulent M. tuberculosis H37Rv. Six weeks after M. tuberculosis infection, separate groups of mice were left untreated (negative controls) or were treated with either rifampicin, isoniazid (INH) or PA-824. Culture-positive relapse was assessed in subgroups of mice after 2 months and 4 months of treatment. Human-equivalent doses of PA-824 given five times weekly showed similar bactericidal activity as INH at Months 1, 2 and 4 of treatment, and 15/15 mice treated with either PA-824 or INH showed lung-culture relapse 3 months after completion of treatment. To the best of our knowledge, this is the first report examining the sterilising activity of PA-824 in an animal model of LTBI. This model may be useful for screening the efficacy of novel drugs against LTBI, particularly those with specific activity against bacilli residing within necrotic lung granulomas.

RBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents

During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.

Cloning and Expression of Mycobacterium tuberculosis Major Secreted Protein Antigen 85B (Ag85B) in Escherichia coli

Background: The 30 kDa major secretory protein of Mycobacterium tuberculosis (antigen 85B) is a primary vaccine candidate. This secreted antigen induces a protective immune response and stimulates the production of IFN-γ in animal models. Objectives: The aim of this study was cloning and expression of Ag 85B of M. tuberculosis in Escherichia coli. Materials and Methods: To produce recombinant Ag85B, the fbpB gene was amplified by PCR method. Then inserted into the pET101/D vector and transported into E. coli strain TOPO10. Plasmid containing pET101/D: Ag85B was transformed into competence E.coli BL21 (DE3). The transformed E.coli strain BL21 was effectively expressed recombinant Ag85B. Results: The expressed fusion protein was found almost entirely in the insoluble form. Followed by sonication to disrupt the cells, Solution of the cell debris was centrifuged and after use of Ni–NTA column and 6 molar urea and 6 M guanidine-HCl solutions recombinant protein was purified. Conclusions: These results could serve as a basis for further studies in endemic regions of tuberculosis on the usefulness of this gene and its expression product in the development of subunit vaccine and DNA vaccine against tuberculosis.

Some fine-structural findings on the thyroid gland in Apc 1638T/1638T mice that express a C-terminus lacking truncated Apc

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc(1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc(1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc(1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc(1638T/1638T) and Apc(+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc(1638T/1638T) mice were significantly different whereas the Apc(+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc(1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc(+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc(1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc(+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc(1638T/1638T) mice and suggest suppressed secretory activities of these cells.

Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis

Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella.