Functional characterization of short-type peptidoglycan recognition proteins (PGRPs) from silkworm Bombyx mori in innate immunity.

Peptidoglycan recognition proteins (PGRPs) are members of an important class of pattern recognition receptors in insects that can specifically recognize peptidoglycan (PGN) in bacterial cell walls and participate in immune regulation and bacterial clearance. Although the role of PGRPs in regulating the innate immune response in Drosophila melanogaster has been studied, little is known regarding PGRPs in Lepidoptera species. In this study, five short (S)-type Bombyx mori PGRPs (BmPGRPs) were cloned, expressed, and evaluated for their function in innate immunity. B. mori larvae that were injected with the gram-positive bacterium Bacillus megaterium or the gram-negative bacterium Escherichia coli exhibited a rapid and significant upregulation in S-type BmPGRP expression. The results showed that the five evaluated BmPGRPs have significant agglutination activity toward E. coli and B. megaterium and more notable amidase activity toward meso-diaminopimelic acid peptidoglycan (DAP-PGN). Furthermore, only in the presence of BmPGRP-S5 did B. mori larval hemocytes exhibit significant phagocytosis against E. coli and B. megaterium.

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains.

BACKGROUND: Different strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv. RESULTS: Extracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L-1) and highest space-time-yield (8 mg L-1 h-1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac). CONCLUSIONS: The genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space-time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.

Dietary inclusion of protease producing novel Pontibacter spp. and Bacillus megaterium as a probiotic enhances immune responses in Labeo rohita.

Abstract: This study stresses the key role which can be played by Tannery Fleshing (TF) hydrolyzing probiotic Pontibacter spp. in aqua feed formulation and identifies the probiotic strains in the fish gut capable of enhancing the overall growth and immune responses. Probiotics included are Pontibacter species (Pb) and Bacillus megaterium (BM) wherein Lactobacillus (LB) served as control. Experimental diets includes tannery fleshing (TF1), TF+LB strain (TF2), TF+BM strain (TF3), TF+Pb strain (TF4), Fishmeal+BM(TF5), Fishmeal+Pb and Control fish meal based diet (TF6). Compared with control, total weight gain (TWG), Specific Growth Rate (SGR), Feed Conversion Ratio (FCR) and Protein Efficiency Ratio (PER) in fish fed with diets supplemented with probiotics were significantly increased (p < 0.05). NBT, lysozyme activity, total protein and globulin content were highest in TF4 diet. After challenge with Aeromonas hydrophila, TF4 recorded highest survival and TF1 lowest survival in comparison with the control. Growth and related parameters reveals the effective utilization potential of tannery fleshing probiotic as a feed source. Comparative studies with standard fish meal diets reveals that the fish fed with Pontibacter spp. and Bacillus megaterium included feeds enhanced both assimilating capacity and immunological responses in Labeo rohita.

Association with soil bacteria enhances p38-dependent infection resistance in Caenorhabditis elegans.

The importance of our inner microbial communities for proper immune responses against invading pathogens is now well accepted, but the mechanisms underlying this protection are largely unknown. In this study, we used Caenorhabditis elegans to investigate such mechanisms. Since very little is known about the microbes interacting with C. elegans in its natural environment, we began by taking the first steps to characterize the C. elegans microbiota. We established a natural-like environment in which initially germfree, wild-type larvae were grown on enriched soil. Bacterial members of the adult C. elegans microbiota were isolated by culture and identified using 16S rRNA gene sequencing. Using pure cultures of bacterial isolates as food, we identified two, Bacillus megaterium and Pseudomonas mendocina, that enhanced resistance to a subsequent infection with the Gram-negative pathogen Pseudomonas aeruginosa. Whereas protection by B. megaterium was linked to impaired egg laying, corresponding to a known trade-off between fecundity and resistance, the mechanism underlying protection conferred by P. mendocina depended on weak induction of immune genes regulated by the p38 MAPK pathway. Disruption of the p38 ortholog, pmk-1, abolished protection. P. mendocina enhanced resistance to P. aeruginosa but not to the Gram-positive pathogen Enterococcus faecalis. Furthermore, protection from P. aeruginosa was similarly induced by a P. aeruginosa gacA mutant with attenuated virulence but not by a different C. elegans-associated Pseudomonas sp. isolate. Our results support a pivotal role for the conserved p38 pathway in microbiota-initiated immune protection and suggest that similarity between microbiota members and pathogens may play a role in such protection.

Antibody production in Bacillus megaterium: strategies and physiological implications of scaling from microtiter plates to industrial bioreactors.

Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a µ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.

Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system.

Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglycans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.

Diptericin expression in bacteria infected Drosophila mbn-2 cells - effect of infection dose and phagocytosis.

Drosophila haemocytes play a key role in defence against microbial aggression. Their capacity to sense and dispose of bacteria and also to signal to other immune tissues is probably vital to overcome an infection. In this work we used the haemocyte-like mbn-2 cell line to investigate how expression of the antimicrobial peptide diptericin is affected after a high dose bacterial challenge with diaminopimelic acid (DAP)-peptidoglycan Gram-positive and Gram-negative bacteria. We report that diptericin expression is negatively affected by high infection dose and rapid bacterial growth regardless of the type of infection and bacterial virulence and occurs in the absence of mbn-2 cell death. Furthermore we show that the mbn-2 cell population is heterogeneous, containing both phagocytic and nonphagocytic cells and that contact with large numbers of bacteria decreases diptericin expression in the phagocytic cell population.

Internal defenses of the snail Biomphalaria glabrata.

The three populations of hemocytes and the fixed phagocytes of Biomphalaria glabrata show different affinities for the following injected foreign materials: ferritin, horseradish peroxidase, zymosan particles, latex beads, and live bacteria. Ferritin and horseradish peroxidase, which are internalized by micropinocytosis, induce a moderate activation of hemocytes. Particles trigger a strong activation that consists of mobilization of the cytoskeleton, with formation of pseudopodia and filopodia and modifications of the plasma membrane that enable hemocytes to adhere together, as in encapsulations. Phagocytosis of zymosan and live bacteria induces alterations in mitochondria that may reflect changes in the respiratory activity. Phagocytosis of particles follows different processes: fixed phagocytes ingest latex beads of two different sizes according to the "zipper model", which implies sequential binding between phagocyte receptors and opsonized ligands; zymosan particles are ingested by medium-size hemocytes by a variant of the zipper model in which a limited close contact between cell and particle resembles focal contacts in cell spreading; bacteria or large latex beads are ingested by large hemocytes without apparent close contact following a "trigger model" which resembles macropinocytosis.

Production of interleukin-12 by murine macrophages in response to bacterial peptidoglycan.

Peptidoglycan (PG), a component of the bacterial cell wall, has various immunomodulating activities, including the capacity to induce delayed-type hypersensitivity reactions to antigens administered in Freund's adjuvant. We report that PG induces interleukin-12 (IL-12) mRNA production and IL-12 secretion by mouse macrophages. The capacity of PG to induce IL-12 production, like its previously reported immunomodulating activities, was dependent on the structure of its peptide subunit. PG from Bacillus megaterium and Staphylococcus aureus induced IL-12 production, whereas PG from Micrococcus luteus and Corynebacterium poinsettiae did not. The ability of most bacterial PGs to induce IL-12 production suggests that they play an important role in triggering host defense mechanisms against bacterial infections.

Immunological crossreactivity to the catabolite control protein CcpA Bacillus megaterium is found in many gram-positive bacteria.

The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, lactic acid bacteria, and some actinomycetes.

[Antibodies to Bacillus megaterium H glycoprotein in patients with lung diseases]. / Antitela k glikoproteinu Bacillus megaterium H u bol'nykh s legochnoi patologii.

Enzyme immunoassay performed in patients with malignant and benign affections of the lungs determined antibodies (Abs) to glycoprotein isolated from saprophyte microorganism Bacilus megaterium H. In pulmonary tuberculosis and nonspecific pulmonary inflammation the above Abs occurred in patients with fibrous-proliferative changes and associated neoplastic conditions. In lung cancer the Abs were detected in well and moderately differentiated tumors. Nondifferentiated and poorly differentiated variants did not exhibit any rise in Abs to glycoprotein.

[Determination of antibodies to cross-reacting antigens of microorganisms in oncological diseases]. / Opredelenie antitel k perekrestno reagiruiushchim antigenam mikroorganizmov pri onkologicheskikh zabolevaniiakh.

It was studied antibodies to saprophytic microorganism Bacillus megaterium H glycoprotein in healthy, oncological, non-oncological gastrointestinal patients and animals. High level of antibodies to microbial glycoprotein in blood sera was revealed in cancer and precancer patients. Analogical results were obtained in mice A/Sn and Balb/c with inductive or transplanted tumors. It has been suggested the use of Bacillus megaterium H glycoprotein (M. m. 65-70 kD) for immunological monitoring.

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium.

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.

Ultrastructural characterization of leucocytes in the pronephros of carp (Cyprinus carpio, L.).

In the pronephros of carp (Cyprinus carpio, L.) the following cells were found and ultrastructurally characterized: erythrocytes; lymphocytes and plasma cells; thrombocytes; neutrophilic, eosinophilic and basophilic granulocytes; phagocytic reticular cells and monocytes and non-phagocytic reticular cells. The cells could be separated on a Percoll continuous density gradient and relatively pure fractions could be obtained. In vitro phagocytosis of bacteria (Bacillus megaterium) was found in monocytes and neutrophilic granulocytes, while basophilic and eosinophilic granulocytes engulfed bacterial cells without actual endocytotic uptake.

Stimulation of human B lymphocytes by Nocardia-delipidated cell mitogen and derived fractions from N. opaca. Structure-activity relationship.

Nocardia-delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, is able to stimulate the proliferation of small resting human B lymphocytes and their differentiation into Ig-secreting cells. This fraction contains two active structures: the cell wall peptidoglycan (PG) and a fraction (Cy I) derived from the cytoplasmic compartment. Treatment of insoluble PG with various bacteriolytic enzymes showed that the minimal structure required for mitogenic activity is more complex than that required for the differentiation of human lymphocytes. The mitogenic activity of cell wall fractions varies in different bacterial species; that prepared from N. opaca is the more potent. Both mitogenic structures of N. opaca induce higher responses in infant and adult PBL as compared to cord lymphocytes. The differentiation of B lymphocytes into Ig-secreting cells induced by PG fractions is T-dependent.

[Modification of 3-methylcholanthrene-induced blastomogenesis in BALB/c mice by using the subcellular components of Bac. megaterium H]. / Modifikatsiia blastomogeneza, indutsirovannogo 3-metilkholantrenom u myshei BALB/c, pri pomoshchi subkletochnykh komponentov Bac. megaterium H.

It is shown that the cytoplasm of Bac. megaterium H possessing the antigenic affinity with the malignant tumour cells, being injected 7 days before the 3-methylcholanthrene injection promotes intensification of its blastomogenic effect, reduces both the period of tumour appearance and longevity of the tumour-bearing animals. On the contrary the cell wall preparation under the similar conditions, produces a therapeutic effect. An assumption is advanced that the effect of these preparations is associated with their different influence on the immune system.

Protein analysis of earthworm coelomic fluid. III. Isolation and characterization of several bacteriostatic molecules from Eisenia fetida andrei.

A bacteriostatic activity in Eisenia fetida andrei cell free coelomic fluid is described. This activity is detected by growth inhibition of a bacteria Bacillus megaterium. Gel filtration analysis revealed eleven coelomic fluid protein fractions designated A, B,..J. Antibacterial activity was mainly found within fractions B and C. Chromatofocusing resolved fractions B-C into five different peaks named alpha BC, beta BC,... epsilon BC. Antibacterial activity appeared mediated by three different proteins characterized by their molecular weights (20,000, 40,000 and 45,000) and their isoelectric points (4.9, 5.75 and 6.0). These bacteriostatic proteins possess either hemolysis or hemagglutination activities. The polymorphic aspect of this humoral antibacterial defense is discussed.