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Acinetobacter spp. porin Omp33-36: Classification and transcriptional response to carbapenems and host cells
Get PDFAcinetobacter baumannii has been recognized as one of the most challeging pathogens in clinical settings worldwide. Outer membrane porins play a significant role in Acinetobacter antibiotic resistance and virulence. A. baumannii carbapenem resistance and virulence factor porin Omp33-36 was the subject of this study. We investigated the omp33-36 gene transcriptional response in the growth phase, its response to carbapenems, and the effect of contact with host cells. Additionally, the cytotoxic effect of A. baumannii towards keratinocytes was assessed, as well as correlation between omp33-36 gene transcription and cytotoxicity. Further, Acinetobacter spp. Omp33-36 was classified and its characteristics relevant for vaccine candidature were determined. The level of the omp33-36 gene transcription varied between growth phases, but a common pattern could not be established among different strains. Treatment with subinhibitory concentrations of carbapenems decreased, while contact with keratinocytes increased omp33-36 expression in the analysed A. baumannii strains. Variations in omp33-36 mRNA levels did not correlate with cytotoxicity levels. Decrease of omp33-36 mRNA during treatment with subinhibitory concentrations of carbapenems, indicated the importance of transcriptional changes in reversible resistance to carbapenems due to the absence of Omp33-36. The transcription of omp33-36 increased after contact with keratinocytes, indicating the important role of de novo transcription during the initial phase of A. baumannii infection. Primary structural analysis of Acinetobacter spp. Omp33-36 revealed three distinct groups (among four A. baumannii variants). Although we have shown that Omp33-36 was highly polymorphic, we propose a potential antigen (PLAEAAFL motif) for vaccine development. According to PROVEAN analysis, the highly polymorphic structure of Omp33-36 porin should not influence its function significantly
Gastric digestome of whole peanut grains from the aspect of immunoproteomics: Characterization of digested allergens in the real food matrix
Get PDFObjective: Major peanut allergens, Ara h 2 and Ara h 6, are known to be resistant to pepsindigestion, and they sensitize individual via the gastrointestinal tract. Mikenus et al. published astandardized static digestion method for food, based on physiological conditions emphasizing theimpact of food matrices. Immunoreactive proteins (large fragments) and peptides (short digestionresistant peptides SDRPs; <10 kDa), to which the immune system of the gastrointestinal tract isexposed during digestion of peanut proteins, has not been investigated under pure physiologicalconditions suggested by this protocol.Matherial and methods: Whole grain of grounded raw peanut was incubated with human α-amylase, and pepsin, mimicking the effects of oral and gastric digestion, in total duration of 2h.Bottom up proteomic approach, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, wereused to identify and characterize peanut digesta.Results: After 2h of oral/gastric phase we got, intact proteins, large, digestion resistant peptides(DRP) and SDRPs, as well. Ara h 2 and Ara h 6 remained mostly intact, and short DRPs from Ara h2 and Ara h 6 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h3 showed preserved allergenic capacity, as well. Almost all of identified short DRPs from Ara h 1,Ara h 2 and Ara h 3, with preserved allergenic potential, were constituents of continuous epitopesequences found via Immune Epitope Database (www.iedb.org).Conclusion: Processes of protein extraction from the matrix and their enzymatic digestion occursimultaneously. Oral and gastric phase digestion products of raw peanut are intact proteins, largeand short digestion resistant peptides. Under these conditions Ara h 2 and Ara h 6 are expectedlyBook of Abstract
Blokada β-adrenergičkih receptora polno dimorfno utiče na primarni odgovor CD4+ T-limfocita u eksperimentalnom autoimunskom encefalomijelitisu (EAE)
No full textIstraživanja su koncipirana imajući u vidu da: 1) se polni diergizam
u primarnom odgovoru CD4+ T-limfocita DA pacova na imunizaciju
homogenatom kičmene moždine (KM) u kompletnom Frojndovom adjuvansu
povezuje sa manjom incidencom EAE i odloženim početkom bolesti kod mužjaka
u poređenju sa ženkama, 2) kateholamini, posredstvom β-adrenergičkih
receptora (AR), modulišu aktivnost ćelija urođene i stečene imunosti i 3)
postoji polni diergizam u aktivnosti simpatičkog nervnog sistema i gustini
simpatičkih nervnih vlakana u limfnim čvorovima (LČ). Cilj istraživanja
je bio da se ispita značaj kateholamina za polni diergizam u primarnm
odgovoru CD4+ T-limfocita u modelu EAE kod DA pacova. Sedmog dana posle
imunizacije bila veća je veća koncentracija noradrenalina u drenirajućem
LČ (dLČ) mužjaka, odražavajući njegov veći sadržaj u nervnim vlaknima
i ćelijama, uključujući i CD4+ T-limfocite, i procenat β-AR+ ćelija u
populaciji CD4+ T-limfocita i CD11b+ antigen prezentujućih ćelija
(APĆ). Iako je blokada β-AR propranololom u kulturi ćelija dLČ povećavala
sintezu IL-2 i proliferaciju CD4+ limfocita, kao i diferencijaciju
IL-17+CD4+ T-limfocita ushodno regulišući ekspresiju IL-1β i IL23 kod životinja oba pola (veći efekat kod mužjaka), sedmodnevni tretman
propranololom počevši od dana imunizacije je smanjivao broj IL17+CD4+
T-limfocita u dLČ drastično redukujući migraciju aktivisanih CD11b+
APĆ u dLČ smanjenjem ekspresije CCL19/21. Ovo je korelirao s manjom
infiltracijom KM pacova IL-17+CD4+ T-limfocitima u induktivnoj fazi
bolesti, manjom incidencom EAE i odloženom pojavom kliničkih znakova
kod životinja koje su razvile bolest, što je bilo više izraženo kod mužjaka.
Istraživanja ukazuju na kompleksno, polno dimorfno imunomodulatorno
delovanje kateholamina i pokazuju da se negativan uticaj propranolola
na migraciju i moguće pozicioniranje APĆ u dLČ ne može kompenzovati
njegovim stimulatornim efektima na proliferaciju CD4+ T-limfocita i
polarizacioni kapacitet APĆ. (Finasiralo MPNTR, pr.175050
The involvement of estrogen receptors alpha and beta in the in vitro effects of 17 beta-estradiol on secretory profile of peritoneal macrophages from naturally menopausal female and middle-aged male rats
No full textThe systemic and extra- gonadal levels of 17 beta-estradiol (E2) change during aging, and affect the expression of estrogen receptors (ERs) in the immune cells of both females and males. The age-related cessation of ovarian function in females, as well as the tissue-specific expression of enzyme aromatase (estrogen synthase which significantly rises with the advancing age) in both males and females, both determine the concentration of E2 to which immune cells may be exposed. The present study was set up to investigate the direct influence of E2 in vitro on the secretory profile of peritoneal macrophages from young and naturally menopausal female rats, and from young and middle-aged male rats. The involvement of receptor(s) responsible for mediating the effects of E2 in vitro was examined by use of antagonists specific for ERa or ER beta. Whereas in macrophages from young female rats E2 treatment diminished interleukin (IL)-1 beta secretion, it increased it in young males, and the middleaged females. The in vitro E2 treatment increased tumor necrosis factor (TNF)-alpha release by macrophages from young rats of both sexes, while it increased macrophage IL-6 release independently of both sex and age. At the same time, E2 decreased hydrogen peroxide (H2O2) production in macrophages from females, and increased it in male rats of both ages, whereas it diminished nitric oxide (NO) release in all experimental groups. Inspite of the sex-and age-specific effects of E2 on macrophage urea release, E2 did not affect the NO/urea ratio in macrophages from female rats, and diminished it in macrophages from both young and middle-aged male rats. Independently of the sex and age, E2 stimulated the release of inflammatory cytokines predominantly via macrophage ER alpha, and inhibited the IL-1 beta release in young females via ER beta. In contrast, E2 increased macrophage H2O2 and urea production by activating ER beta, but diminished their release via ER alpha. Our study may contribute to better understanding of the complex role(s) that E2 may play in innate immunity during aging, and that are dependent of sex
Mouse strain and sex as determinants of immune response to trivalent influenza vaccine
No full textAims: The study examined the influence of sex and mouse strain on germinal center (GC) reaction and antibody responses to seasonal split trivalent influenza vaccine (TIV). Main methods: C57BL/6 and BALB/c mice of both sexes were immunized with TIV and examined for specific antibody response by ELISA. Splenic T follicular regulatory (Tfr), T follicular helper (Tfh) and GC B cells are detected by flow cytometry. The proliferative response of splenocytes, and concentrations of IFN-gamma and IL-4 upon restimulation with vaccine antigens were examined by 7-AAD staining and ELISA, respectively. Key findings: BALB/c mice developed more robust IgG responses to vaccine type A antigens than their sexmatched C57BL/6 counterparts, while that to B antigen did not differ between strains. In both strains IgG responses against type A vaccine antigens were greater in females than in males. The greater IgG responses correlated with lower splenic Tfr/Tfh and Tfr/GC B cell ratios and greater vaccine antigen-specific proliferative responses of CD4+ and B cells in splenocyte cultures. In both mouse strains IgG2a(c)/IgG1 ratios were comparable between sexes, but lower in BALB/c than in C57BL/6 mice indicating a shift in Th1/Th2 balance towards Th2 response in BALB/c ones. Consistently, splenocytes from BALB/c mice produced more IL-4 and less IFN-gamma than those from C57BL/6 mice. Significance: The study indicated that magnitude of humoral response to influenza type A haemagglutinins depends on mouse strain and sex, and thereby set background for the vaccination strategies taking into account biological sex, and in a longterm perspective individual differences in immune reactivity
Newly designed hemagglutinin-Der p 2 chimera is a potential candidate for allergen specific immunotherapy
Get PDFAim To investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). Main methods: H1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized-metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. Key findings: Purified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. Significance: H1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype.Peer-reviewed manuscript: [http://intor.torlakinstitut.com/handle/123456789/646
Characterisation of peanut allergens and possible post-translational modifications (PTMs)
Get PDFBackgroundPeanut allergy is the most common type of food allergy causing severe reactions or even fatal anaphylaxis in sensitised individuals. The major peanut allergens are Ara h 1, Ara h 2, Ara h 3, and Ara h 6 which cause the most severe responses. Their molecular properties have been characterised but possible post-translational modifications (PTMs) that might explain their severe allergenicity are not well understood. The goal of this study was to utilize a combination of nanoLC-Mass Spectrometry (MS)/MS methods and PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Ontario, Canada) program to evaluate PTMs in the major peanut allergens.MethodAcquired MS data of purified peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6 were analysed and identified via hybridized databases obtained from UniProt (www.uniprot.org).More than 1200 reviewed (Swiss-Prot) and unreviewed (TrEMBL) entries from peanut were combined with common MS contaminants, the Repository of Adventitious Proteins (cRAP), to create a hybridized database. We then focused on Ara h 2 (Conglutin-7) and Ara h 6 (Conglutin) because of their propensity to cause severe anaphylactic reactions. Epitopes found in the Immune Epitope Database (www.iedb.org) were analysed for possible PTMs by matching PEAKS PTM results with mapped positions of epitope sequences.ResultsWe identified 37 proteins from the purified peanut allergens. There were 33 peanut proteins and 4 contaminants originating from human keratin and pig trypsin. Ara h 2 had 242 epitopes, 29 potential PTMs and 4 mutations. Eight of the epitopes had up to 8 possible PTMs. Several relevant PTMs were discovered, including tryptophan oxidation to oxolactone in position 25, sulfonation of N-terminus of cysteine in position 116 and oxidation of methionine in position 50 and 125. Notably, all had either a “NNQRCMCEALQ” or “QQIMENQSD” motif, which are linked to Th2 cytokines and T cell proliferation. We observed 8 epitopes, 9 likely PTMs and no mutations for Ara h 6 and half of the epitopes had possible PTMs and a maximum of 4 PTMs was found on one epitope.ConclusionThe analysis of relevant peanut allergens by nanoLC-MS/MS methods and PEAKS Studio 8.0 program revealed several PTMs, which might have important ramifications due to their influence on allergenicity and digestibility resulting from modification properties by trypsin and other food protein enzymes. These data suggest that PTMs on certain peanut epitopes could be involved in the pathogenesis of severe food allergy to peanuts
The specificity of MccC to peptide-nucleotide antibiotics
No full textMicrocin C-like compounds (McC) are a family of peptidenucleotide
antibiotics, with some of having additional post-translational
modifications. Bioinformatic searches revealed mcc-like
gene clusters in a diverse range of Gram-negative and Grampositive
bacteria. One of these discovered clusters is from S. equinus.
There are four genes in the cluster: mccBSeq, mccASeq,
mccE2S and mccCSeq. A 19 amino-acid peptide precursor molecule
is encoded by mccASeq. The N-terminal domain of MccBSeq
demonstrates an ability to attach a terminal cytidine monophosphate
to the cognate precursor peptide MccASeq. The C-terminal
domain of MccE2SSeq is a carboxy-S-adenosyl-L-methionine
(cx-SAM) synthetase. MccSSeq is responsible for the synthesis of
cx-SAM in the presence of SAM and prephenic acid. Next, the
C-terminal domain of MccBSeq uses cx-SAM as a donor of a carboxymethyl
group for the modification reaction of the cytidine
residue of MccASeq-cytidylate. The entire biosynthetic pathway
of the carboxymethylated peptide-cytidylate (MccASeq-cxCMP)
from S. equinus was reconstructed in vitro. However, this compound
did not demonstrate any antimicrobial activity against
E. coli. MccCSeq is a member of the superfamily of membrane
transport proteins, and is responsible for microcin export from
producing cells. The N-terminal domain of MccE2SSeq is a predicted
acetyltransferase, and may possibly play a role in selfimmunity
to produced microcin. To test this, cells overexpressing
MccE2
Seq from S. equinus were obtained, and conferred immunity
to McC from both E. coli and Y. pseudotuberculosis. This work
was supported by Russia Science Foundation RSF 16-14-10356
Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4
Get PDFThe gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, IliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene IliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two rho-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the IliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci