Inhibits Pore Formation Research Articles

TRIM21 inhibits irradiation-induced mitochondrial DNA release and impairs antitumour immunity in nasopharyngeal carcinoma tumour models

Although radiotherapy can promote antitumour immunity, the mechanisms underlying this phenomenon remain unclear. Here, we demonstrate that the expression of the E3 ubiquitin ligase, tumour cell-intrinsic tripartite motif-containing 21 (TRIM21) in tumours, is inversely associated with the response to radiation and CD8+ T cell-mediated antitumour immunity in nasopharyngeal carcinoma (NPC). Knockout of TRIM21 modulates the cGAS/STING cytosolic DNA sensing pathway, potentiates the antigen-presenting capacity of NPC cells, and activates cytotoxic T cell-mediated antitumour immunity in response to radiation. Mechanistically, TRIM21 promotes the degradation of the mitochondrial voltage-dependent anion-selective channel protein 2 (VDAC2) via K48-linked ubiquitination, which inhibits pore formation by VDAC2 oligomers for mitochondrial DNA (mtDNA) release, thereby inhibiting type-I interferon responses following radiation exposure. In patients with NPC, high TRIM21 expression was associated with poor prognosis and early tumour relapse after radiotherapy. Our findings reveal a critical role of TRIM21 in radiation-induced antitumour immunity, providing potential targets for improving the efficacy of radiotherapy in patients with NPC.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Antimicrobial peptides inhibit pore formation by redistribution across the membrane

With the increase of resistance to conventional antibiotics development of antimicrobial peptide (AMP) therapy becomes an extremely relevant. To define the balance between AMP toxicity and efficiency in medical treatment it is utterly important to gain a full picture of peptide activity. Using the patch-clamp technique and planar lipid bilayers, we clarified mechanisms of pore formation induced by melittin and magainin-I, and its subsequent inhibition. Kinetics of change of membrane conductivity under action of these antimicrobial peptides demonstrated the transient nature of the pores.

Antibiotic Effects of Volatiles Produced by Bacillus tequilensis XK29 against the Black Spot Disease Caused by Ceratocystis fimbriata in Postharvest Sweet Potato.

Black spot disease caused by Ceratocystis fimbriata is destructive to the production, transportation, and storage of sweet potato. The antifungal effects of Bacillus tequilensis XK29 against C. fimbriata through volatile organic compounds (VOCs) were evaluated in this study. The activated carbon assay proved that XK29 could exert antibiotic effects through volatiles. By optimizing the wheat seed weight, inoculation method, concentration, volume, and time, the antifungal activity of XK29 was significantly improved. XK29 fumigation inhibited spore formation and germination and changed the cell morphology of C. fimbriata. During the storage of sweet potato tuber roots, XK29 effectively controlled black spot disease and reduced the weight loss and malondialdehyde content. Metabolomic analysis revealed that 21 volatile compounds were released from XK29. Isovaleric acid, isobutyric acid, and 2-methylbutanoic acid effectively inhibited the growth of C. fimbriata. These results indicate that B. tequilensis XK29 has a good potential to be developed as a microbial fumigation agent.

Role of cholesterol flip-flop in oxidized lipid bilayers

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0–50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol’s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.

Antifungal properties of recombinant Puroindoline B protein against aflatoxigenic Aspergillus flavus

Aspergillus flavus contamination is of great concern to food safety. Our previous studies demonstrated that recombinant Puroindoline B protein (rPINB) has an inhibitory effect on A. flavus mycelium development. However, fungal contamination often begins with spore germination within food shelf life. Here, rPINB was evaluated for its antifungal properties against A. flavus spores in vivo and in vitro. The results showed that rPINB could inhibit spore formation and aflatoxin B1 of A. flavus on maize. In vitro analysis revealed its dose-dependent antifungal effect on spore germination and germ tube growth of A. flavus. Scanning electron microscopy revealed that rPINB exposure altered spore morphology, indicating it damaged the cell wall. Transmission electron microscopy and the inhibition of ergosterol synthesis further revealed that rPINB exposure significantly damaged the cell membrane. The mitochondrial membrane potential decreased, and DNA fragments were scattered in the cytoplasm as detected using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide and 4′,6-diamidino-2-phenylindole staining. The direct antifungal activity of rPINB observed both in vivo and in vitro suggests that rPINB may be used to prevent fungal contamination of stored foods and agricultural products in the future.

Anti-fungal activity of lactic acid bacterial isolates against aflatoxigenic fungi inoculated on peanut kernels

Aflatoxigenic fungi infect various agricultural commodities and produce a class I carcinogen aflatoxin B1 (AFB1). Sixteen bacterial isolates were screened from idli batter and other fermented foods, for antagonistic effect against Aspergillus spp. Based on the preliminary observation, three bacterial isolates from idli batter namely, IB1, IB4, IB7, and one from mango wine namely, MW2 were selected and further characterized. IB1 was found to be closely related to Lactobacillus plantarum argentoratensis while IB4, IB7, and MW2 were closely related to Enterococcus faecium according to their 16sRNA sequencing. Invitro and invivo antifungal tests in peanut revealed that cell free supernatant (CFS) obtained from these isolates inhibited fungal growth and significantly suppressed AFB1 production, and expression of aflD, aflP, OrdB and aflR genes. Further, microscopic examination showed that all isolates could reduce the conidial development and inhibits spore formation. The highest antifungal activity was observed in CFS of L. plantarum MN880325 and E. faecium MN880376, with an inhibition zone of ≥28 mm in PDA and ≥97% reduction in aflatoxin production on peanut kernels. ESI-MS and NMR spectroscopy revealed the active compounds from L. plantarum MN880325 and E. faecium MN880376 to be 3-phenyllactic acid and 4-hydroxyphenyllactic acid respectively.

Neuroprotective effects of natural cordycepin on LPS-induced Parkinson’s disease through suppressing TLR4/NF-κB/NLRP3-mediated pyroptosis

• LPS resulted in TLR4-mediated NF-κB activation and NLRP3-dependent pyroptosis both in vivo and in vitro. • Cordycepin treatment ameliorated locomotor impairments and reversed DA degeneration. • Treatment with cordycepin inhibited the activation of NLRP3 inflammasome through TLR4/NF-κB signaling pathways. • Cordycepin administration suppressed LPS-induced NLRP3-depedent pyroptosis and inflammatory cascades. Cordycepin extracted from Cordyceps militaris , an edible and rare caterpillar fungus, possess neuroprotective and anti-inflammatory activity. Neuroinflammation has been implicated in the onset and development of Parkinson’s disease (PD). Notably, pyroptosis is a new form of programmed cell death which plays an important role in inflammation. However, the roles of pyroptosis and cordycepin on anti-pyroptosis in PD remain unclear. This study was designed to investigate neuroprotective effects of cordycepin in LPS-treated C57BL/6J mice and LPS- treated BV2. The results showed that cordycepin ameliorated LPS-induced PD symptoms and suppressed TLR4/NF-κB-mediated NLRP3 inflammasome activation and GSDMD-related pyroptosis. Additionally, cordycepin remarkably inhibited pore formation in the plasma membrane and reduced the release of proinflammatory mediators in vitro , which is associated with the inhibition of NLRP3-dependent pyroptosis. Collectively, cordycepin exerts neuroprotective activity by regulating TLR4/NF-κB/NLRP3-dependent pyroptosis, which should be developed as healthcare food or natural medicine for the treatment of PD in the future.

Biocontrol of Colletotrichum falcatum with volatile metabolites produced by endophytic bacteria and profiling VOCs by headspace SPME coupled with GC–MS

A total of 49 endophytic bacteria isolated from sugarcane were screened in vitro for antagonistic property against C. falcatum through production of volatile organic compounds (VOCs). Among them, 27 bacteria produced volatiles with moderate inhibitory level, i.e. 30 to 50% and 9 produced volatiles with strong inhibitory properties, i.e. > 50% mycelial growth inhibition over control. The volatile compounds produced by B. axarquiensis—ESR 7 inhibited C. falcatum mycelia growth to the tune of 59.2% followed by B. licheniformis—ESR 26 (57.8%) and B. subtilis—ESR 24 (54.8%), respectively. The volatiles produced by bacteria not only inhibited the radial growth of mycelium but also suppressed the vertical expansion of mycelia and caused deformation in mycelia growth. The VOCs produced by 24 endophytic bacteria completely inhibited spore formation in C. falcatum culture. Profiling of antagonistic VOCs produced by bacterial strains ESR 7, ESR 24 and ESR 26 was done by head space-solid phase microextraction (SPME) coupled with gas chromatography mass spectral analysis. The analysis showed the presence of 63 compounds belonging to chemical groups of alcohols, esters, hydrocarbons, ketones, acids, amino acid, carbohydrates, ethers, aldehydes, amines and amides. Among the identified microbial volatiles, 6 compounds viz., acetic acid, methoxy-phenyl-oxime, octamethyl-cyclotetrasiloxane, 5,7-dimethyl-undecane, hexamethyl-cyclotrisiloxane and dodecane were reported in VOCs produced by all three bacteria. However, among 63 volatiles, only 31 were already reported to be produced by many bacteria and fungi and 11 compounds viz., acetic acid, hexanal, 2-ethyl-1-hexanol, undecane 5,7-dimethyl, undecane 3,7-dimethyl, 2-decanone, dodecane, 2-undecanone, 2-dodecanone, 1,2-Benzenedicarboxylic acid, diisooctyl ester and 2-methyl-hexadecanol were reported with antagonistic property against many plant pathogens. The study revealed that many VOCs produced by B. axarquiensis—ESR 7, B. subtilis—ESR 24 and B. licheniformis—ESR 26 play role in mediating antagonism against C. falcatum.

Effects of SpoIVA on the formation of spores and crystal protein in Bacillus thuringiensis

In addition to forming spores, Bacillus thuringiensis (Bt) 4.0718 can produce toxins, insecticidal crystal protein (ICP) and vegetative insecticidal protein (Vip). The Bt spoIVA was successfully knocked out by gene recombination and was shown to inhibit sporulation. The mutant strain also exhibited significantly decreased growth and crystal formation, which inhibited spore formation and partially reduced the rate of crystal synthesis.The 50 % lethal concentrations (LC50) values of Bt 4.0718, replacement, complementation and multi-copy mutant strains against the fourth larval stage of H. armigera was determined as 5.422, 6.776, 6.223 and 5.018 μg/mL, respectively. A total of 1814 proteins were identified through isobaric tags for relative and absolute protein (iTRAQ), with 41 and 54 up and downregulated proteins observed. Gene ontology enrichment analysis showed that differentially expressed proteins were primarily involved in the biological process and molecular function. Quantitative real-time PCR analysis confirmed that 9 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The results of this study provide a basis for further studies of the metabolic regulatory network of spores and crystal protein formation. Moreover, they can be used to ecologically safe insecticide of farmland production because the constructed Bt spoIVA mutants did not produce spores.Provides new ideas for the targeted improvement and application of environmentally friendly spore-free strains.

Inhibitory effect of fidaxomicin on biofilm formation in Clostridioides difficile

Clostridioides difficile infection results from a disturbance of the normal microbial flora of the colon, allowing proliferation of C. difficile and toxin production by toxigenic strains. Fidaxomicin, a macrocyclic antibiotic that prevents RNA synthesis in C. difficile and inhibits spore formation, toxin production, and cell proliferation, is clinically effective in treating C. difficile infection. As recent studies have suggested that biofilm formation influences C. difficile colonization and infection in the colon, we undertook the present study to determine the effects of fidaxomicin on C. difficile biofilm formation. Sub-minimum inhibitory concentrations (MICs) of fidaxomicin inhibited biofilm formation by C. difficile UK027 and delayed planktonic growth. Sub-MICs of vancomycin did not inhibit biofilm formation or affect planktonic growth. In C. difficile UK027 exposed to sub-MICs of fidaxomicin, mRNA expression of biofilm-related flagellin gene fliC was slightly increased compared with that of other biofilm-related genes (pilA1, cwp84, luxS, dccA, and spo0A). In conclusion, this study indicates that sub-MICs of fidaxomicin inhibit C. difficile UK027 biofilm formation by influencing cell growth and fliC transcription.

Kinetoplastid Membrane Protein-11 Induces Pores in Anionic Phospholipid Membranes: Effect of Cholesterol

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.

Sounding the Alarm and Putting Out the Fire: New Mechanistic Insights into Inflammatory Cell Death

Inflammasomes are multi‐protein signaling scaffolds that assemble in response to invasive pathogens and sterile danger signals to activate inflammatory caspases, which trigger inflammatory death (pyroptosis) and processing and release of pro‐inflammatory cytokines. Inflammasome activation contributes to many human diseases, including sepsis, the often‐fatal response to systemic infection. We recently found that gasdermin D (GSDMD) causes pyroptosis by forming pores in the plasma membrane after it is cleaved by the inflammatory caspases. The N‐terminal fragment binds to acidic phospholipids on the inner leaflet. GSDMD pores also assemble on bacterial membranes to kill the bacteria that trigger pyroptosis. Cryo‐electron microscopy was used to solve the structure of the pore formed by 27 monomers of the related gasdermin, gasdermin A3. The N‐terminal fragment undergoes radical conformational changes after it binds to lipids to assemble into a 108‐stranded b‐barrel that inserts into the membrane to form the pore. A small molecule screen identified several compounds that inhibit pore formation by covalently binding to a reactive cysteine in GSDMD and also inhibit other steps in inflammasome priming and assembly and caspase activation. One of the inhibitors, disulfiram, which is a drug used to treat alcohol dependence, promoted survival and reduced inflammatory cytokine secretion in LPS‐induced sepsis in mice.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection.

Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein (ACP) reductase II (FabK), which catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed that C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using ClosTron mutagenesis and by growth inhibition upon gene silencing with CRISPR interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazole-derived inhibitor (1). The inability of fatty acids to bypass FASII inhibition is likely due to the function of the transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti- C. difficile drugs that treat CDI but avoid collateral damage to the gut microbiota.

Identification of Cyclic Dipeptides from Escherichia coli as New Antimicrobial Agents against Ralstonia Solanacearum.

Ralstonia solanacearum is a causative agent of bacterial wilt in many important crops throughout the world. How to control bacterial wilt caused by R. solanacearum is a major problem in agriculture. In this study, we aim to isolate the biocontrol agents that have high efficacy in the control of bacterial wilt. Three new bacterial strains with high antimicrobial activity against R. solanacearum GMI1000 were isolated and identified. Our results demonstrated that these bacteria could remarkably inhibit the disease index of host plant infected by R. solanacearum. It was indicated that strain GZ-34 (CCTCC No. M 2016353) showed an excellent protective effect to tomato under greenhouse conditions. Strain GZ-34 was characterized as Escherichia coli based on morphology, biochemistry, and 16S rRNA analysis. We identified that the main antimicrobial compounds produced by E. coli GZ-34 were cyclo(l-Pro-d-Ile) and cyclo(l-Pro-l-Phe) using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) analysis. The two active compounds also interfered with the expression levels of some pathogenicity-contributors of R. solanacearum. Furthermore, cyclo(l-Pro-l-Phe) effectively inhibited spore formation of Magnaporthe grisea, which is a vital pathogenesis process of the fungal pathogen, suggesting cyclic dipeptides from E. coli are promising potential antimicrobial agents with broad-spectrum activity to kill pathogens or interfere with their pathogenesis.

Alpha-tocopherol inhibits pore formation in oxidized bilayers.

In biological membranes, alpha-tocopherols (α-toc; vitamin E) protect polyunsaturated lipids from free radicals. Although the interactions of α-toc with non-oxidized lipid bilayers have been studied, their effects on oxidized bilayers remain unknown. In this study, atomistic molecular dynamics (MD) simulations of oxidized lipid bilayers were performed with varying concentrations of α-toc. Bilayers with 1-palmitoyl-2-lauroyl-sn-glycero-3-phosphocholine (PLPC) lipids and their aldehyde derivatives at a 1 : 1 ratio were studied. Our simulations show that oxidized lipids self-assemble into aggregates with a water pore rapidly developing across the bilayer. The free energy of transporting an α-toc molecule in a bilayer suggests that α-tocs can passively adsorb into it. When α-toc molecules were present at low concentrations in bilayers containing oxidized lipids, water pore formation was slowed down. At high α-toc concentrations, no pores were observed. Based on the simulations, we propose that the mechanism of how α-toc inhibits pore formation in bilayers with oxidized lipids is the following: α-tocs trap the polar groups of the oxidized lipids at the membrane-water interface resulting in a decreased probability of the oxidized lipids making contact with the two leaflets and initiating pore formation. This demonstrates that α-toc molecules not only protect the bilayer from oxidation but also help to stabilize the bilayer after lipid peroxidation occurs. These results will help in designing more efficient molecules to protect membranes from oxidative stress.

Sulforaphane is a Nrf2-independent inhibitor of mitochondrial fission

The KEAP1-Nrf2-ARE antioxidant system is a principal means by which cells respond to oxidative and xenobiotic stresses. Sulforaphane (SFN), an electrophilic isothiocyanate derived from cruciferous vegetables, activates the KEAP1-Nrf2-ARE pathway and has become a molecule-of-interest in the treatment of diseases in which chronic oxidative stress plays a major etiological role. We demonstrate here that the mitochondria of cultured, human retinal pigment epithelial (RPE-1) cells treated with SFN undergo hyperfusion that is independent of both Nrf2 and its cytoplasmic inhibitor KEAP1. Mitochondrial fusion has been reported to be cytoprotective by inhibiting pore formation in mitochondria during apoptosis, and consistent with this, we show Nrf2-independent, cytoprotection of SFN-treated cells exposed to the apoptosis-inducer, staurosporine. Mechanistically, SFN mitigates the recruitment and/or retention of the soluble fission factor Drp1 to mitochondria and to peroxisomes but does not affect overall Drp1 abundance. These data demonstrate that the beneficial properties of SFN extend beyond activation of the KEAP1-Nrf2-ARE system and warrant further interrogation given the current use of this agent in multiple clinical trials.

CBid, Bax and Bcl-xL exhibit opposite membrane remodeling activities.

The proteins of the Bcl-2 family have a crucial role in mitochondrial outer membrane permeabilization during apoptosis and in the regulation of mitochondrial dynamics. Current models consider that Bax forms toroidal pores at mitochondria that are responsible for the release of cytochrome c, whereas Bcl-xL inhibits pore formation. However, how Bcl-2 proteins regulate mitochondrial fission and fusion remains poorly understood. By using a systematic analysis at the single vesicle level, we found that cBid, Bax and Bcl-xL are able to remodel membranes in different ways. cBid and Bax induced a reduction in vesicle size likely related to membrane tethering, budding and fission, besides membrane permeabilization. Moreover, they are preferentially located at highly curved membranes. In contrast, Bcl-xL not only counterbalanced pore formation but also membrane budding and fission. Our findings support a mechanism of action by which cBid and Bax induce or stabilize highly curved membranes including non-lamellar structures. This molecular activity reduces the energy for membrane remodeling, which is a necessary step in toroidal pore formation, as well as membrane fission and fusion, and provides a common mechanism that links the two main functions of Bcl-2 proteins.

α-Defensins partially protect human neutrophils against Panton-Valentine leukocidin produced by Staphylococcus aureus.

α-Defensins produced by neutrophils are important effector molecules of the innate immune system. In addition to their microbicidal effects, α-defensins have the ability to neutralize bacterial toxins. Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus. Staphylococcus aureus that produce PVL are responsible for severe diseases, including necrotizing pneumonia. Polymorphonuclear neutrophils (PMNs) are the target cells of PVL action. The goal of this study was to elucidate the effect of a group of α-defensins known as the human neutrophil peptides (HNPs) on the interactions between LukS-PV and LukF-PV, which compose PVL, and human PMNs. We observed that HNPs bound to both subunits of PVL and significantly decreased PVL pore formation in PMNs, with a maximum inhibition of 27%. When various HNP molecules were tested individually under the same conditions, we observed that HNP3, but not HNP1 or 2, decreased pore formation. Similarly, HNP3 significantly decreased PVL-induced PMN lysis, with a maximum inhibition of 31%. Interestingly, HNPs did not affect LukS-PV LukF-PV oligomerization, LukS-PV LukF-PV binding to PMNs or calcium influx induced by PVL in PMNs. Our results suggest that HNP3 partially protects neutrophils against PVL by interfering with the conformational changes of PVL required to form a functional pore. Panton-Valentine leukocidin (PVL) is a pore-forming toxin produced by Staphylococcus aureus, responsible for neutrophil damage and key player of severe staphylococcal diseases. Antimicrobial peptides produced by neutrophils (HNP1-3) neutralize several other bacterial cytotoxins. We examined the impact of human neutrophil peptides (HNPs) on PVL cytotoxicity against human neutrophils and we found that HNPs bind to both LukS and LukF components of PVL, thereby inhibiting pore formation and neutrophil lysis. Our results suggest that HNP3 may impair PVL conformational changes required to form a functional pore and provide insight into the pathogenesis of PVL-related staphylococcal infection, with potential impact on the disease outcome.

Bacillus megaterium 501rif INTRODUCED INTO THE SOIL: FACTORS AFFECTING THE RATE OF SURVIVAL, SPORULATION AND DECOMPOSITION OF THE HERBICIDE PROMETRYN

Introduction of microorganisms into the soil is a fundamental problem in application of microbial preparations in agriculture, crop production and ecology. Its effectiveness depends on many factors (i.e. the type and strain of microorganism, the physical and chemical properties of the soil, plant cover, climate and so on), which are not well understood, leading to a high variability of the results of application of microbial preparations. In this article, there is reported the first investigation of survival and sporulation dynamics of Bacillus megaterium 501 rif introduced into sodpodzolic soil, depending on the temperature of the soil, organic matter content and herbicide prometryn, the 2-methylthio-4,6-bis (iso-propylamino)-sim-triazine, application. In laboratory experiments it was found that the optimal conditions for the B. megaterium 501 rif introduced into the soil are achieved at a temperature of 20 °N. After the third day after inoculation the titer of bacteria in the soil increased almost 10 times and remained at that level until the end of the experiment (32 days). B. megaterium 501 rif throughout the experiment was mainly in the form of a physiologically active cells and fits well into the soil microbiom. Under these conditions an additional incorporation into the soil of the organic matter, such as cereal straw and corn flour, leads to 10-100-fold increase of the amount of B. megaterium 501 rif . At 37 °N the total number of B. megaterium 501 rif reduced several times, and stabilized at this level until the end of the experiment. In this case, 100 % of B. megaterium 501rif revert into spores and are physiologically inactive. The temperature below the minimum for physiological reproduction of B. megaterium (4 °N) inhibits spore formation and leads to a rapid loss of physiologically active bacterial cells introduced into the soil. The total number of bacteria decreases 1000 times within a month. These data lead to the conclusion that the «low-temperature shock» reduces the competitiveness of B. megaterium to the indigenous microflora and its resistance to bactericidal metabolic products of soil biome. It is shown that at the optimum temperature (20 °N) of soil the B. megaterium inoculation and the use of additional organic matter such as straw accelerate the decomposition of herbicide prometryn, can potentially be used in a biotechnology of remediation of the soil, contaminated by herbicide.