When cells and microbes meet in Krakow

The sixth edition of the biannual 'At the Joint Edge of Cellular Microbiology & Cell Biology' meeting took place in October 2010 in Krakow with the support of EMBO. The meeting highlighted that research at the interface between cell biology and microbiology continues to bloom, and that cell biologists still have a lot to learn from bugs, as do microbiologists from cell biology.     

Exogenous ATP enhances calcium influx in intact thymocytes

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.     

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.     

Sorafenib-induced mitochondrial complex I inactivation and cell death in human neuroblastoma cells

Sorafenib is a multikinase inhibitor that is approved for use against renal cell and hepatocellular carcinoma. We found that sorafenib potently induced cell death in human neuroblastoma cells. To understand the molecular basis of sorafenib-mediated cell death in human SH-SY5Y cells, we performed a temporal quantitative proteome analysis. The results showed significant quantitative changes of 193 unique proteins. Bioinformatics-assisted pathway analysis of the regulated proteins revealed that mitochondrial proteins, especially components of the electron transport chain and the mitochondrial ribosomes, were significantly affected upon exposure to sorafenib. The observed down-regulation of the respiratory chain complex I (NADH dehydrogenase) was accompanied with loss of mitochondrial transmembrane potential (Δψm) and complete impairment of complex I enzyme activity. The destabilization of complex I subunits was consistent, rapid, and independent of caspase activation as well as Bcl-2 overexpression. This study provides an overview of the molecular machinery driving sorafenib-mediated cell death in neuroblastoma cells and suggests that sorafenib could be a potential therapeutic drug for the treatment of neuroblastoma.     

Poly-N-Acetylglucosamine Matrix Polysaccharide Impedes Fluid Convection and Transport of the Cationic Surfactant Cetylpyridinium Chloride through Bacterial Biofilms

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix (7). The main function of the biofilm matrix is to provide a structural framework that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In addition to having a structural role, the matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients, secreted enzymes, DNA, and phages. The matrix may also contribute to the increased antimicrobial resistance exhibited by biofilm cells, either by providing a diffusion barrier or by directly binding to antimicrobial agents and preventing their penetration into the biofilm (19).Polysaccharides are a major matrix component in most bacterial biofilms (26). Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm cohesion in numerous gram-negative members of the Proteobacteria family, including Escherichia coli, Yersinia pestis, Pseudomonas fluorescens, Bordetella spp., Xenorhabdus nematophila, Aggregatibacter actinomycetemcomitans, and Actinobacillus pleuropneumoniae (4, 8, 15, 22), and in the gram-positive species Staphylococcus aureus and Staphylococcus epidermidis (3, 17). Specific biofilm-related functions ascribed to PNAG include abiotic surface attachment (1), epithelial cell attachment (23, 28), intercellular adhesion (15, 17), and resistance to killing by antibiotics, detergents, antimicrobial peptides, and mammalian phagocytic cells (9, 10, 16, 27, 29).In the present study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced by the porcine respiratory pathogen A. pleuropneumoniae and the device-associated pathogen S. epidermidis. By using a novel centrifugal filter device assay, we obtained evidence that PNAG significantly inhibits fluid convection and solute transport through A. pleuropneumoniae and S. epidermidis biofilms.     

Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors.

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.     

Negative modulation of human NK cell activity by purinoceptors. 1. Effect of exogenous adenosine triphosphate.

A variety of receptors, including purinergic receptors (for adenosine and adenosine nucleotides), are present on lymphoid cells. We have investigated the negative modulatory action of ATP, a high-energy purinergic compound, on the human peripheral blood natural killer (NK) cells. Micromolar doses of exogenous ATP produced a strong, direct, reversible, and dose-dependent (2.5 x 10(-5) to 8 x 10(-4) M) inhibition of NK cell-mediated cytotoxic activity in 98% of the 131 healthy donors tested. The inhibitory effect of ATP could be demonstrated in unseparated total lymphocytes and enriched NK cells (Percoll density gradient or negative selection by complement lysis) in 2- or 16-hr NK assays, at various effector:target ratios, at suboptimal and saturating concentrations of the K562 targets. The inhibitory constants ID20 and ID50 max for ATP (2 hr), which were 5.6 x 10(-5) and 8 x 10(-5) M, respectively, appear to be a relatively stable feature of an individual. The mechanism of action of ATP was also explored. It is not due to a simple chelation by ATP of the divalent cations Mg2+ and Ca2+ which play a role in the binding/lytic reactions during NK cell-mediated oncolysis. ATP treatment of lymphocytes neither nonspecifically decreased the number of viable cells nor specifically eliminated the NK cells, including their subsets, CD57+16-, CD57+16+, and CD57-16+ as revealed by immunofluorescence staining. Use of an enzyme-like kinetic approach to define the nature of ATP-induced inhibition suggests that it may be an "uncompetitive" type (Lineweaver-Burke reciprocal plot), because both Vmax (maximal velocity of oncolysis) and Km (Michaelis constant) were reduced simultaneously from 1.53 x 10(4) and 2.64 x 10(4) to 0.44 x 10(4) and 0.73 x 10(4), respectively, in presence of ATP. Other methods (Eadie-Hofstee's and Hane's equations) confirmed that ATP does elicit a qualitatively and quantitatively similar shift in these kinetic parameters. Finally, an 18-hr preactivation of effectors by interleukin 2 (IL2) resulted in partial protection from the negative modulation of NK activity by ATP, while the presence of ATP during preincubation did not interfere with the desensitizing effect of IL2. The biological relevance of these findings are discussed and it is concluded that by interacting with appropriate (non-FcRIII?) cell-surface binding sites such as P2 purinergic receptors, ATP is capable of negatively modulating the antitumor cytotoxic activity of human NK cells. This down-regulation can be partially overcome by IL2 that up-regulates NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transconjugation studies in <Emphasis Type="Italic">Azospirillum</Emphasis> sp. negative to mineral phosphate solubilization

It has been previously shown that certain gram-negative bacteria do not have the ability to solubilize insoluble phosphates due to the lack of pyrroloquinoline-quinone synthesis genes (pqq). PQQ is required as cofactor for the assembly of the glucose dehydrogenase (GDH) holoenzyme, which acts in the oxidation of glucose to gluconic acid. In this context the transconjugation and expression of pqq genes in Azospirillum sp. was studied using the construct pMCG 898. pMCG 898 containing pqq gene/s was mobilized into an Azospirillum strain negative to mineral phosphate solubilization by biparental mating. The presence of the construct was also confirmed by minipreps of the transconjugants. The transconjugants were able to solubilize dicalcium phosphate while the wild type was not able to do so. The nitrogen-fixing ability of the transconjugants was also examined and they retained the ability to fix nitrogen. Further detailed studies are required to confirm the utility of such strains in releasing inorganic P from fixed phosphates in soil.     

ERK5 modulates IL-6 secretion and contributes to tumor-induced immune suppression

Tumors exhibit a variety of strategies to dampen antitumor immune responses. With an aim to identify factors that are secreted from tumor cells, we performed an unbiased mass spectrometry-based secretome analysis in lung cancer cells. Interleukin-6 (IL-6) has been identified as a prominent factor secreted by tumor cells and cancer-associated fibroblasts isolated from cancer patients. Incubation of dendritic cell (DC) cultures with tumor cell supernatants inhibited the production of IL-12p70 in DCs but not the surface expression of other activation markers which is reversed by treatment with IL-6 antibody. Defects in IL-12p70 production in the DCs inhibited the differentiation of Th1 but not Th2 and Th17 cells from naïve CD4⁺ T cells. We also demonstrate that the classical mitogen-activated protein kinase, ERK5/MAPK7, is required for IL-6 production in tumor cells. Inhibition of ERK5 activity or depletion of ERK5 prevented IL-6 production in tumor cells, which could be exploited for enhancing antitumor immune responses.Subject terms: Cancer microenvironment, Extracellular signalling molecules