Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species.

OBJECTIVES: Invasive mould infections are associated with a high mortality rate and the emergence of MDR moulds is of particular concern. Calcineurin and its chaperone, the heat shock protein 90 (Hsp90), represent an important pathway for fungal virulence that can be targeted at different levels. We investigated the antifungal activity of compounds directly or indirectly targeting the Hsp90-calcineurin axis against different mould species. METHODS: The in vitro antifungal activity of the anticalcineurin drug FK506 (tacrolimus), the Hsp90 inhibitor geldanamycin, the lysine deacetylase inhibitor trichostatin A and the Hsp70 inhibitor pifithrin-μ was assessed by the standard broth dilution method against 62 clinical isolates of Aspergillus spp. and non-Aspergillus moulds (Mucoromycotina, Fusarium spp., Scedosporium spp., Purpureocillium/Paecilomyces spp. and Scopulariopsis spp.) RESULTS: FK506 had variable antifungal activity against different Aspergillus spp. and was particularly active against Mucor spp. Geldanamycin had moderate antifungal activity against Fusarium spp. and Paecilomyces variotii. Importantly, trichostatin A had good activity against the triazole-resistant Aspergillus ustus and the amphotericin B-resistant Aspergillus terreus as well as the MDR Scedosporium prolificans. Moreover, trichostatin A exhibited synergistic interactions with caspofungin against A. ustus and with geldanamycin against Rhizopus spp. for which none of the other agents showed activity. Pifithrin-μ exhibited little antifungal activity. CONCLUSIONS: Targeting the Hsp90-calcineurin axis at different levels resulted in distinct patterns of susceptibility among different fungal species. Lysine deacetylase inhibition may represent a promising novel antifungal strategy against emerging resistant moulds.

Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK.

The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase activity. In between the substrate-binding domain and the J-domain anchor to DnaK, DnaJ has a unique domain with four conserved CXXC motives that bind two Zn(2+) and partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, which is characteristic to type I but not of type II or III J-proteins. This caused a loss of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest that in addition to their general ability to target misfolded polypeptide substrates to the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds. Whereas the unfoldase can remain ineffective without the transient untying of disulfide bonds by the foldase, the foldase can remain ineffective without the transient ATP-fuelled unfolding of wrong local structures by the unfoldase.

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.

Immune regulation and function of the NOD-like receptors NLRC5 and NLRP3

AbstractThe vertebrate immune system is composed of the innate and the adaptive branches. Innate immune cells represent the first line of defense and detect pathogens through pattern recognition receptors (PRRs), detecting evolutionary conserved pathogen- and danger- associated molecular patterns. Engagement of these receptors initiates the inflammatory response, but also instructs antigen-specific adaptive immune cells. NOD-like receptors (NLRs) are an important group of PRRs, leading to the production of inflammatory mediators and favoring antigen presentation to Τ lymphocytes through the regulation of major histocompatibility complex (MHC) molecules.In this work we focused our attention on selected NOD-like receptors (NLRs) and their role at the interface between innate and adaptive immunity. First, we describe a new regulatory mechanism controlling IL-1 production. Our results indicate that type I interferons (IFNs) block NLRP1 and NLRP3 inflammasome activity and interfere with LPS-driven proIL-Ια and -β induction. As type I IFNs are produced upon viral infections, these anti-inflammatory effects of type I IFN could be relevant in the context of superinfections, but could also help explaining the efficacy of IFN-β in multiple sclerosis treatment.The second project addresses the role of a novel NLR family member, called NLRC5. The function of this NLR is still matter of debate, as it has been proposed as both an inhibitor and an activator of different inflammatory pathways. We found that the expression of this protein is restricted to immune cells and is positively regulated by IFNs. We generated Nlrc5-deficient mice and found that this NLR plays an essential role in Τ, NKT and, NK lymphocytes, in which it drives the expression of MHC class I molecules. Accordingly, we could show that CD8+ Τ cell-mediated killing of target lymphocytes lacking NLRC5 is strongly impaired. Moreover, NLRC5 expression was found to be low in many lymphoid- derived tumor cell lines, a mechanism that could be exploited by tumors to escape immunosurveillance.Finally, we found NLRC5 to be involved in the production of IL-10 by CD4+ Τ cells, as Nlrc5- deficient Τ lymphocytes produced less of this cytokine upon TCR triggering. In line with these observations, Mrc5-deficient CD4+ Τ cells expanded more than control cells when transferred into lymphopenic hosts and led to a more rapid appearance of colitis symptoms. Therefore, our work gives novel insights on the function of NLRC5 by using knockout mice, and strongly supports the idea that NLRs direct not only innate, but also adaptive immune responses.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Reversible behavioural autistic-like regression: A manifestation of a special (new?) epileptic syndrome in a 28-month-old child. A 2-year longitudinal study .

A 28-month-old boy was referred for acute onset of abnormal head movements. History revealed an insidious progressive regression in behaviour and communication over several months. Head and shoulder 'spasms' with alteration of consciousness and on one occasion ictal laughter were seen. The electroencephalograph (EEG) showed repeated bursts of brief generalized polyspikes and spike-wave during the 'spasms', followed by flattening, a special pattern which never recurred after treatment. Review of family videos showed a single 'minor' identical seizure 6 months previously. Magnetic resonance imaging was normal. Clonazepam brought immediate cessation of seizures, normalization of the EEG and a parallel spectacular improvement in communication, mood and language. Follow-up over the next 10 months showed a new regression unaccompained by recognized seizures, although numerous seizures were discovered during the videotaped neuropsychological examination, when stereotyped subtle brief paroxysmal changes in posture and behaviour could be studied in slow motion and compared with the 'prototypical' initial ones. The EEG showed predominant rare left-sided fronto-temporal discharges. Clonazepam was changed to carbamazepin with marked improvement in behaviour, language and cognition which has been sustained up to the last control at 51 months. Videotaped home observations allowed the documentation of striking qualitative and quantitative variations in social interaction and play of autistic type in relation to the epileptic activity. We conclude that this child has a special characteristic epileptic syndrome with subtle motor and vegetative symptomatology associated with an insidious catastrophic 'autistic-like' regression which could be overlooked. The methods used to document such fluctuating epileptic behavioural manifestations are discussed.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production.

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C(10)OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

Mutation analysis of the different tfd genes for degradation of chloroaromatic compounds in Ralstonia eutropha JMP134.

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfdI cluster) and tfdDII CII EII FII BII (tfdII cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd gene's reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdDII CII EII FII BII genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA ( tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfdI cluster and those in tfdDII, tfdCII, tfdEII and tfdBII were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfdI cluster but also the tfdII cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdDII resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdDII activity.

Ultraviolet induction of antifungal activity in plants.

Ultraviolet-C irradiation as a method to induce the production of plant compounds with antifungal properties was investigated in the leaves of 18 plant species. A susceptibility assay to determine the antifungal susceptibility of filamentous fungi was developed based on an agar dilution series in microtiter plates. UV irradiation strongly induced antifungal properties in five species against a clinical Fusarium solani strain that was responsible for an onychomycosis case that was resistant to classic pharmacological treatment. The antifungal properties of three additional plant species were either unaffected or reduced by UV-C irradiation. This study demonstrates that UV-C irradiation is an effective means of modulating the antifungal activity of very diverse plants from a screening perspective.

Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor.

Activation of the hepatoportal glucose sensors by portal glucose infusion leads to increased glucose clearance and induction of hypoglycemia. Here, we investigated whether glucagon-like peptide-1 (GLP-1) could modulate the activity of these sensors. Mice were therefore infused with saline (S-mice) or glucose (P-mice) through the portal vein at a rate of 25 mg/kg. min. In P-mice, glucose clearance increased to 67.5 +/- 3.7 mg/kg. min as compared with 24.1 +/- 1.5 mg/kg. min in S-mice, and glycemia decreased from 5.0 +/- 0.1 to 3.3 +/- 0.1 mmol/l at the end of the 3-h infusion period. Coinfusion of GLP-1 with glucose into the portal vein at a rate of 5 pmol/kg. min (P-GLP-1 mice) did not increase the glucose clearance rate (57.4 +/- 5.0 ml/kg. min) and hypoglycemia (3.8 +/- 0.1 mmol/l) observed in P-mice. In contrast, coinfusion of glucose and the GLP-1 receptor antagonist exendin-(9-39) into the portal vein at a rate of 0.5 pmol/kg. min (P-Ex mice) reduced glucose clearance to 36.1 +/- 2.6 ml/kg. min and transiently increased glycemia to 9.2 +/- 0.3 mmol/l at 60 min of infusion before it returned to the fasting level (5.6 +/- 0.3 mmol/l) at 3 h. When glucose and exendin-(9-39) were infused through the portal and femoral veins, respectively, glucose clearance increased to 70.0 +/- 4.6 ml/kg. min and glycemia decreased to 3.1 +/- 0.1 mmol/l, indicating that exendin-(9-39) has an effect only when infused into the portal vein. Finally, portal vein infusion of glucose in GLP-1 receptor(-/-) mice failed to increase the glucose clearance rate (26.7 +/- 2.9 ml/kg. min). Glycemia increased to 8.5 +/- 0.5 mmol/l at 60 min and remained elevated until the end of the glucose infusion (8.2 +/- 0.4 mmol/l). Together, our data show that the GLP-1 receptor is part of the hepatoportal glucose sensor and that basal fasting levels of GLP-1 sufficiently activate the receptor to confer maximum glucose competence to the sensor. These data demonstrate an important extrapancreatic effect of GLP-1 in the control of glucose homeostasis.

Daily physical activity assessment: what is the importance of upper limb movements vs whole body movements?

OBJECTIVE: The movement of the upper limbs (eg fidgeting-like activities) is a meaningful component of nonexercise activity thermogenesis (NEAT). This study examined the relationship between upper limb movements and whole body trunk movements, by simultaneously measuring energy expenditure during the course of the day. DESIGN: A cross-sectional study consisting of 88 subjects with a wide range in body mass index (17.3-32.5 kg/m(2)). The energy expenditure over a 24-h period was measured in a large respiratory chamber. The body movements were assessed by two uniaxial-accelerometers during daytime, one on the waist and the other on the dominant arm. The accelerometry scores from level 0 (=immobile) up to level 9 (=maximal intensity) were recorded. The activities of subjects were classified into eight categories: walking at two speeds on a horizontal treadmill (A & B), ambling (C), self-care tasks (D), desk work (E), meals (F), reading (G), watching TV (H). RESULTS: There was a significant relationship between the accelerometry scores from the waist (ACwaist) and that from the wrist (ACwrist) over the daytime period (R(2)=0.64; P<0.001). The ACwrist was systematically higher than the ACwaist during sedentary activities, whereas it was the reverse for walking activities. ACwrist to ACwaist ratio of activities E-H were above 1.0 and for walking activities (A-C) were below 1.0. A multiple regression analysis for predicting daytime energy expenditure revealed that the explained variance improved by 2% only when the ACwrist was added as a second predictor in addition to the ACwaist. This indicates that the effect of the ACwrist for predicting energy expenditure was of limited importance in our conditions of measurement. CONCLUSIONS: The acceleration of the upper limbs which includes fidgeting is more elevated than that of the whole body for sitting/lying down activities. However, their contribution to energy expenditure is lower than whole body trunk movements, thus indicating that the weight-bearing locomotion activities may be a key component of NEAT. However, its contribution may depend on the total duration of the upper limb movements during the course of the day.

Stochastic time-concentration activity models for cytotoxicity in 3D brain cell cultures.

BACKGROUND: In vitro aggregating brain cell cultures containing all types of brain cells have been shown to be useful for neurotoxicological investigations. The cultures are used for the detection of nervous system-specific effects of compounds by measuring multiple endpoints, including changes in enzyme activities. Concentration-dependent neurotoxicity is determined at several time points. METHODS: A Markov model was set up to describe the dynamics of brain cell populations exposed to potentially neurotoxic compounds. Brain cells were assumed to be either in a healthy or stressed state, with only stressed cells being susceptible to cell death. Cells may have switched between these states or died with concentration-dependent transition rates. Since cell numbers were not directly measurable, intracellular lactate dehydrogenase (LDH) activity was used as a surrogate. Assuming that changes in cell numbers are proportional to changes in intracellular LDH activity, stochastic enzyme activity models were derived. Maximum likelihood and least squares regression techniques were applied for estimation of the transition rates. Likelihood ratio tests were performed to test hypotheses about the transition rates. Simulation studies were used to investigate the performance of the transition rate estimators and to analyze the error rates of the likelihood ratio tests. The stochastic time-concentration activity model was applied to intracellular LDH activity measurements after 7 and 14 days of continuous exposure to propofol. The model describes transitions from healthy to stressed cells and from stressed cells to death. RESULTS: The model predicted that propofol would affect stressed cells more than healthy cells. Increasing propofol concentration from 10 to 100 μM reduced the mean waiting time for transition to the stressed state by 50%, from 14 to 7 days, whereas the mean duration to cellular death reduced more dramatically from 2.7 days to 6.5 hours. CONCLUSION: The proposed stochastic modeling approach can be used to discriminate between different biological hypotheses regarding the effect of a compound on the transition rates. The effects of different compounds on the transition rate estimates can be quantitatively compared. Data can be extrapolated at late measurement time points to investigate whether costs and time-consuming long-term experiments could possibly be eliminated.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

PEGylating a bacteriophage endolysin inhibits its bactericidal activity.

ABSTRACT: Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.