ABSTRACT
Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)-producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A-subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re-evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS-pathogenesis and to develop therapeutic approaches.
Subject(s)
Escherichia coli/chemistry , Shiga Toxin 2/chemistry , Shiga Toxin 2/metabolism , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Chlorocebus aethiops , Circular Dichroism , Complement Factor H/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescence , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Protein Binding , Protein Conformation , Shiga Toxin 2/genetics , Trihexosylceramides/metabolism , Trypsin , Vero CellsABSTRACT
Candida species were tested for susceptibility to caspofungin, anidulafungin, and micafungin in order to evaluate the roles of Etest and Sensititre YeastOne in antifungal susceptibility testing for daily routines and to survey resistance. A total of 104 Candida species isolates detected from blood cultures were investigated. With EUCAST broth microdilution as the reference method, essential agreement (EA), categorical agreement (CA), very major errors (VME), major errors (ME), and minor (MIN) errors were assessed by reading MICs at 18, 24, and 48 h. By use of EUCAST broth microdilution and species-specific clinical breakpoints (CBPs), echinocandin resistance was not detected during the study period. Using EUCAST CBPs, MIC readings at 24 h for the Etest and Sensititre YeastOne resulted in CA levels of 99% and 93% for anidulafungin and 99% and 97% for micafungin. Using revised CLSI CBPs for caspofungin, CA levels were 92% and 99% for Etest and Sensititre YeastOne. The Etest proved an excellent, easy-to-handle alternative method for testing susceptibility to anidulafungin and micafungin. Due to misclassifications, the Etest is less suitable for testing susceptibility to caspofungin (8% of isolates falsely tested resistant). The CA levels of Sensititre YeastOne were 93% and 97% for anidulafungin and micafungin (24 h) by use of EUCAST CBPs and increased to 100% for both antifungals if CLSI CBPs were applied and to 100% and 99% if Sensititre YeastOne epidemiological cutoff values (ECOFFs) were applied. No one echinocandin could be demonstrated to be superior to another in vitro Since resistance was lacking among our Candida isolates, we cannot derive any recommendation from accurate resistance detection by the Etest and Sensititre YeastOne.
Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Candida/isolation & purification , Echinocandins/pharmacology , Lipopeptides/pharmacology , Anidulafungin , Austria , Candida/classification , Candidiasis/drug therapy , Candidiasis/microbiology , Caspofungin , Disk Diffusion Antimicrobial Tests , Drug Resistance, Fungal/genetics , Humans , MicafunginABSTRACT
Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.
