LPS-primed CD11b+ leukocytes serve as an effective carrier of Shiga toxin 2 to cause hemolytic uremic syndrome in mice.

Shiga toxin (Stx)-induced hemolytic uremic syndrome (HUS) is a life-threatening complication associated with Stx-producing Escherichia coli infection. One critical barrier of understanding HUS is how Stx transports from infected intestine to kidney to cause HUS. Passive dissemination seems unlikely, while circulating blood cells have been debated to serve as the toxin carrier. Employing a murine model of Stx2-induced HUS with LPS priming (LPS-Stx2), we investigate how Stx causes HUS and identify possible toxin carrier. We show that peripheral white blood cells (WBC), but not other blood cells or cell-free plasma, carry Stx2 in LPS-Stx2-treated mice. The capability of WBC binding to Stx2 is confirmed in brief ex vivo Stx2 incubation, and adoptively transferring these Stx2-bound WBC into mice induces HUS. Cell separation further identifies a subpopulation in the CD11b+ myeloid leukocytes not the CD11b- lymphocytes group act as the toxin carrier, which captures Stx2 upon exposure and delivers the toxin in vivo. Interestingly, LPS-induced inflammation significantly augments these leukocytes for binding to Stx2 and enhances HUS toxicity. Our results demonstrate that a specific fraction of circulating leukocytes carry Stx2 and cause HUS in vivo, and that LPS priming enhances the carrier capacity and aggravates organ damage.

Loss of Cell Surface CD47 Clustering Formation and Binding Avidity to SIRPα Facilitate Apoptotic Cell Clearance by Macrophages.

CD47, a self recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies suggested that cells may lose surface CD47 during aging or apoptosis to enable phagocytic clearance. In the current study, we demonstrate that the level of cell surface CD47 is not decreased, but the distribution pattern of CD47 is altered, during apoptosis. On nonapoptotic cells, CD47 molecules are clustered in lipid rafts forming punctates on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicits SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated with a significant reduction in the binding avidity to SIRPα and a failure to trigger SIRPα signal transduction. Disruption of plasma membrane lipid rafts with methyl-ß-cyclodextrin diffuses CD47 clusters, leading to a decrease in the cell binding avidity to SIRPα and a concomitant increase in cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on nonapoptotic cells but is diffused in the plasma membrane when apoptosis occurs; this transformation of CD47 greatly reduces the strength of CD47-SIRPα engagement, resulting in the phagocytosis of apoptotic cells.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits.

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

Biochemical and functional characterization of CpMuc4, a Cryptosporidium surface antigen that binds to host epithelial cells.

Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a Cryptosporidium parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in Escherichia coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in Toxoplasma gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.

Genetic prerequisites for additive or synergistic actions of 5-fluorocytosine and fluconazole in baker's yeast.

During applications of 5-fluorocytosine (5FC) and fluconazole (FLC), additive or synergistic action may even occur when primary resistance to 5FC is established. Here, we analysed conjoint drug action in Saccharomyces cerevisiae strains deficient in genes known to be essential for 5FC or FLC function. Despite clear primary resistance, residual 5FC activity and additive 5FC+FLC action in cells lacking cytosine permease (Fcy2p) or uracil phosphoribosyl transferase (Fur1p) were detected. In contrast, Deltafcy1 mutants, lacking cytosine deaminase, became entirely resistant to 5FC, concomitantly losing 5FC+FLC additivity. Disruption of the orotate phosphoribosyltransferase gene (URA5) in the wild-type led to low-level 5FC tolerance, while an alternative orotate phosphoribosyltransferase, encoded by URA10, contributed to 5FC toxicity only in the Deltaura5 background. Remarkably, combination of Deltaura5 and Deltafur1 resulted in complete 5FC resistance. Thus, yeast orotate phosphoribosyltransferases are involved in 5FC metabolism. Similarly, disruption of the ergosterol Delta(5,6)-desaturase-encoding gene ERG3 resulted only in partial resistance to FLC, and concomitantly a synergistic effect with 5FC became evident. Full resistance to FLC occurred in Deltaerg3 Deltaerg11 double mutants and, simultaneously, synergism or even an additive effect with FLC and 5FC was no longer discernible. Since the majority of spontaneously occurring resistant yeast clones displayed residual sensitivity to either 5FC or FLC and those strains responded to combined drug treatment in a predictable manner, careful resistance profiling based on the findings reported here may help to address yeast infections by combined application of antimycotic compounds.

The primary target of the killer toxin from Pichia acaciae is tRNA(Gln).

The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S-phase of the cell cycle and induces DNA double-strand breaks (DSBs). Surprisingly, loss of the tRNA-methyltransferase Trm9 - along with the Elongator complex involved in synthesis of 5-methoxy-carbonyl-methyl (mcm(5)) modification in certain tRNAs - conferred resistance against PaT. Overexpression of mcm(5)-modified tRNAs identified tRNA(Gln)((UUG)) as the intracellular target. Consistently, toxin-challenged cells displayed reduced levels of tRNA(Gln) and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA(Gln)((UUG)). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA(Glu)((UUC)), affecting its target in a mcm(5)-dependent manner, PaT exerts activity also on tRNA(Gln) lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNA(Gln)((UUG)), tRNA(Gln)((CUG)) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA(Gln)((CUG)) as an additional TRM9-independent target, overexpression of PaT's tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.

Pichia acaciae killer system: genetic analysis of toxin immunity.

The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.

Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae.

5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues.