A rivet model for channel formation by aerolysin-like pore-forming toxins

The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. Surprisingly, we found that the turn of the beta-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.     

Membrane channel formation by antimicrobial protegrins.

Protegrins are small, arginine- and cysteine-rich, beta-sheet peptides with potent activity against bacteria, fungi, and certain enveloped viruses. We report that protegrins form weakly anion-selective channels in planar phospholipid bilayers, induce potassium leakage from liposomes and form moderately cation-selective channels in planar lipid membranes that contain bacterial lipopolysaccharide. The disruption of microbial membranes may be a central attribute related to the host defense properties of protegrins.     

Early changes in cytosolic calcium and membrane potential induced by Actinobacillus actinomycetemcomitans leukotoxin in susceptible and resistant target cells

Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.     

Fitness comparison inPhytoseiulus persimilis strains resistant and susceptible to methidathion

The effect of selection for methidathion resistance on fitness components of aP. persimilis strain was analysed by different means. The resistant strain was compared with the susceptible one from which it was selected. The life history and some parameters essential to the successful use of this species in biological control (voracity, resistance to starvation and drought) were analysed. Methidathion resistance was followed for 6 months under rearing conditions free of insecticide in the resistant and in a mixed strain. The investigations showed that the fitness of resistant mites did not seem altered by methidathion selection. It thus appears possible to increase pesticide resistance in beneficial arthropods without adversely affecting their main fitness components.     

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.     

Leishmania major: analysis of lymphocyte and macrophage cellular phenotypes during infection of susceptible and resistant mice

Genetically susceptible BALB/c and resistant C57BL/6 mice were infected with Leishmania major and the phenotypes of the responding cells in the draining lymph nodes and cutaneous lesions were analyzed. As early as 1 week, significantly increased numbers of L3T4+ cells as compared to Lyt-2+ cells were present in BALB/c mice lymph nodes (P less than 0.005). Increases in L3T4+ and Lyt-2+ cells were comparable in C57BL/6 mice, resulting in threefold lower L3T4/Lyt-2 ratio than in BALB/c mice. T cell subsets were activated in both strains to express interleukin-2 receptor (IL2R) above resting values, although greater numbers of activated L3T4+ cells were present in the draining lymph nodes from BALB/c at 1 and 3 weeks of infection than in C57BL/6 (P = 0.02). Despite the presence of activated L3T4+ cells in both strains, macrophages differed in the expression of immunologically important surface molecules during infection. Tissue macrophages from BALB/c mice were IgG1/G2b Fc receptor (FcR)+ and Ia- late in disease, whereas macrophages in C57BL/6 became FcR and Ia during healing. BALB/c mice, treated with monoclonal antibody GK1.5 to transiently deplete L3T4+ cells, became resistant to subsequent infection and developed a macrophage phenotype that was FcR- and Ia+. These differences in macrophage phenotype were closely linked to susceptibility during infection with L. major and may play a role in the pathophysiology of murine leishmaniasis.     

The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point

The murine lymphocyte pore-forming protein (PFP) was purified to apparent homogeneity by successive steps of liquid chromatography. Monospecific antibodies were raised against purified PFP that detect only one protein band in murine CTL lines. 25% of the primary sequence of PFP (134 amino acids) was determined by amino terminal analysis of the purified protein and of some of its enzymatic cleavage products. These primary sequences were identical to sequences deduced by cDNA cloning. By isoelectric focusing, PFP was found to have a pI of 6.4. On the chromatofocusing column Mono P, however, PFP was found to elute at pH 4.7. This suggests a tertiary structure for monomeric PFP that is enriched in surface acidic amino acids.     

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30’s fusogenic activity depends on Mg²⁺ binding to IM30; yet, it still is unclear how Mg²⁺-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg²⁺ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg²⁺. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg²⁺. Thus, Mg²⁺-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.     

Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation

The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.     

Differential intra-epithelial lymphocyte phenotypes following Cryptosporidium parvum challenge in susceptible and resistant athymic strains of mice

The lack of immunocompetent laboratory animal models has limited our understanding of functional immune responses to Cryptosporidium parvum infection, but such responses have been studied in susceptible laboratory rodents with genetic, acquired, or induced immunodeficiencies. We previously observed that athymic C57BL/6J nude mice inoculated with C. parvum oocysts had lower or absent fecal oocyst excretion when compared to inoculated athymic BALB/cJ nude mice. This discrepancy prompted us to explore potential differences in intestinal immune responses in both strains. Prior to and after C. parvum challenge, BALB/cJ nude and C57BL/6J nude mice did not differ in either spleen cell numbers or in parasite-specific proliferation. However, both strains of mice exhibited a significant increase in intra-epithelial lymphocyte (IEL) numbers prior to and following C. parvum inoculation when compared to uninoculated controls (P<0.05). Prior to challenge, C57BL/6J nude mice had a higher percentage of both CD8+ and CD8+ gammadelta+ IEL than BALB/cJ nude mice. Following challenge, resistant C57BL/6J nude mice had a higher percentage of gammadelta+, CD4+, and CD8+ gammadelta+ IEL than uninoculated C57BL/6J nude mice and than susceptible BALB/cJ nude mice (P<0.05). Conversely, inoculated C57BL/6J nude mice had a significantly lower percentage of alphabeta+ IEL than inoculated BALB/cJ nude mice (P<0.05). We conclude that gammadelta+, CD4+, and/or CD8+ gammadelta+ IEL may influence responses to cryptosporidiosis in athymic murine models, and that the increased percentage of alphabeta+ IEL in susceptible BALB/cJ nude mice could reflect a preferential expression during chronic C. parvum infection and/or might downregulate local protective responses.     

Expression of genes for cytokines and cytokine-related functions in leukocytes infected with Herpes simplex virus: comparison between resistant and susceptible mouse strains

Cytokines and chemokines play an important role in the first line of defence against viral infections. Moreover, these groups of proteins also contribute significantly to regulation of the acquired immune response. Therefore, knowledge of the expression of cytokines, chemokines and factors involved in their action may provide information about the immune reaction responsible for elimination of viral infections and for immune-mediated pathology. Using cDNA arrays, we have evaluated the expression of cytokines and genes related to cytokine function in resting murine peritoneal cells and in inflammatory macrophages infected with Herpes simplex virus (HSV)-1 and -2. To allow comparison, the experiments were performed using both the resistant mouse strain C57BL/6 and the susceptible strain BALB/c. The work identified a group of genes that is differentially expressed during HSV infection of cells from the two strains. Another group of genes was affected by HSV-1 but not HSV-2 infection and vice versa. Further analysis of these genes may provide new information about host defense against viral infections and could also lead to identification of the molecular basis for the pathological differences between infections with HSV-1 and -2.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.