2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli.

Escherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.

PstS-1, the 38-kDa Mycobacterium tuberculosis glycoprotein, is an adhesin, which binds the macrophage mannose receptor and promotes phagocytosis.

Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin-labelled mycobacterial cell wall proteins revealed five Concanavalin A-reactive proteins that bind macrophages. Among these proteins, we identified PstS-1, a 38-kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS-1 binds the mannose receptor. We purified PstS-1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS-1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m-periodate and α-D-mannosidase. Gas chromatography revealed that purified PstS-1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS-1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre-incubation of cells with GlcNAc, mannan and α-methyl mannoside. The interaction of PstS-1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.

Escherichia coli-mediated impairment of ureteric contractility is uropathogenic E. coli specific.

BACKGROUND: Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. METHODS: Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. RESULTS: Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. CONCLUSIONS: We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved.

Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: variation of valency, aglycon and scaffolding.

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-D-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial arrangement of attached mannosyl residues. They were tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were referenced to the inhibitory potency of methyl α-D-mannopyranoside. It was shown, that besides the nature of the mannoside aglycon moiety, scaffolding of α-D-mannopyranosides on a peptide backbone was important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.

Crystallographic complexes of surfactant protein A and carbohydrates reveal ligand-induced conformational change.

Surfactant protein A (SP-A), a C-type lectin, plays an important role in innate lung host defense against inhaled pathogens. Crystallographic SP-A·ligand complexes have not been reported to date, limiting available molecular information about SP-A interactions with microbial surface components. This study describes crystal structures of calcium-dependent complexes of the C-terminal neck and carbohydrate recognition domain of SP-A with d-mannose, D-α-methylmannose, and glycerol, which represent subdomains of glycans on pathogen surfaces. Comparison of these complexes with the unliganded SP-A neck and carbohydrate recognition domain revealed an unexpected ligand-associated conformational change in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D and mannan-binding lectin. The net result of the conformational change is that the SP-A lectin site and the surrounding loop region become more compact. The Glu-202 side chain of unliganded SP-A extends out into the solvent and away from the calcium ion; however, in the complexes, the Glu-202 side chain translocates 12.8 Å to bind the calcium. The availability of Glu-202, together with positional changes involving water molecules, creates a more favorable hydrogen bonding environment for carbohydrate ligands. The Lys-203 side chain reorients as well, extending outward into the solvent in the complexes, thereby opening up a small cation-friendly cavity occupied by a sodium ion. Binding of this cation brings the large loop, which forms one wall of the lectin site, and the adjacent small loop closer together. The ability to undergo conformational changes may help SP-A adapt to different ligand classes, including microbial glycolipids and surfactant lipids.

Association of Lactobacillus acidophilus with mice Peyer's patches.

OBJECTIVE: To clarify the adhesion mechanism of Lactobacillus acidophilus to Peyer's patches. METHODS: Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the differing effects of physical, chemical, and enzymatic pre-treatments of the bacteria and the inhibitory effects of sugars on the adhesion. The presence of lectin-like proteins on the cell surface was determined by hemagglutination assay. The effect of L. acidophilus FN001 on the inhibition of adhesion of pathogens to Peyer's patches was also studied. RESULTS: The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-α-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the adhesive capacity indicating that some cell surface proteins might be involved in the adhesion. L. acidophilus FN001 showed agglutinating activity toward the rabbit red cells in a mannose specific manner, which was decreased after protease pretreatment, suggesting possible occurrence of mannose specific lectin(s) on the L. acidophilus FN001 surface. In adhesion inhibition assay, L. acidophilus NF001, when applied to Peyer's patches first or at the same time with pathogen, significantly inhibited adhesion of Escherichia coli ATCC25922 to Peyer's patches. CONCLUSION: L. acidophilus FN001 contains some mannose-specific protein(s) on its surface that mediates its adhesion to the Peyer's patches. FN001 inhibits the adhesion of E. coli, which also contains mannose specific lectin.

The secretion and uptake of lysosomal phospholipase A2 by alveolar macrophages.

Macrophages have long been known to secrete a Phospholipase A(2) with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A(2) (LPLA(2)) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA(2). LPLA(2) is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA(2) produced by HEK293 cells was applied to LPLA(2)-deficient (LPLA(2)(-/-)) mouse alveolar macrophages. The uptake of exogenous LPLA(2) into LPLA(2)(-/-) alveolar macrophages occurred in a concentration-dependent manner. The LPLA(2) taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of alpha-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA(2) is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA(2)(-/-) alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA(2) rescued the LPLA(2)(-/-) alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA(2) revealed that the enzymatic activity of LPLA(2) was required for the phospholipid reduction. These studies identify LPLA(2) as a high m.w.-secreted Phospholipase A(2).

Inhibition of duck hepatitis B virus infection of liver cells by combined treatment with viral e antigen and carbohydrates.

The e antigen (eAg) of duck hepatitis B virus (DHBV) is a glycosylated secretory protein with a currently unknown function. We concentrated this antigen from the supernatants of persistently infected primary duck liver cell cultures by ammonium sulphate precipitation, adsorption chromatography over concanavalin A Sepharose, preparative isoelectric focusing and molecular sieve chromatography. The combined treatment of duck liver cells with DHBV eAg (DHBe) concentrate and alpha-methyl-d-mannopyranoside strongly inhibited DHBV replication at de novo infection. When DHBe was added to non-infected primary duck liver cells, it was found to be associated with liver sinusoidal endothelial cells. This binding could be inhibited by the addition of alpha-methyl-d-mannopyranoside and other sugar molecules. The inhibitory effect of DHBe on infection could play a role in maintaining viral persistence.

Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip.

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds.

AIMS: The ability of 31 Lactobacillus plantarum strains to adhere to biological matrixes was evaluated, and the molecules involved in adherence were studied. METHODS AND RESULTS: Mucin, basement membrane proteins and Caco-2 cells were used in adhesion tests. These in vitro assays, together with a yeast agglutination test, were found to be discriminative for screening Lact. plantarum strains for adhesion. Some strains, such as 299v, CBE, BMCM12, Col4S and T25, were shown to possess interesting adhesion properties in at least two models. The adhesion of these strains was strongly inhibited when the bacterial cells were pretreated with trypsin. Lithium chloride and methyl-alpha-D-mannoside also inhibited adhesion to a lower extent. CONCLUSIONS: The adhesion of Lact. plantarum depends on both the model and the strain used. The chemical and enzymatic pretreatments applied to the bacterial cells suggested that lectin-like adhesins and other proteinaceous cell-surface structures are involved in adhesion of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We found a great diversity in the adhesion properties between Lact. plantarum strains. Based upon the adhesive property of these strains interesting candidates were identified, that will undergo further study as potential probiotics.

Characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches.

The characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches was studied in vitro. The adhesion of L. fermentum 003 was strongly inhibited in the presence of d-mannose and methyl-alpha-d-mannoside although other carbohydrates tested, such as N-acetyl-glucosamine, d-galactose, d-glucose and l-fucose, did not affect the adhesion. Lactobacillus fermentum 003 was shown to strongly attach to mannose immobilized on a surface using BSA, suggesting that L. fermentum 003 specifically adhered to mannose-containing molecule(s). Pretreatment of L. fermentum 003 with proteinase K and trypsin decreased the adhesive capacity and bacterial surface extracts diminished adhesion of L. fermentum 003 indicating that cell surface proteins are involved in adhesion to Peyer's patches. It was concluded that a mannose-specific protein mediated adhesion of L. fermentum 003 to the Peyer's patches.

Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.

Importance of complement 3 and mannose receptors in phagocytosis of Paracoccidioides brasiliensis conidia by Nramp1 congenic macrophages lines.

Genetic factors influence susceptibility to Paracoccidioidomycosis, a Latin American endemic mycosis. The pattern of susceptibility of congenic mouse strains infected with Paracoccidioides brasiliensis resembles the pattern of the Nramp1 gene. Thus, congenic murine bone-marrow-derived macrophage lines B10R (Nramp1rGly169) and B10S (null Nramp1 protein expression, Nramp1sAsp169) were infected with P. brasiliensis conidia and compared, under opsonic and nonopsonic conditions. Opsonization increased the percentage of phagocytosis by both cell lines. B10R macrophages exhibited a higher percentage of cells with associated conidia and higher number of conidia per macrophage than B10S. Heat-inactivation and EDTA treatment of serum used for opsonization, and treatment of macrophages with anti-complement receptor 3 (CR3) decreased phagocytosis by both cell lines. alpha-methyl-d-mannoside reduced phagocytosis by B10R macrophages, suggesting that the mannose receptor participates in phagocytosis by these cells. The CR3 expression was similar on both cell lines and B10R expressed more mannose receptors, but neither cell line expressed CR1. IFNgamma decreased the conversion of conidia to the yeast form of P. brasiliensis in B10R, but not in B10S macrophages.

Elevated shear stress protects Escherichia coli cells adhering to surfaces via catch bonds from detachment by soluble inhibitors.

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.

A novel approach to study adhesion mechanisms by isolation of the interacting system.

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.

The 19-kDa antigen of Mycobacterium tuberculosis is a major adhesin that binds the mannose receptor of THP-1 monocytic cells and promotes phagocytosis of mycobacteria.

Identification of mycobacterial adhesins is needed to understand better the pathogenesis of tuberculosis and to develop new strategies to fight this infection. In this work, THP-1 monocytic cells were incubated with Mycobacterium tuberculosis culture filtrate proteins labelled with biotin and a dominant 19-kDa adhesin was found. This adhesin was characterized as the glycosylated and acylated 19-kDa antigen (Rv 3763). These findings were confirmed in assays with culture filtrate proteins and cell-wall fractions from a recombinant Mycobacterium smegmatis strain that overexpresses the 19-kDa antigen. Further, fluorescent microspheres coated with recombinant culture filtrate proteins adhere to cells in higher numbers than microspheres coated with native M. smegmatis proteins. The binding of the 19-kDa antigen to cells was inhibited with mannose receptor competitor sugars, Ca(2+) chelators and with a monoclonal antibody to the human mannose receptor. Phagocytosis assays showed high-level binding of bacilli to THP-1 cells that was inhibited with alpha-methyl-mannoside, mannan, EDTA and mAbs to the mannose receptor and to the 19-kDa M. tuberculosis antigen. Immunoprecipitation, cell-surface ELISA and immunostaining confirmed the expression of the mannose receptor by THP-1 cells. In conclusion, here we show that the macrophage mannose receptor, considered a pathogen pattern recognition receptor, may interact with mannose residues of mycobacterial glycoproteins that could promote the phagocytosis of mycobacteria.

Dietary lectin lowers serum cholesterol and raises fecal neutral sterols in cholesterol-fed rats.

This study examined the influence of a low level of dietary lectin (0.34%), at a dose that did not affect body weight or food intake, on the concentration of serum cholesterol and fecal excretion of neutral sterols in rats fed a diet containing 0.50% cholesterol and 0.13% sodium cholate for 12 d. In experiment 1, rats fed a diet with 0.34% lectin, concanavalin A, had significantly lower concentrations of serum total cholesterol and hepatic cholesterol, a higher ratio of HDL-cholesterol to total cholesterol, enhanced excretion of fecal neutral sterols and reduced apparent cholesterol absorption or digestibility as compared with rats fed a diet without lectin. Fecal excretion of acidic sterols was unaffected by dietary lectin. In contrast, dietary 0.34% lectin had no significant effect on concentrations of serum total protein or glucose. In experiment 2, we examined whether the cholesterol-lowering activity of the lectin was responsibility for its carbohydrate-binding activity. The effect of dietary lectin on concentrations of serum and hepatic cholesterol and excretion of fecal neutral sterols was prevented by simultaneous administration of methyl-alpha-D-mannopyranoside with specific affinity for the carbohydrate-binding sites of the lectin. These results suggest that dietary lectins might reduce concentrations of serum and hepatic cholesterol by a mechanism involving higher excretion of neutral sterols and that these alterations might be associated with the carbohydrate-binding activity of lectin.

Genetic evidence that the Vibrio cholerae monolayer is a distinct stage in biofilm development.

Biofilm development is conceived as a developmental process in which free swimming cells attach to a surface, first transiently and then permanently, as a single layer. This monolayer of immobilized cells gives rise to larger cell clusters that eventually develop into the biofilm, a three-dimensional structure consisting of large pillars of bacteria interspersed with water channels. Previous studies have shown that efficient development of the Vibrio cholerae biofilm requires a combination of pili, flagella and exopolysaccharide. Little is known, however, regarding the requirements for monolayer formation by wild-type V. cholerae. In this work, we have isolated the wild-type V. cholerae monolayer and demonstrated that the environmental signals, bacterial structures, and transcription profiles that induce and stabilize the monolayer state are unique. Cells in a monolayer are specialized to maintain their attachment to a surface. The surface itself activates mannose-sensitive haemagglutinin type IV pilus (MSHA)-mediated attachment, which is accompanied by repression of flagellar gene transcription. In contrast, cells in a biofilm are specialized to maintain intercellular contacts. Progression to this stage occurs when exopolysaccharide synthesis is induced by environmental monosaccharides. We propose a model for biofilm development in natural environments in which cells form a stable monolayer on a surface. As biotic surfaces are degraded with subsequent release of carbohydrates, the monolayer develops into a biofilm.

Valency conversion in the type 1 fimbrial adhesin of Escherichia coli.

FimH protein is a lectin-like adhesive subunit of type 1, or mannose-sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose-specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185-279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose-specific binding property accompanied by the acquisition of a distinct monomannose-specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells -- functions readily accomplished by the E. coli-expressing mannotriose-specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH-FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose-only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor-binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c.

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypodiploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by alpha-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.