Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-Ad. Binding of anti-CD48 mAb and cholera toxin B subunit decreased significantly in P388D1(I), and consequently, colocalization with I-Ad was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-Ad and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2+ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune system.

Vibrio cholerae hemolysin is an extracellular pore-forming monomeric protein with a native molecular weight of about 60,000. In this study, we showed that the hemolysin interacted with immobilized phospholipids and cholesterol and formed oligomers in vesicles constituted from phospholipids alone with a stoichiometry identical to those produced in rabbit erythrocyte membrane. However, the hemolysin bound to glycoproteins with terminal beta1-galactosyl residues and an association constant of 9.4 x 10(7) M(-1) was estimated for the hemolysin-asialofetuin complex by solid phase binding assay. Oligomerization of the hemolysin in lipid bilayer converted the sugar-binding monomer to a lectin with strong carbohydrate-dependent hemagglutinating activity accompanied by inactivation of hemolytic activity and loss in ability to interact with phospholipids. There was no evidence for erythrocyte surface carbohydrates playing an essential role in interaction of the hemolysin with the cell. However, specific glycoproteins inhibited hemolysis of rabbit erythrocytes as well as interaction of the hemolysin with phospholipid. The results suggest (i) V. cholerae hemolysin is a monomer with distinct domains associated with specific binding to carbohydrates and interaction with lipids, (ii) the pore-forming property depends solely on the protein-lipid interaction with no evidence for involvement of sugars, and (iii) specific sugars can down-regulate the ability of the hemolysin to form pores in lipid bilayers.

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Vibrio cholerae is a gram-negative bacterium that can cause a severe, acute secretory diarrhea. Serological differentiation of V. cholerae strains is based on the O-side chain of the lipopolysaccharide (LPS) component of the outer membrane. Of the more than 200 serogroups of V. cholerae identified, only the O1 and O139 serogroups can cause epidemic cholera (44). These pathogens are noninvasive and colonize the mucosal surface of the small intestine (44).Natural infection with V.

When bodies are brought for autopsy it is sometimes unclear whether the injuries are due to a fall from a height or due to blunt trauma from other causes, especially when the bodies are found near buildings with no eyewitnesses available. Studies discussing the injury patterns in adults after falls exclusively from buildings are rare. A five-year retrospective study was carried out on 151 cases of fatal falls from buildings brought for autopsy. The aim was to assess the pattern of these injuries and identify features helpful in discriminating between these and injuries due to blunt trauma from other causes. The majority of cases comprised subjects who fell from heights of 10-20 feet (3-6m) with most falls occurring late at night or in the early morning. The pattern that emerged is quite distinct from ground level falls and pedestrian injuries. Abrasions are the commonest injury and bruises very rare. Lacerations are mostly on the head and skull fractures are evenly distributed between the vertex, base and vertex plus base. Subarachnoid haemorrhage is the commonest intracranial lesion. Extradural haemorrhage alone is rare. Fractures of ribs and cervical vertebrae are common and fractures of thoracic vertebrae and long bones are uncommon. Safety measures to prevent such falls have been suggested.

Vibrio cholerae hemolysin (HlyA) is a pore-forming toxin that exists in two stable forms: a hemolytically active water-soluble monomer with a native molecular weight of 65,000 and a hemolytically inactive SDS-stable heptamer with the configuration of a transmembrane diffusion channel. Transformation of the monomer into the oligomer is spontaneous but very slow in the absence of interaction with specific membrane components like cholesterol and sphingolipids. In this report, we show that mild disruption of the native tertiary structure of HlyA by 1.75 M urea triggered rapid and quantitative conversion of the monomer to an oligomer. Furthermore, the HlyA monomer when unfolded in 8 M urea refolded and reconstituted on renaturation into the oligomer biochemically and functionally similar to the heptamer formed in target lipid bilayer, suggesting that the HlyA polypeptide had a strong propensity to adopt the oligomer as the stable native state in preference to the monomer. On the basis of our results, we propose that (a) the hemolytically active HlyA monomer represents a quasi-stable conformation corresponding to a local free energy minimum and the transmembrane heptameric pore represents a stable conformation corresponding to an absolute free energy minimum and (b) any perturbation of the native tertiary structure of the HlyA monomer causing relaxation of conformational constraints tends to promote self-assembly to the oligomer with membrane components playing at most an accessory role.Vibrio cholerae hemolysin (HlyA), 1 an extracellular membrane-damaging protein with a native monomeric molecular weight of 65,000 (1-4), belongs to a unique class of dimorphic proteins that can exist in two stable states, a water-soluble monomer and an oligomeric integral membrane protein (5). These proteins are commonly known as pore-forming toxins (PFTs) (6) for their ability to lyse target eucaryotic cells by punching holes in the plasma membrane and are released as water-soluble monomeric proteins by a wide variety of pathogenic bacteria. PFTs interact with specific target membrane components and self-assemble by noncovalent interactions involving oligomerization domains into exceptionally stable and rigid ␤-barrel oligomers that eventually insert into the membrane by using amphiphilic ␤-hairpins as anchors to the nonpolar core of the lipid bilayer (5, 7). High resolution structural data are available for only two of the PFTs, viz. Staphylococcus aureus ␣-toxin (8) and aerolysin of Aeromonas hydrophila (9). Our understanding of the mode of action of PFTs is inadequate in at least two aspects: the driving force for self-assembly of the toxin monomer and the precise role of membrane components in triggering this transition.HlyA is synthesized as an 82-kDa preprohemolysin by V. cholerae El Tor O1 and non-O1 strains and exported to the culture medium as the 79-kDa prohemolysin (proHlyA) (10). Proteolytic removal of the 132-residue N-terminal stretch (Proregion) generates the mature 65-kDa HlyA with a specific hemolytic activity of app...

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A comparative study was undertaken of clinical and environmental isolates of non-O1 Vibrio cholerae with respect to their hemagglutinating, hemolytic, enterotoxigenic, and enteropathogenic activities. Cell-associated hemagglutinin titers of the clinical and environmental isolates did not differ much, although the clinical isolates displayed higher cell-free hemagglutinin titers compared with those of environmental isolates. Culture supernatants of 61.5% (24 of 39) of clinical isolates showed hemolytic activity (greater than or equal to 10% lysis of rabbit erythrocytes), while only 33.3% (10 to 30) of the environmental group had such activity. Furthermore, hemolytic activities of the clinical isolates showed a good correlation with their cell-associated hemagglutinin titers which was not true for the environmental group. Culture supernatants of 45.8% (11 of 25) of the clinical and 20% (2 of 10) of the environmental isolates exhibited enterotoxigenic activity in the rabbit ileal loop assay. Such activity was mediated mainly by cholera toxin-like substances, although some of the isolates produced fluid-accumulating factors unrelated to cholera toxin. Experimental animal studies demonstrated that the enteropathogenic potential of the environmental isolates was significantly lower than that of the clinical group. Further analysis of our data showed that phenotypic expression of cholera toxin-like products by the non-O1 V. cholerae isolates was accompanied by their enteropathogenicity. The latter effect was also noted with some of the cholera toxin-negative isolates, particularly in those having high hemagglutinating and hemolytic titers.