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The protozoan parasite Leishmania donovani, the causative agent of visceral leishmaniasis, is transmitted to humans through bites by infected sand flies. Flagellated Leishmania promastigotes are phagocytosed by host macrophages, where they survive inside phagosomes (9, 27). Lipophosphoglycan (LPG), which is the major surface glycoconjugate of promastigotes, is essential for intracellular survival (10, 15, 23, 30). Phagosomes containing L. donovani mutants that lack the repeating sugars of LPG (lpg2−/− knockout [KO]) proceed through the phagolysosomal pathway, and the parasites are killed (10). The mechanism by which LPG arrests phagosomal maturation in macrophages is partially clarified and includes accumulation of F-actin around newly formed phagosomes (17). The effects of LPG on actin are in part related to inhibition of protein kinase Cα (PKCα), an enzyme implicated in F-actin depolymerization at the phagosomal membrane (2, 16, 17, 23, 25). Results from our group indicate that LPG from L. donovani interacts with GM1-enriched lipid microdomains (lipid rafts, detergent-resistant membranes) in the macrophage plasma membrane and that active PKCα colocalizes to a lesser degree with GM1 in these cells (unpublished data). Moreover, it was recently shown that LPG causes a disorganization of the phagosomal membrane, preventing normal assembly of GM1-enriched microdomains (8). In line with this, we found that disrupting membrane microdomains by cholesterol depletion, thereby mimicking the effect of LPG, raised the levels of periphagosomal F-actin around LPG-deficient lpg2−/− KO mutants. However, the levels of periphagosomal F-actin in cholesterol-depleted cells did not reach the levels observed around wild-type (WT) promastigotes (unpublished data). The present study investigates the involvement of the Rho family members Cdc42 and Rac1 in the generation of the F-actin shell formed around phagosomes containing L. donovani promastigotes. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has emerged as main regulators of the cytoskeleton (11, 14). GTP-bound Rho GTPases drive cellular processes such as cytoskeletal reorganization, migration, and proliferation (7, 14, 28). Importantly, Rho GTPases play a profound role in phagocytosis of opsonized prey (6, 22, 26, 29); however, their role in nonopsonic phagocytosis is poorly understood. Dominant negative (N17) and constitutively active (V12) forms of Cdc42 and Rac1, expressed as fusion proteins with the human immunodeficiency virus-derived cell permeable peptide TAT, were used to study the effects of these proteins on periphagosomal F-actin and phagosomal maturation in RAW264.7 macrophages. We found that simultaneous introduction of N17Cdc42 and N17Rac1 prevented the accumulation of F-actin around phagosomes with WT L. donovani. Conversely, introduction of V12Cdc42 and V12Rac1 restored the accumulation of periphagosomal F-actin around phagosomes with LPG-defective promastigotes, resulting in inhibition of phagosomal maturation. Interestingly, introduction of N17Cdc42 and N17Rac1 into the cells, which reduced the levels of periphagosomal F-actin around WT L. donovani, did not promote maturation of these phagosomes. This result points toward a direct role for Cdc42 and Rac1 in phagosomal maturation. In conclusion, our results show that L. donovani requires functional Cdc42 and Rac1 to build up a coat of F-actin around its phagosome to prevent phagosomal maturation.

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA -deficient mutants. Initially, all bacteria except the invasin-deficient ( inv ) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed β1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, β1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with β1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to β1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.

Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca 2+ ] i ), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz ., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.

When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection.

An aqueous, two-polymer phase system was employed in an attempt to separate and characterize a series of R mutants, derived from Salmonella typhimurium 395 MS, with differing lengths of lipopolysaccharide chains on their surfaces. R mutants with varying degrees of virulence and phagocytic resistance were unresolved in this system. However, the smooth MS bacteria were clearly separated from the rough cells and showed a high affinity for the polyethylene glycol-rich top phase. A uridine 5′-diphosphate- gal -4-epimeraseless mutant, phenotypically in R or S form depending on the growth medium, partitioned as the R mutants and S bacteria, respectively. These results demonstrate the great influence of long polysaccharide chains on the physicochemical properties of the cell surface.

Sensitization of smooth Salmonella typhimurium 395 MS bacteria with hyper-immune anti-MS immunoglobulin G (IgG) antibodies increased the liability to hydrophobic interaction as assessed by the affinity for a column of Octyl-Sepharose. After sensitization, the material originally eluted with 1 M (NH4)SO4 in a 0.01 M phosphate buffer (pH 6.8) was not desorbed until the ionic strength was reduced to nil, and 0.1% (vol/vol) Triton X-100 in the 0.01 M phosphate buffer was used as eluant. Furthermore, by including positively charged bis-trimethylamino-polyethylene glycol (PEG) or negatively charged bis-sulfoamino-PEG in an aqueous two-phase system of dextran T500 and PEG 6000, the partition of the IgG-sensitized bacteria was affected by either of the polymers, whereas that of the parent bacteria was not. The hydrophobic effect of IgG binding was enhanced by complement. With heat-inactivated complement, the effect of IgG was diminished. The F(ab')2 fragment showed a much lower capacity to promote a hydrophobic interaction than the complete IgG molecule.