The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification

Diversity of Raft-Like Domains in Late Endosomes

BACKGROUND: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties. CONCLUSIONS/SIGNIFICANCE: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed

Trypanosome Lytic Factor, an Antimicrobial High-Density Lipoprotein, Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway

Stage-Specific Pathways of Leishmania infantum chagasi Entry and Phagosome Maturation in Macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival

Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

CD14 Signaling Restrains Chronic Inflammation through Induction of p38-MAPK/SOCS-Dependent Tolerance

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes

Increased Abeta production leads to intracellular accumulation of Abeta in flotillin-1-positive endosomes

Extracellular accumulation of Abeta in beta-amyloid plaques is thought to be associated with the neurodegeneration observed in Alzheimer's disease (AD) patients, although a lack of correlation with cognitive decline raised doubts on this hypothesis. In different transgenic mouse models Abeta accumulates inside the cells and mice develop behavioral deficits well before visible extracellular beta-amyloid accumulation. Here we show that intracellular Abeta accumulates in flotillin-1 positive endocytic vesicles. We also demonstrate that flotillin-1 is not only associated with intracellular Abeta in transgenic mice but also with extracellular beta-amyloid plaques in AD patient brain sections