Joachim Clos

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37°C ± pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-α. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite.

In Leishmania major a 100-kDa heat shock protein, Hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. A replacement of both clpB alleles which encode Hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. In experimental infections of BALB/c inbred mice, the lack of Hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with wild-type L. major. Overexpression of exogenous clpB gene copies can partly restore virulence to the gene replacement mutants. Genetic-selection experiments also reveal a strong pressure for Hsp100 expression in the mammalian stage. This requirement for Hsp100 was also observed in in vitro infection experiments with mouse peritoneal macrophages. These experiments indicated a role for Hsp100 during the development from the promastigote to the amastigote stage. Our results suggest an important role for this parasite heat shock protein during the initial stages of a mammalian infection.

Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1−/− null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.

Leishmania donovani Heat Shock Protein 100 CHARACTERIZATION AND FUNCTION IN AMASTIGOTE STAGE DIFFERENTIATION

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5′- and 3′-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5′-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.

Inhibition of HSP90 in Trypanosoma cruzi Induces a Stress Response but No Stage Differentiation

ABSTRACT The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite Leishmania donovani. We investigated the role of HSP90 in the related parasite Trypanosoma cruzi by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G1 phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in T. cruzi and serves as a feedback inhibitor in the cellular stress response. In contrast to L. donovani cells, however, T. cruzi cells treated with GA do not begin to differentiate into relevant life cycle stages.

A member of the clpb family of stress proteins is expressed during heat shock in Leishmania spp

We have identified and isolated the Leishmania major homologue to the bacterial ClpB gene and to the yeast Hsp104 gene. ClpB in Leishmania major is a single-copy gene and encodes a low-abundance mRNA which is induced several-fold during a heat stress. We raised antibodies against the product of the recombinant gene and show that the leishmanial ClpB encodes a predominantly cytoplasmic protein of approx. 100 kDa which is detectable in Leishmania promastigotes of various species after exposure to elevated temperatures. We, therefore, term this protein Hsp100.

A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani.

Heat shock proteins of the 100 kD family have been known to confer general stress tolerance in yeast and plants. Several protozoan parasites possess genes for Hsp100 proteins. In Leishmania species the protein is expressed under heat stress and during the mammalian stage, the amastigote. We show here that replacement of the clpB gene which encodes Hsp100 does not affect thermotolerance or general viability in Leishmania donovani insect stages (promastigotes) nor in axenically cultured mammalian stages (amastigotes). However, its expression is required for normal development of the parasite inside mammalian host cells. Hsp100 appears to function as an antagonist of amastigote-to-promastigote differentiation and a promoter of full amastigote development.

Leishmania infantum expresses a mitochondrial nuclease homologous to EndoG that migrates to the nucleus in response to an apoptotic stimulus

It is increasingly accepted that single-celled organisms, such as Leishmania parasites, are able to undergo a cell death process that resembles apoptosis in metazoans and is induced by a variety of stimuli. However, the molecular mechanisms that participate and regulate this death process are still very poorly described, and very few of the participating molecules have been identified. Because DNA degradation is probably the most frequently characterized event during programmed cell death in Leishmania parasites, we have focused on identifying a candidate nuclease responsible for this effect during the cell death process. The results presented herein demonstrate that Leishmania infantum promastigotes express a nuclease similar to the endonuclease G of higher eukaryotes which, according to its predicted structure, belongs to the beta beta alpha metal superfamily of nucleases. Its cation dependence resembles that of the EndoGs present in other organisms and, similarly to them, it is inhibited by moderate concentrations of K+ or Na+. L. infantum EndoG contains a signal peptide that causes its translocation to the mitochondrion where it is maintained under normal growth conditions. However, under the pressure of a death stimulus such as edelfosine treatment, L. infantum EndoG is released from the single mitochondrion and translocates to the nucleus, where it is thought to participate in the process of DNA degradation that is associated with programmed cell death. Our results also demonstrate that overexpression of the nuclease in edelfosine-treated promastigotes causes a significant increase in the percentage of TUNEL-positive parasites.

Secreted virulence factors and immune evasion in visceral leishmaniasis

Evasion or subversion of host immune responses is a well‐established paradigm in infection with visceralizing leishmania. In this review, we summarize current findings supporting a model in which leishmania target host regulatory molecules and pathways, such as the PTP SHP‐1 and the PI3K/Akt signaling cascade, to prevent effective macrophage activation. Furthermore, we describe how virulence factors, secreted by leishmania, interfere with macrophage intracellular signaling. Finally, we discuss mechanisms of secretion and provide evidence that leishmania use a remarkably adept, exosome‐based secretion mechanism to export and deliver effector molecules to host cells. In addition to representing a novel mechanism for trafficking of virulence factors across membranes, recent findings indicate that leishmania exosomes may have potential as vaccine candidates.