Integrity of the Actin Cytoskeleton of Host Macrophages is Essential for Leishmania donovani Infection

Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis.     

Leishmania major intracellular survival is not altered in SHP-1 deficient mev or CD45-/- mice

Protozoan parasites of the genus Leishmania escape from the immune response by interfering with signal transduction pathways of its host cell, the macrophage, thereby establishing permissive conditions for intracellular survival. Inhibition of macrophage activation after Leishmania infection has been suggested to require activation of the host cell phosphatase SHP-1. However, by utilizing infections of SHP-1 deficient (me^v) and CD45 null mutant mice or macrophages, we provide evidence that intracellular survival of Leishmania major is not generally dependent on these cellular phosphatases.     

Leishmania donovani Prevents Oxidative Burst-mediated Apoptosis of Host Macrophages through Selective Induction of Suppressors of Cytokine Signaling (SOCS) Proteins

One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H₂O₂ for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H₂O₂-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H₂O₂ treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.     

MicroRNA expression profiling of Leishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.     

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4^?/? mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-β with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.     

Toll-like receptor signaling: a perspective to develop vaccine against leishmaniasis

The toll-like receptors (TLRs) are the sentinel factor of the innate immunity, which are essential for host defense. These receptors detect the presence of conserved molecular patterns of potentially pathogenic microorganisms and contribute in both, cellular as well as humoral immune responses. Leishmania is an intracellular pathogen that silently invades host immune system. After phagocytosis, it divides and proliferates in the harmful environment of host macrophages by down-regulating its vital effector functions. In leishmaniasis, the outcome of the infection basically relies on the skewed balance between Th1/Th2 immune responses. Lots of work have been done and on progress but still characterization of either preventive or prophylactic candidate antigen/s is far from satisfactory. How does Leishmania regulate host innate immune system? Still it is unanswered. TLRs play very important role during inflammatory process of various diseases such as cancer, bacterial and viral infections but TLR signaling is comparatively less explained in leishmanial infection. In the context to Th1/Th2 dichotomy, identification of leishmanial antigens that modulate toll-like receptor signaling will certainly help in the development of future vaccine. This review will initially describe global properties of TLRs, and later will discuss their role in the pathogenesis of leishmaniasis.     

Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease.     

Complement C3 opsonization of Chlamydia trachomatis facilitates uptake in human monocytes

Chlamydia trachomatis is an obligate intracellular bacterium that causes severe infections, which can lead to infertility and ectopic pregnancy. Although both innate and adaptive immune responses are elicited during chlamydial infection the bacterium succeeds to evade host defense mechanisms establishing chronic infections. Thus, studying the host–pathogen interaction during chlamydial infection is of importance to understand how C. trachomatis can cause chronic infections. Both the complement system and monocytes play essential roles in anti-bacterial defense, and, therefore, we investigated the interaction between the complement system and the human pathogens C. trachomatis D and L2.Complement competent serum facilitated rapid uptake of both chlamydial serovars into monocytes. Using immunoelectron microscopy, we showed that products of complement C3 were loosely deposited on the bacterial surface in complement competent serum and further characterization demonstrated that the deposited C3 product was the opsonin iC3b. Using C3-depleted serum we confirmed that complement C3 facilitates rapid uptake of chlamydiae into monocytes in complement competent serum. Complement facilitated uptake did not influence intracellular survival of C. trachomatis or C. trachomatis-induced cytokine secretion. Hence, C. trachomatis D and L2 activate the complement system leading to chlamydial opsonization by iC3b and subsequent phagocytosis, activation and bacterial elimination by human monocytes.     

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background

Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Methodology/Principal Findings

We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain.

Conclusions/Significance

The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.     

A genomic-based approach combining in vivo selection in mice to identify a novel virulence gene in Leishmania

Background

Infection with Leishmania results in a broad spectrum of pathologies where L. infantum and L. donovani cause fatal visceral leishmaniasis and L. major causes destructive cutaneous lesions. The identification and characterization of Leishmania virulence genes may define the genetic basis for these different pathologies.

Methods and Findings

Comparison of the recently completed L. major and L. infantum genomes revealed a relatively small number of genes that are absent or present as pseudogenes in L. major and potentially encode proteins in L. infantum. To investigate the potential role of genetic differences between species in visceral infection, seven genes initially classified as absent in L. major but present in L. infantum were cloned from the closely related L. donovani genome and introduced into L. major. The transgenic L. major expressing the L. donovani genes were then introduced into BALB/c mice to select for parasites with increased virulence in the spleen to determine whether any of the L. donovani genes increased visceral infection levels. During the course of these experiments, one of the selected genes (LinJ32_V3.1040 (Li1040)) was reclassified as also present in the L. major genome. Interestingly, only the Li1040 gene significantly increased visceral infection in the L. major transfectants. The Li1040 gene encodes a protein containing a putative component of an endosomal protein sorting complex involved with protein transport.

Conclusions

These observations demonstrate that the levels of expression and sequence variations in genes ubiquitously shared between Leishmania species have the potential to significantly influence virulence and tissue tropism.     

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.     

Localization and induction of the A2 virulence factor in Leishmania: evidence that A2 is a stress response protein

Leishmaniasis is a vector‐borne infectious disease with a wide range of pathologies depending on the species of Leishmania. Leishmania parasites are transmitted by the sand fly vector as promastigotes; within the mammalian host, Leishmania parasites differentiate into amastigotes and replicate in macrophages. The A2 protein from Leishmania donovani is expressed predominantly in amastigotes and therefore likely plays a role in survival in the mammalian host. In the present study, we have determined that the A2 protein colocalized with the Leishmania endoplasmic reticulum binding protein, BiP, was induced by stress and complexed with BiP following heat shock. The A2 gene in Leishmania major is a non‐expressed pseudogene, and we present evidence that ectopic expression of a transfected A2 gene in L. major enhanced its viability following heat shock. A2 may therefore play a role in protecting L. donovani from stress associated with infection in visceral organs, including the fever typically associated with visceral leishmaniasis. Interestingly, when comparing A2 protein localization, we also observed that the Leishmania secreted acid phosphatase SAcP protein was transported out of the parasite‐containing phagolysosome and was located throughout the macrophage cytoplasm in vesicles, providing the first example of a secreted Leishmania‐derived protein exiting the parasite‐containing phagolysosome.     

Staphylococcus aureus promotes autophagy by decreasing intracellular cAMP levels

Staphylococcus aureus is an intracellular bacterium responsible for serious infectious processes. This pathogen escapes from the phagolysosomal pathway into the cytoplasm, a strategy that allows intracellular bacterial replication and survival with the consequent killing of the eukaryotic host cell and spreading of the infection. S. aureus is able to secrete several virulence factors such as enzymes and toxins. Our recent findings indicate that the main virulence factor of S. aureus, the pore-forming toxin α-hemolysin (Hla), is the secreted factor responsible for the activation of an alternative autophagic pathway. We have demonstrated that this noncanonical autophagic response is inhibited by artificially elevating the intracellular levels of cAMP. This effect is mediated by RAPGEF3/EPAC (Rap guanine nucleotide exchange factor (GEF)3/exchange protein activated by cAMP), a cAMP downstream effector that functions as a GEF for the small GTPase Rap. We have presented evidence that RAPGEF3 and RAP2B, through calpain activation, are the proteins involved in the regulation of Hla and S. aureus-induced autophagy. In addition, we have found that both, RAPGEF3 and RAP2B, are recruited to the S. aureus–containing phagosome. Of note, adding purified α-toxin or infecting the cells with S. aureus leads to a decrease in intracellular cAMP levels, which promotes autophagy induction, a response that favors pathogen intracellular survival, as previously demonstrated. We have identified some key signaling molecules involved in the autophagic response upon infection with a bacterial pathogen, which have important implications in understanding innate immune defense mechanisms.     

Impact of Neutrophil-Secreted Myeloid Related Proteins 8 and 14 (MRP 8/14) on Leishmaniasis Progression

The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.     

Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS^-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments encountered during infection and can be targeted for chemotherapeutic purpose to treat visceral leishmaniasis.