Modelling the impact of nucleolin expression level on the activity of F3 peptide-targeted pH-sensitive pegylated liposomes containing doxorubicin.

Strategies targeting nucleolin have enabled a significant improvement in intracellular bioavailability of their encapsulated payloads. In this respect, assessment of the impact of target cell heterogeneity and nucleolin homology across species (structurally and functionally) is of major importance. This work also aimed at mathematically modelling the nucleolin expression levels at the cell membrane, binding and internalization of pH-sensitive pegylated liposomes encapsulating doxorubicin and functionalized with the nucleolin-binding F3 peptide (PEGASEMP), and resulting cytotoxicity against cancer cells from mouse, rat, canine, and human origin. Herein, it was shown that nucleolin expression levels were not a limitation on the continuous internalization of F3 peptide-targeted liposomes, despite the saturable nature of the binding mechanism. Modeling enabled the prediction of nucleolin-mediated total doxorubicin exposure provided by the experimental settings of the assessment of PEGASEMP's impact on cell death. The former increased proportionally with nucleolin-binding sites, a measure relevant for patient stratification. This pattern of variation was observed for the resulting cell death in nonsaturating conditions, depending on the cancer cell sensitivity to doxorubicin. This approach differs from standard determination of cytotoxic concentrations, which normally report values of incubation doses rather than the actual intracellular bioactive drug exposure. Importantly, in the context of development of nucleolin-based targeted drug delivery, the structural nucleolin homology (higher than 84%) and functional similarity across species presented herein, emphasized the potential to use toxicological data and other metrics from lower species to infer the dose for a first-in-human trial.

Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells.

Microglia are the immune competent cell of the central nervous system (CNS), promoting brain homeostasis and regulating inflammatory response against infection and injury. Chronic or exacerbated neuroinflammation is a cause of damage in several brain pathologies. Endogenous carbon monoxide (CO), produced from the degradation of heme, is described as anti-apoptotic and anti-inflammatory in several contexts, including in the CNS. Neuroglobin (Ngb) is a haemoglobin-homologous protein, which upregulation triggers antioxidant defence and prevents neuronal apoptosis. Thus, we hypothesised a crosstalk between CO and Ngb, in particular, that the anti-neuroinflammatory role of CO in microglia depends on Ngb. A novel CO-releasing molecule (ALF826) based on molybdenum was used for delivering CO in microglial culture.BV-2 mouse microglial cell line was challenged with lipopolysaccharide (LPS) for triggering inflammation, and after 6 h ALF826 was added. CO exposure limited inflammation by decreasing inducible nitric oxide synthase (iNOS) expression and the production of nitric oxide (NO) and tumour necrosis factor-α (TNF-α), and by increasing interleukine-10 (IL-10) release. CO-induced Ngb upregulation correlated in time with CO's anti-inflammatory effect. Moreover, knocking down Ngb reversed the anti-inflammatory effect of CO, suggesting that dependents on Ngb expression. CO-induced Ngb upregulation was independent on ROS signalling, but partially dependent on the transcriptional factor SP1. Finally, microglial cell metabolism is also involved in the inflammatory response. In fact, LPS treatment decreased oxygen consumption in microglia, indicating a switch to glycolysis, which is associated with a proinflammatory. While CO treatment increased oxygen consumption, reverting LPS effect and indicating a metabolic shift into a more oxidative metabolism. Moreover, in the absence of Ngb, this phenotype was no longer observed, indicating Ngb is needed for CO's modulation of microglial metabolism. Finally, the metabolic shift induced by CO did not depend on alteration of mitochondrial population. In conclusion, neuroglobin emerges for the first time as a key player for CO signalling against exacerbated inflammation in microglia.

Identification of SPRY4 as a Novel Candidate Susceptibility Gene for Familial Nonmedullary Thyroid Cancer.

Background: The molecular basis of familial nonmedullary thyroid cancer (FNMTC) is still poorly understood, representing a limitation for molecular diagnosis and clinical management. In this study, we aimed to identify new susceptibility genes for FNMTC through whole-exome sequencing (WES) analysis of leukocyte DNA of patients from a highly informative FNMTC family. Methods: We selected six affected family members to conduct WES analysis. Bioinformatic analyses were undertaken to filter and select the genetic variants shared by the affected members, which were subsequently validated by Sanger sequencing. To select the most likely pathogenic variants, several studies were performed, including family segregation analysis, in silico impact characterization, and gene expression (messenger RNA and protein) depiction in databases. For the most promising variant identified, we performed in vitro studies to validate its pathogenicity. Results: Several potentially pathogenic variants were identified in different candidate genes. After filtering with appropriate criteria, the variant c.701C>T, p.Thr234Met in the SPRY4 gene was prioritized for in vitro functional characterization. This SPRY4 variant led to an increase in cell viability and colony formation, indicating that it confers a proliferative advantage and potentiates clonogenic capacity. Phosphokinase array and Western blot analyses suggested that the effects of the SPRY4 variant were mediated through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, which was further supported by a higher responsiveness of thyroid cancer cells with the SPRY4 variant to a MEK inhibitor. Conclusions: WES analysis in one family identified SPRY4 as a likely novel candidate susceptibility gene for FNMTC, allowing a better understanding of the cellular and molecular mechanisms underlying thyroid cancer development.

Arl13b Regulates Breast Cancer Cell Migration and Invasion by Controlling Integrin-Mediated Signaling.

Breast cancer is the first cause of cancer-related mortality among women worldwide, according to the most recent estimates. This mortality is mainly caused by the tumors' ability to form metastases. Cancer cell migration and invasion are essential for metastasis and rely on the interplay between actin cytoskeleton remodeling and cell adhesion. Therefore, understanding the mechanisms by which cancer cell invasion is controlled may provide new strategies to impair cancer progression. We investigated the role of the ADP-ribosylation factor (Arf)-like (Arl) protein Arl13b in breast cancer cell migration and invasion in vitro, using breast cancer cell lines and in vivo, using mouse orthotopic models. We show that Arl13b silencing inhibits breast cancer cell migration and invasion in vitro, as well as cancer progression in vivo. We also observed that Arl13b is upregulated in breast cancer cell lines and patient tissue samples. Moreover, we found that Arl13b localizes to focal adhesions (FAs) and interacts with ß3-integrin. Upon Arl13b silencing, ß3-integrin cell surface levels and FA size are increased and integrin-mediated signaling is inhibited. Therefore, we uncover a role for Arl13b in breast cancer cell migration and invasion and provide a new mechanism for how ARL13B can function as an oncogene, through the modulation of integrin-mediated signaling.

Rab35 controls cilium length, function and membrane composition.

Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.

Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells.

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

Inhibition of fucosylation in human invasive ductal carcinoma reduces E-selectin ligand expression, cell proliferation, and ERK1/2 and p38 MAPK activation.

Breast cancer tissue overexpresses fucosylated glycans, such as sialyl-Lewis X/A (sLeX/A ), and α-1,3/4-fucosyltransferases (FUTs) in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E-selectin, initiating tumor extravasation. However, their role in breast carcinogenesis is still unknown. Here, we aimed to define the contribution of the fucosylated structures, including sLeX/A , to cell adhesion, cell signaling, and cell proliferation in invasive ductal carcinomas (IDC), the most frequent type of breast cancer. We first analyzed expression of E-selectin ligands in IDC tissue and established primary cell cultures from the tissue. We observed strong reactivity with E-selectin and anti-sLeX/A antibodies in both IDC tissue and cell lines, and expression of α-1,3/4 FUTs FUT4, FUT5, FUT6, FUT10, and FUT11. To further assess the role of fucosylation in IDC biology, we immortalized a primary IDC cell line with human telomerase reverse transcriptase to create the 'CF1_T cell line'. Treatment with 2-fluorofucose (2-FF), a fucosylation inhibitor, completely abrogated its sLeX/A expression and dramatically reduced adherence of CF1_T cells to E-selectin under hemodynamic flow conditions. In addition, 2-FF-treated CF1_T cells showed a reduced migratory ability, as well as decreased cell proliferation rate. Notably, 2-FF treatment lowered the growth factor expression of CF1_T cells, prominently for FGF2, vascular endothelial growth factor, and transforming growth factor beta, and negatively affected activation of signal-regulating protein kinases 1 and 2 and p38 mitogen-activated protein kinase signaling pathways. These data indicate that fucosylation licenses several malignant features of IDC, such as cell adhesion, migration, proliferation, and growth factor expression, contributing to tumor progression.

Inefficient recruitment of kinesin-1 to melanosomes precludes it from facilitating their transport.

Microtubules and F-actin, and their associated motor proteins, are considered to play complementary roles in long- and short-range organelle transport. However, there is growing appreciation that myosin/F-actin networks can drive long-range transport. In melanocytes, myosin-Va and kinesin-1 have both been proposed as long-range centrifugal transporters moving melanosomes into the peripheral dendrites. Here, we investigated the role of kinesin-1 heavy chain (Kif5b) and its suggested targeting factor Rab1a in transport. We performed confocal microscopy and subcellular fractionation, but did not detect Kif5b or Rab1a on melanosomes. Meanwhile functional studies, using siRNA knockdown and dominant negative mutants, did not support a role for Kif5b or Rab1a in melanosome transport. To probe the potential of Kif5b to function in transport, we generated fusion proteins that target active Kif5b to melanosomes and tested their ability to rescue perinuclear clustering in myosin-Va-deficient cells. Expression of these chimeras, but not full-length Kif5b, dispersed melanosomes with similar efficiency to myosin-Va. Our data indicate that kinesin and microtubules can compensate for defects in myosin-Va and actin-based transport in mammals, but that endogenous Kif5b does not have an important role in transport of melanocytes due to its inefficient recruitment to melanosomes.

The role of galectin-1 in in vitro and in vivo photodynamic therapy with a galactodendritic porphyrin.

Conventional photodynamic agents used in clinic are porphyrin-based photosensitizers. However, they have low tumour selectivity, which may induce unwanted side-effects and damage to healthy tissues. In this study, we used a porphyrin with dendritic units of galactose (PorGal8) developed by us, which can target the galactose-binding protein, galectin-1, known to be overexpressed in many tumour tissues. In vitro and in vivo studies had been conducted for the validation of PorGal8 effectiveness. We showed a specific uptake of PorGal8 and induction of apoptotic cell death by generating oxidative stress and alterations in the cytoskeleton of bladder cancer cells overexpressing galectin-1. We further validated the photodynamic efficiency of PorGal8 in athymic nude mice (Balb/c nu/nu) bearing subcutaneously implanted luciferase-positive bladder cancer xenografts, overexpressing galectin-1 protein. PorGal8 (5 µmol/kg, intraperitoneal), injected 24 h before light delivery (50.4 J/cm2), inhibited tumour growth. We conclude that the use of PorGal8 enables selective target and cytotoxicity by photodynamic therapy in cancer cells overexpressing galectin-1, preventing undesired phototoxicity in the surrounding healthy tissues.

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity.

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

Functional and molecular characterization of cancer stem-like cells in bladder cancer: a potential signature for muscle-invasive tumors.

Striking evidence associates cancer stem cells (CSCs) to the high recurrence rates and poor survival of patients with muscle-invasive bladder cancer (BC). However, the prognostic implication of those cells in risk stratification is not firmly established, mainly due to the functional and phenotypic heterogeneity of CSCs populations, as well as, to the conflicting data regarding their identification based on a single specific marker. This emphasizes the need to exploit putative CSC-related molecular markers with potential prognostic significance in BC patients.This study aimed to isolate and characterize bladder CSCs making use of different functional and molecular approaches. The data obtained provide strong evidence that muscle-invasive BC is enriched with a heterogeneous stem-like population characterized by enhanced chemoresistance and tumor initiating properties, able to recapitulate the heterogeneity of the original tumor. Additionally, a logistic regression analysis identified a 2-gene stem-like signature (SOX2 and ALDH2) that allows a 93% accurate discrimination between non-muscle-invasive and invasive tumors.Our findings suggest that a stemness-related gene signature, combined with a cluster of markers to more narrowly refine the CSC phenotype, could better identify BC patients that would benefit from a more aggressive therapeutic intervention targeting CSCs population.

Challenges in Antibody Development against Tn and Sialyl-Tn Antigens.

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment.

Rab27a GTPase modulates L-type Ca2+ channel function via interaction with the II-III linker of CaV1.3 subunit.

In a variety of cells, secretory processes require the activation of both Rab27a and L-type channels of the Ca(V)1.3 subtype. In the retinal pigment epithelium (RPE), Rab27a and Ca(V)1.3 channels regulate growth-factor secretion towards its basolateral side. Analysis of murine retina sections revealed a co-localization of both Rab27a and Ca(V)1.3 at the basolateral membrane of the RPE. Heterologously expressed Ca(V)1.3/ß3/α2δ1 channels showed negatively shifted voltage-dependence and decreased current density of about 70% when co-expressed with Rab27a. However, co-localization analysis using α(5)ß(1) integrin as a membrane marker revealed that Rab27a co-expression reduced the surface expression of Ca(V)1.3 only about 10%. Physical binding of heterologously expressed Rab27a with Ca(V)1.3 channels was shown by co-localization in immunocytochemistry as well as co-immunoprecipitation which was abolished after deletion of a MyRIP-homologous amino acid sequence at the II-III linker of the Ca(V)1.3 subunit. Rab27a over-expression in ARPE-19 cells positively shifted the voltage dependence, decreased current density of endogenous Ca(V)1.3 channels and reduced VEGF-A secretion. We show the first evidence of a direct functional modulation of an ion channel by Rab27a suggesting a new mechanism of Rab and ion channel interaction in the control of VEGF-A secretion in the RPE.

Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection.

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5-kinase (PIKfyve) that converts phosphatidylinositol 3-phosphate [PI(3)P] into phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2 ] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non-pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P2 effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P2 -dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.

STUB1/CHIP is required for HIF1A degradation by chaperone-mediated autophagy.

The transcription factor HIF1 is mostly regulated by the oxygen-dependent proteasomal degradation of the labile subunit HIF1A. Recent data showed degradation of HIF1A in the lysosome through chaperone-mediated autophagy (CMA). However the molecular mechanism involved has not been elucidated. This study shows that the KFERQ-like motif, that has been identified in all CMA substrates, is required to mediate the interaction between HIF1A and the chaperone HSPA8. Moreover, mutations in the KFERQ-like motif of HIF1A preclude the interaction with the CMA receptor LAMP2A, thus inhibiting its lysosomal degradation. Importantly, we show for the first time that the ubiquitin ligase STUB1 is required for degradation of HIF1A in the lysosome by CMA. Indeed, mutations in STUB1 that inhibit either the ubiquitin ligase activity or its ability to bind to HSPA8, both prevent degradation of HIF1A by CMA. Moreover, we show that HIF1A binds to and is translocated into intact lysosomes isolated from rat livers. This new pathway for degradation of HIF1A does not depend on the presence of oxygen and is activated in response to nutrient deprivation such that the levels of HIF1A bound to CMA positive lysosomes significantly increase in starved animal livers and the binding of HIF1A to LAMP2A increases in response to serum deprivation. Moreover, excessive degradation of HIF1A by CMA compromises cells' ability to respond to and survive under hypoxia, suggesting that this pathway might be of pathophysiological importance in conditions that combine hypoxia with starvation.

Impact of anti-PLK1 siRNA-containing F3-targeted liposomes on the viability of both cancer and endothelial cells.

We have previously described the development of novel sterically stabilized F3-targeted pH-sensitive liposomes, which exhibited the ability to target both cancer and endothelial cells. Herein, the therapeutic potential of those liposomes was assessed upon encapsulation of a siRNA against a well-validated molecular target, PLK1. Treatment of prostate cancer (PC3) and angiogenic endothelial (HMEC-1) cells with F3-targeted liposomes containing anti-PLK1 siRNA resulted in a significant decrease in cell viability, which was mediated by a marked PLK1 silencing, both at the mRNA and protein levels. Furthermore, pre-treatment of PC3 cells with F3-targeted liposomes containing anti-PLK1 siRNA enabled a 3-fold reduction of paclitaxel IC50 and a 2.5-fold augment of the percentage of cancer cells in G2/mitosis arrest, which ultimately culminated in cell death. Overall, the F3-targeted nanocarrier containing an anti-PLK1 siRNA might constitute a valuable system for prostate cancer treatment, either applied in a single schedule or combined with conventional chemotherapy.

Impact of PLK-1 silencing on endothelial cells and cancer cells of diverse histological origin.

The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a molecular target within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelial cells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 downregulation on the cancer cells chemosensitization to paclitaxel was further assessed. Downregulation of Plk-1 reduced cancer cells viability from 40 to 85% and up to 59% in endothelial cells. Regarding the latter, it compromised their ability to form new tube-like structures, decreasing the formation of network projections up to 46%. This suggested for the first time, PLK-1 as a valuable angiogenic molecular target. In combination with paclitaxel, anti-PLK-1 siRNA chemosensitized non-small cell lung cancer (NSCLC) and prostate carcinoma cell lines, leading up to a 2-fold increase in the drug cytotoxic effect. Moreover, the sequential incubation of anti-PLK-1 siRNA and paclitaxel led to a decrease in the IC50 of the latter up to 2.7- and 4.1-fold, in A-549 and PC-3 cells, respectively. The combination of anti-PLK-1 siRNA with paclitaxel led to cell cycle arrest, increasing the number of cells at the G2/M and S phases to 1.5 and 1.3-fold in PC-3 cells, and to 1.6 and 1.4-fold in A-549 cells, respectively. Overall, it has been demonstrated that PLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunity to interfere with different hallmarks of cancer, in tumors with diverse histological origin.

Efficient intracellular delivery of siRNA with a safe multitargeted lipid-based nanoplatform.

AIM: The design of novel F3-targeted liposomes with adequate features for systemic administration, to enable efficient intracellular delivery of siRNA toward both cancer and endothelial cells from angiogenic blood vessels. MATERIALS & METHODS: Cellular association studies were performed by flow cytometry. Gene silencing was evaluated with eGFP-overexpressing cells, by flow cytometry and real-time reverse-transcription PCR. Safety and immunogenicity was assessed in CD1 mice. RESULTS: A strong improvement on siRNA internalization by the target cells was achieved, which was correlated with effective downregulation of eGFP. In addition, the F3-targeted liposomes were nonimmunogenic, even in a multiadministration schedule. CONCLUSION: Overall, the developed F3-targeted nanocarrier constitutes a valuable tool for the specific and safe systemic delivery of siRNA to solid tumors.

Bacteria and protozoa differentially modulate the expression of Rab proteins.

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.

The host endocytic pathway is essential for Plasmodium berghei late liver stage development.

The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively little is known about the parasite interactions with the host hepatocyte. Using immunofluorescence, live cell imaging and electron microscopy, we show that Plasmodium berghei parasites are surrounded by vesicles from the host late endocytic pathway. We found that these vesicles are acidic and contain the membrane markers Rab7a, CD63 and LAMP1. When host cell vesicle acidification was disrupted using ammonium chloride or Concanamycin A during the late liver stage of infection, parasite survival was not affected, but schizont size was significantly decreased. Furthermore, when the host cell endocytic pathway was loaded with BSA-gold, gold particles were found within the parasite cytoplasm, showing the transport of material from the host endocytic pathway toward the parasite interior. These observations reveal a novel Plasmodium-host interaction and suggest that vesicles from the host endolysosomal pathway could represent an important source of nutrients exploited by the fast-growing late liver stage parasites.