Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile.

Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence.

Suppression of CD4+ Effector Responses by Naturally Occurring CD4+ CD25+ Foxp3+ Regulatory T Cells Contributes to Experimental Cerebral Malaria.

The role of naturally occurring CD4(+) CD25(+) Foxp3(+) regulatory T cells (nTreg) in the pathogenesis of cerebral malaria (CM), which involves both pathogenic T cell responses and parasite sequestration in the brain, is still unclear. To assess the contribution and dynamics of nTreg during the neuropathogenesis, we unbalanced the ratio between nTreg and naive CD4(+) T cells in an attenuated model of Plasmodium berghei ANKA-induced experimental CM (ECM) by using a selective cell enrichment strategy. We found that nTreg adoptive transfer accelerated the onset and increased the severity of CM in syngeneic C57BL/6 (B6) P. berghei ANKA-infected mice without affecting the level of parasitemia. In contrast, naive CD4(+) T cell enrichment prevented CM and promoted parasite clearance. Furthermore, early during the infection nTreg expanded in the spleen but did not efficiently migrate to the site of neuroinflammation, suggesting that nTreg exert their pathogenic action early in the spleen by suppressing the protective naive CD4(+) T cell response to P. berghei ANKA infection in vivo in both CM-susceptible (B6) and CM-resistant (B6-CD4(-/-)) mice. However, their sole transfer was not sufficient to restore CM susceptibility in two CM-resistant congenic strains tested. Altogether, these results demonstrate that nTreg are activated and functional during P. berghei ANKA infection and that they contribute to the pathogenesis of CM. They further suggest that nTreg may represent an early target for the modulation of the immune response to malaria.

A key role for Plasmodium subtilisin-like SUB1 protease in egress of malaria parasites from host hepatocytes.

In their mammalian host, Plasmodium parasites have two obligatory intracellular development phases, first in hepatocytes and subsequently in erythrocytes. Both involve an orchestrated process of invasion into and egress from host cells. The Plasmodium SUB1 protease plays a dual role at the blood stage by enabling egress of the progeny merozoites from the infected erythrocyte and priming merozoites for subsequent erythrocyte invasion. Here, using conditional mutagenesis in P. berghei, we show that SUB1 plays an essential role at the hepatic stage. Stage-specific sub1 invalidation during prehepatocytic development showed that SUB1-deficient parasites failed to rupture the parasitophorous vacuole membrane and to egress from hepatocytes. Furthermore, mechanically released parasites were not adequately primed and failed to establish a blood stage infection in vivo. The critical involvement of SUB1 in both pre-erythrocytic and erythrocytic developmental phases qualifies SUB1 as an attractive multistage target for prophylactic and therapeutic anti-Plasmodium intervention strategies.

In Silico screening on the three-dimensional model of the Plasmodium vivax SUB1 protease leads to the validation of a novel anti-parasite compound.

Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50 µM for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial.

Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia.

The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.

Impact of Green Gold Nanoparticle Coating on Internalization, Trafficking, and Efficiency for Photothermal Therapy of Skin Cancer.

Skin cancer is a global health issue and mainly composed of melanoma and nonmelanoma cancers. For the first clinical proof of concept on humans, we decided to study good prognosis skin cancers, i.e., carcinoma basal cell. In UE, the first-line treatment remains surgical resection, healing most of the tumors, but presents aesthetic disadvantages with a high reoccurrence rate on exposed areas. Moreover, the therapeutic indications could extend to melanoma and metastasis, which is a different medical strategy that could combine this treatment. Indeed, patients with late-stage melanoma are in a therapeutic impasse, despite recent targeted and immunological therapies. Photothermal therapy using gold nanoparticles is the subject of many investigations due to their strong potential to treat cancers by physical, thermal destruction. We developed gold nanoparticles synthesized by green chemistry (gGNPs), using endemic plant extract from Reunion Island, which have previously showed their efficiency at a preclinical stage. Here, we demonstrate that these gGNPs are less cytotoxic than gold nanoparticles synthesized by Turkevich's method. Furthermore, our work describes the optimization of gGNP coating and stabilization, also taking into consideration the gGNP path in cells (endocytosis, intracellular trafficking, and exocytosis), their specificity toward cancerous cells, their cytotoxicity, and their in vivo efficiency. Finally, based on the metabolic switch of cancerous cells overexpressing Glut transporters in skin cancers, we demonstrated that glucose-stabilized gGNP (gGNP@G) enables a quick internalization, fourfold higher in cancerous cells in contrast to healthy cells with no side cytotoxicity, which is particularly relevant to target and treat cancer.

Three-dimensional images reveal the impact of the endosymbiont Midichloria mitochondrii on the host mitochondria.

The hard tick, Ixodes ricinus, a main Lyme disease vector, harbors an intracellular bacterial endosymbiont. Midichloria mitochondrii is maternally inherited and resides in the mitochondria of I. ricinus oocytes, but the consequences of this endosymbiosis are not well understood. Here, we provide 3D images of wild-type and aposymbiotic I. ricinus oocytes generated with focused ion beam-scanning electron microscopy. Quantitative image analyses of endosymbionts and oocyte mitochondria at different maturation stages show that the populations of both mitochondrion-associated bacteria and bacterium-hosting mitochondria increase upon vitellogenisation, and that mitochondria can host multiple bacteria in later stages. Three-dimensional reconstructions show symbiosis-dependent morphologies of mitochondria and demonstrate complete M. mitochondrii inclusion inside a mitochondrion. Cytoplasmic endosymbiont located close to mitochondria are not oriented towards the mitochondria, suggesting that bacterial recolonization is unlikely. We further demonstrate individual globular-shaped mitochondria in the wild type oocytes, while aposymbiotic oocytes only contain a mitochondrial network. In summary, our study suggests that M. mitochondrii modulates mitochondrial fragmentation in oogenesis possibly affecting organelle function and ensuring its presence over generations.

Parvimonas micra, an oral pathobiont associated with colorectal cancer, epigenetically reprograms human colonocytes.

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here, we characterize and investigate one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we show that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylation alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra's possible role in the carcinogenic process.

Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori.

Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction. IMPORTANCE Bacterial resistance to antibiotics is a major threat to human health. Treatments combining antibiotics with metals were proposed to circumvent this hurdle. Only one such combination is successfully used in clinics associating antibiotics with the metal bismuth to treat infections by the human pathogen Helicobacter pylori. H. pylori causes 800,000 deaths by gastric cancer yearly. How bismuth impacts H. pylori and its response to this toxic metal were ill defined. We discovered that upon bismuth exposure, H. pylori secretes membrane vesicles that are enriched in bismuth. Bismuth also induces the formation of intracellular polyphosphate granules associated with compaction of the chromosome. Upon bismuth exposure, H. pylori displays both defense and protection mechanisms, with bismuth extrusion by vesicles and shielding of the chromosome.

Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A2 (PLA2) activity. Inhibition of PRDX6 with a PLA2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance.

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Characterization of Extracellular Vesicles Produced by Aspergillus fumigatus Protoplasts.

Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells.IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.

Tetanus Toxin Synthesis is Under the Control of A Complex Network of Regulatory Genes in Clostridium tetani.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C.tetani fermentation and subsequent purification and chemical inactivation. C.tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C.tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

Intracellular offspring released from SFB filaments are flagellated.

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

An early burst of IFN-gamma induced by the pre-erythrocytic stage favours Plasmodium yoelii parasitaemia in B6 mice.

BACKGROUND: In murine models of malaria, an early proinflammatory response has been associated with the resolution of blood-stage infection. To dissect the protective immune mechanism that allow the control of parasitaemia, the early immune response of C57BL/6 mice induced during a non-lethal plasmodial infection was analysed. METHODS: Mice were infected with Plasmodium yoelii 265BY sporozoites, the natural invasive form of the parasite, in order to complete its full life cycle. The concentrations of three proinflammatory cytokines in the sera of mice were determined by ELISA at different time points of infection. The contribution of the liver and the spleen to this cytokinic response was evaluated and the cytokine-producing lymphocytes were identified by flow cytometry. The physiological relevance of these results was tested by monitoring parasitaemia in genetically deficient C57BL/6 mice or wild-type mice treated with anti-cytokine neutralizing antibody. Finally, the cytokinic response in sera of mice infected with parasitized-RBCs was analysed. RESULTS: The early immune response of C57BL/6 mice to sporozoite-induced malaria is characterized by a peak of IFN-gamma in the serum at day 5 of infection and splenic CD4 T lymphocytes are the major producer of this cytokine at this time point. Somewhat unexpected, the parasitaemia is significantly lower in P. yoelii-infected mice in the absence of IFN-gamma. More precisely, at early time points of infection, IFN-gamma favours parasitaemia, whereas helping to clear efficiently the blood-stage parasites at later time points. Interestingly, the early IFN-gamma burst is induced by the pre-erythrocytic stage. CONCLUSION: These results challenge the current view regarding the role of IFN-gamma on the control of parasite growth since they show that IFN-gamma is not an essential mediator of protection in P. yoelii-infected C57BL/6 mice. Moreover, the mice parasitaemia is more efficiently controlled in the absence of an early IFN-gamma production, suggesting that this cytokine promotes parasite's growth. Finally, this early burst of IFN-gamma is induced by the pre-erythrocytic stage, showing the impact of this stage on the immune response taking place during the subsequent erythrocytic stage.

Extract from Aphloia theiformis, an edible indigenous plant from Reunion Island, impairs Zika virus attachment to the host cell surface.

The mosquito-borne Zika virus (ZIKV) belongs to the flavivirus genus of the Flaviviridae family. Contemporary epidemic strains of ZIKV are associated with congenital malformations in infants, including microcephaly, as well as Guillain-Barré syndrome in adults. A risk of human-to-human transmission of ZIKV is also well documented. A worldwide research effort has been undertaken to identify safe and effective strategies to prevent or treat ZIKV infection. We show here that extract from Aphloia theiformis, an edible endemic plant from Indian Ocean islands, exerts a potent antiviral effect against ZIKV strains of African and Asian lineages, including epidemic strains. The antiviral effect of A. theiformis extract was extended to clinical isolates of dengue virus (DENV) of the four serotypes in human hepatocytes. A. theiformis inhibited virus entry in host cells by acting directly on viral particles, thus impairing their attachment to the cell surface. Electron microscopic observations revealed that organization of ZIKV particles was severely affected by A. theiformis. We propose a model of antiviral action for A. theiformis against flaviviruses that highlights the potential of medicinal plants as promising sources of naturally-derived antiviral compounds to prevent ZIKV and DENV infections.

Regulatory CD4+ CD25+ Foxp3+ T cells expand during experimental Plasmodium infection but do not prevent cerebral malaria.

Pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in Plasmodium berghei ANKA (PbA) infected mice. Therefore, we hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+ CD25+ Treg cells. Remarkably, the number of CD4+ CD25high T cells expressing Foxp3 increased in the spleen during the course of infection. These cells displayed an activated phenotype and consistent with that, CD4+ CD25high Treg cells isolated from PbA-infected mice showed an enhanced regulatory activity in vitro. Surprisingly, these cells do not migrate to the brain at the time of neurological symptoms as the conventional CD4+ T cells do. CM was not exacerbated in anti-CD25 treated mice when infected with PbA one month after treatment, even if splenic CD8+ T cells expressing CD69 increased in these mice. Taken together, these results show that P. berghei infection leads to an increase of the number of splenic CD4+ CD25high Treg cells exhibiting in vitro suppressive function, but they do not seem to be involved in vivo in the protection against CM.

Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans.

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.

Sequestration of host metabolism by an intracellular pathogen.

For intracellular pathogens, residence in a vacuole provides a shelter against cytosolic host defense to the cost of limited access to nutrients. The human pathogen Chlamydia trachomatis grows in a glycogen-rich vacuole. How this large polymer accumulates there is unknown. We reveal that host glycogen stores shift to the vacuole through two pathways: bulk uptake from the cytoplasmic pool, and de novo synthesis. We provide evidence that bacterial glycogen metabolism enzymes are secreted into the vacuole lumen through type 3 secretion. Our data bring strong support to the following scenario: bacteria co-opt the host transporter SLC35D2 to import UDP-glucose into the vacuole, where it serves as substrate for de novo glycogen synthesis, through a remarkable adaptation of the bacterial glycogen synthase. Based on these findings we propose that parasitophorous vacuoles not only offer protection but also provide a microorganism-controlled metabolically active compartment essential for redirecting host resources to the pathogens.