The Munich Near-Infrared Cluster Survey (MUNICS) - IX. Galaxy Evolution to z ~ 2 From Optically Selected Catalogues

(Abridged) We present B, R, and I-band selected galaxy catalogues based on the Munich Near-Infrared Cluster Survey (MUNICS) which, together with the K-selected sample, serve as an important probe of galaxy evolution in the redshift range 0 < z < 2. Furthermore, used in comparison they are ideally suited to study selection effects. The construction of the B, R, and I-selected photometric catalogues, containing ~9000, ~9000, and ~6000 galaxies, respectively, is described in detail. The catalogues reach 50% completeness limits for point sources of B ~ 24.5mag, R ~ 23.5mag, and I ~ 22.5mag and cover an area of about 0.3 square degrees. Photometric redshifts are derived for all galaxies with an accuracy of dz/(1+z) ~ 0.057. We investigate the influence of selection band and environment on the specific star formation rate (SSFR). We find that K-band selection indeed comes close to selection in stellar mass, while B-band selection purely selects galaxies in star formation rate. We use a galaxy group catalogue constructed on the K-band selected MUNICS sample to study possible differences of the SSFR between the field and the group environment, finding a marginally lower average SSFR in groups as compared to the field, especially at lower redshifts. The field-galaxy luminosity function in the B and R band as derived from the R-selected sample evolves out to z ~ 2 in the sense that the characteristic luminosity increases but the number density decreases. This effect is smaller at longer rest-frame wavelengths and gets more pronounced at shorter wavelengths. Parametrising the redshift evolution of the Schechter parameters as M*(z) = M*(0) + a ln(1+z) and Phi*(z) = Phi*(0) (1+z)^b we find evolutionary parameters a ~ -2.1 and b ~ -2.5 for the B band, and a ~ -1.4 and b ~ -1.8 for the R band.Comment: 23 pages, 19 figures; accepted for publication in MNRAS; version with high-resolution figures will be made available at http://www.usm.uni-muenchen.de/people/feulner/munics9/preprint_munics9.pd

The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony.

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for > or =6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria

Alpha-v-containing integrins are host receptors for the Plasmodium falciparum sporozoite surface protein, TRAP.

Malaria-causing Plasmodium sporozoites are deposited in the dermis by the bite of an infected mosquito and move by gliding motility to the liver where they invade and develop within host hepatocytes. Although extracellular interactions between Plasmodium sporozoite ligands and host receptors provide important guidance cues for productive infection and are good vaccine targets, these interactions remain largely uncharacterized. Thrombospondin-related anonymous protein (TRAP) is a parasite cell surface ligand that is essential for both gliding motility and invasion because it couples the extracellular binding of host receptors to the parasite cytoplasmic actinomyosin motor; however, the molecular nature of the host TRAP receptors is poorly defined. Here, we use a systematic extracellular protein interaction screening approach to identify the integrin αvβ3 as a directly interacting host receptor for Plasmodium falciparum TRAP. Biochemical characterization of the interaction suggests a two-site binding model, requiring contributions from both the von Willebrand factor A domain and the RGD motif of TRAP for integrin binding. We show that TRAP binding to cells is promoted in the presence of integrin-activating proadhesive Mn2+ ions, and that cells genetically targeted so that they lack cell surface expression of the integrin αv-subunit are no longer able to bind TRAP. P. falciparum sporozoites moved with greater speed in the dermis of Itgb3-deficient mice, suggesting that the interaction has a role in sporozoite migration. The identification of the integrin αvβ3 as the host receptor for TRAP provides an important demonstration of a sporozoite surface ligand that directly interacts with host receptors. Copyright © 2018 the Author(s). Published by PNAS

Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

Palmitoyl transferases have critical roles in the development of mosquito and liver stages of Plasmodium.

As the Plasmodium parasite transitions between mammalian and mosquito host, it has to adjust quickly to new environments. Palmitoylation, a reversible and dynamic lipid post-translational modification, plays a central role in regulating this process and has been implicated with functions for parasite morphology, motility and host cell invasion. While proteins associated with the gliding motility machinery have been described to be palmitoylated, no palmitoyl transferase responsible for regulating gliding motility has previously been identified. Here, we characterize two palmityol transferases with gene tagging and gene deletion approaches. We identify DHHC3, a palmitoyl transferase, as a mediator of ookinete development, with a crucial role for gliding motility in ookinetes and sporozoites, and we co-localize the protein with a marker for the inner membrane complex in the ookinete stage. Ookinetes and sporozoites lacking DHHC3 are impaired in gliding motility and exhibit a strong phenotype in vivo; with ookinetes being significantly less infectious to their mosquito host and sporozoites being non-infectious to mice. Importantly, genetic complementation of the DHHC3-ko parasite completely restored virulence. We generated parasites lacking both DHHC3, as well as the palmitoyl transferase DHHC9, and found an enhanced phenotype for these double knockout parasites, allowing insights into the functional overlap and compensational nature of the large family of PbDHHCs. These findings contribute to our understanding of the organization and mechanism of the gliding motility machinery, which as is becoming increasingly clear, is mediated by palmitoylation

Antibody-Mediated Protection against Plasmodium Sporozoites Begins at the Dermal Inoculation Site

Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity.Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site

Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species‐specific barrier

Abstract Malaria infection starts with the injection of Plasmodium sporozoites into the host’s skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species‐specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre‐clinical screening of next‐generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down‐selection of candidates prior to human vaccine trials

Targeting Plasmodium falciparum protein kinases with adenosine analogue-oligoarginine conjugates.

During the last decade, a vast number of inhibitors, ligands and fluorescent probes have evolved for mammalian protein kinases; however, the suitability of these compounds for studies of evolutionarily divergent eukaryotes has mostly been left beyond the scope of research. Here, we examined whether adenosine analogue-oligoarginine conjugates that had been extensively characterized as efficient inhibitors of the human protein kinases are applicable for targeting Plasmodium protein kinases. We demonstrated that ARCs were not only able to bind to and inhibit a representative member of Plasmodium falciparum kinome (cGMP-dependent protein kinase) in biochemical assay, but also affected the general phosphorylation levels in parasites released from the infected red blood cells upon saponin treatment. These findings urge advantaging of already existing biochemical tools, whose initially generic, but intrinsically "tunable" selectivity profiles could be used for dissection of signaling pathways outside the initially defined group of biological targets