Neta Regev-Rudzki

Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.

Dual Targeting of Nfs1 and Discovery of Its Novel Processing Enzyme, Icp55

In eukaryotes, each subcellular compartment harbors a specific group of proteins that must accomplish specific tasks. Nfs1 is a highly conserved mitochondrial cysteine desulfurase that participates in iron-sulfur cluster assembly as a sulfur donor. Previous genetic studies, in Saccharomyces cerevisiae, have suggested that this protein distributes between the mitochondria and the nucleus with biochemically undetectable amounts in the nucleus (termed “eclipsed distribution”). Here, we provide direct evidence for Nfs1 nuclear localization (in addition to mitochondria) using both α-complementation and subcellular fractionation. We also demonstrate that mitochondrial and nuclear Nfs1 are derived from a single translation product. Our data suggest that the Nfs1 distribution mechanism involves at least partial entry of the Nfs1 precursor into mitochondria, and then retrieval of a minor subpopulation (probably by reverse translocation) into the cytosol and then the nucleus. To further elucidate the mechanism of Nfs1 distribution we determined the N-terminal mitochondrial sequence of Nfs1 by Edman degradation. This led to the discovery of a novel mitochondrial processing enzyme, Icp55. This enzyme removes three amino acids from the N terminus of Nfs1 after cleavage by mitochondrial processing peptidase. Intriguingly, Icp55 protease (like its substrate Nfs1) appears to be dual distributed between the nucleus and mitochondria.

The mitochondrial targeting sequence tilts the balance between mitochondrial and cytosolic dual localization

Dual localization of proteins in the cell has appeared in recent years to be a more abundant phenomenon than previously reported. One of the mechanisms by which a single translation product is distributed between two compartments, involves retrograde movement of a subset of processed molecules back through the organelle-membrane. Here, we investigated the specific contribution of the mitochondrial targeting sequence (MTS), as a cis element, in the distribution of two proteins, aconitase and fumarase. Whereas the cytosolic presence of fumarase is obvious, the cytosolic amount of aconitase is minute. Therefore, we created (1) MTS-exchange mutants, exchanging the MTS of aconitase and fumarase with each other as well as with those of other proteins and, (2) a set of single mutations, limited to the MTS of these proteins. Distribution of both proteins is affected by mutations, a fact particularly evident for aconitase, which displays extraordinary amounts of processed protein in the cytosol. Thus, we show for the first time, that the MTS has an additional role beyond targeting: it determines the level of retrograde movement of proteins back into the cytosol. Our results suggest that the translocation rate and folding of proteins during import into mitochondria determines the extent to which molecules are withdrawn back into the cytosol.

Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis

Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the “find me” and “eat me” signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such “eat me” signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific “find me” and “eat me” signals.

Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs

Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS). KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

Pathogen-derived extracellular vesicles coordinate social behaviour and host manipulation

Infectious diseases are the leading cause of death of children worldwide, causing a tenacious and major public-health burden. The dynamic interplay between pathogens and their host is one of the most complicated themes of the disease progression. Pathogens excel in developing different means to facilitate cell-cell communication via secreted vesicles, among others. The released vesicles are involved in the transfer of biologically active molecules that induce phenotypic changes in the recipient cells. The messages within the vesicles are delivered to coordinate diverse processes, including virulence factor expression, differentiation state and control of their population density. Importantly, production of such vesicles promotes pathogen survival, as it provides a secure means of pathogen-pathogen communication and an ability to manipulate host responses for their own benefits. This review highlights intriguing findings, which show the important role of EVs in the social activity of pathogens, within and in between their communities. We further present examples of how pathogens use EVs to alter host immune and non-immune responses. Advancing our understanding of cell-cell communication in infectious diseases will be particularly useful to decipher the complexity of the cross-talk between pathogens themselves and their hosts, leading to the development of therapeutic strategies for fighting infectious agents.

Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.STING is an intracellular DNA sensor that can alter response to infection, but in the case of malaria it is unclear how parasite DNA in red blood cells (RBCs) reaches DNA sensors in immune cells. Here the authors show that STING in human monocytes can sense P. falciparum nucleic acids transported from infected RBCs via parasite extracellular vesicles.

Identification and classification of the malaria parasite blood developmental stages, using imaging flow cytometry

Malaria is the most devastating parasitic disease of humans, caused by the unicellular protozoa of the Plasmodium genus, such as Plasmodium falciparum (Pf) and is responsible for up to a million deaths each year. Pf life cycle is complex, with transmission of the parasite between humans via mosquitos involving a remarkable series of morphological transformations. In the bloodstream, the parasites undergo asexual multiplications inside the red blood cell (RBC), where they mature through the ring (R), trophozoite (T) and schizont (S) stages, and sexual development, resulting in gametocytes (G). All symptoms of malaria pathology are caused by the asexual blood stage parasites. Flow cytometry methods were previously used to detect malaria infected (i) RBCs, in live or fixed cells, using DNA (Hoechst) and RNA (Thiazole Orange) stains. Here, by using imaging flow cytometry, we developed improved methods of identifying and quantifying each of the four parasite blood stages (R, T, S and G). This technique allows multi-channel, high resolution imaging of individual parasites, as well as detailed morphological quantification of Pf-iRBCs cultures. Moreover, by measuring iRBC morphological properties, we can eliminate corrupted and extracellular (dying) parasites from the analysis, providing accurate quantification and robust measurement of the parasitemia profile. This new method is a valuable tool in malaria molecular biology research and drug screen assays.

Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis

Background Schistosomiasis traditionally has been diagnosed by detecting eggs in stool or urine. However, the sensitivity of these examinations is limited, especially in travelers with a low worm burden. Serologic tests have a greater sensitivity, but their results remain positive regardless of treatment and thus cannot be used for follow-up of patients. We hypothesized that detection of worm microRNAs (miRNAs) in serum can overcome the drawbacks of the existing diagnostic methods. Methods and Results Twenty-six returning travelers with schistosomiasis (based on positive results of serologic tests or detection of ova) and 17 healthy controls were included in the study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification of miRNA extracted directly from 500 µL of serum had limited sensitivity and specificity. However, qRT-PCR analysis of RNA extracted from 200 μL of serum extracellular vesicles detected 4 schistosomal miRNAs; the sensitivity and specificity of the 2 highest expressed miRNAs (bantam and miR-2c-3p) were 86% and 84%, respectively. In 7 patients with posttreatment serum available for analysis, we observed outcomes ranging from a reduction in the schistosomal miRNA level to full recovery from disease. Conclusions qRT-PCR of pathogen miRNAs isolated from extracellular vesicles in sera from infected individuals may provide a new tool for diagnosing schistosomiasis in patients with a low parasite burden. This assay could also be used for evaluating the outcome of therapy, as well as disease-control programs.

Extracellular Vesicles: A Prevalent Tool for Microbial Gene Delivery?

Genetic plasticity of prokaryotic microbial communities is largely dependent on the ongoing exchange of genetic determinants by Horizontal Gene Transfer (HGT). HGT events allow beneficial genetic transitions to occur throughout microbial life, thus promoting adaptation to changing environmental conditions. Here, the significance of secreted vesicles in mediating HGT between microorganisms is discussed, while focusing on the benefits gained by vesicle‐mediated gene delivery and its occurrence under different environmental cues. The potential use of secreted DNA‐harboring vesicles as a mechanism of currently unresolved HGT events in eukaryotic microbes is further discussed.