Publications by authors named "Seamus R Morrone"

9 Publications

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Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector.

mBio 2021 Nov 2:e0257521. Epub 2021 Nov 2.

Institute for Systems Biology, Seattle, Washington, USA.

Gametocytes of the malaria parasite are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.
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http://dx.doi.org/10.1128/mBio.02575-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8561384PMC
November 2021

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period.

Curr Biol 2021 Oct 8. Epub 2021 Oct 8.

Prehistoric Department, Museum of Natural History Vienna, Burgring 7, 1010 Vienna, Austria. Electronic address:

We subjected human paleofeces dating from the Bronze Age to the Baroque period (18 century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.
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http://dx.doi.org/10.1016/j.cub.2021.09.031DOI Listing
October 2021

High flavivirus structural plasticity demonstrated by a non-spherical morphological variant.

Nat Commun 2020 06 19;11(1):3112. Epub 2020 Jun 19.

Program in Emerging Infectious Diseases, Duke-NUS Medical School, KTP Building, 8 College Road, Singapore, 169857, Singapore.

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.
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http://dx.doi.org/10.1038/s41467-020-16925-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305169PMC
June 2020

Structural perspectives of antibody-dependent enhancement of infection of dengue virus.

Curr Opin Virol 2019 06 4;36:1-8. Epub 2019 Mar 4.

Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, KTP Building, 8 College Road, Singapore 169857, Singapore; Centre for BioImaging Sciences, National University of Singapore, Singapore 117557, Singapore. Electronic address:

Dengue virus (DENV) consists of four serotypes. Sequential serotype infections can cause increased disease severity, likely due to antibody-dependent enhancement (ADE) of infection. Here, we review two recent papers showing major advancements in the understanding of the ADE mechanism for both mature and immature DENV. The surface of both mature and immature DENV contains E and another protein - M in mature and prM in immature virus. On mature DENV, the orientation of anti-E antibody with respect to the virus surface determines the antibody enhancement properties. On the immature virus, binding of anti-prM antibody aids the dissociation of pr from the fusion loop of E protein allowing virus-endosomal membrane interaction, thus overcoming the hurdle in the early step of fusion.
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http://dx.doi.org/10.1016/j.coviro.2019.02.002DOI Listing
June 2019

Digital signaling network drives the assembly of the AIM2-ASC inflammasome.

Proc Natl Acad Sci U S A 2018 02 12;115(9):E1963-E1972. Epub 2018 Feb 12.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205

The AIM2-ASC inflammasome is a filamentous signaling platform essential for mounting host defense against cytoplasmic dsDNA arising not only from invading pathogens but also from damaged organelles. Currently, the design principles of its underlying signaling network remain poorly understood at the molecular level. We show here that longer dsDNA is more effective in inducing AIM2 assembly, its self-propagation, and downstream ASC polymerization. This observation is related to the increased probability of forming the base of AIM2 filaments, and indicates that the assembly discerns small dsDNA as noise at each signaling step. Filaments assembled by receptor AIM2, downstream ASC, and their joint complex all persist regardless of dsDNA, consequently generating sustained signal amplification and hysteresis. Furthermore, multiple positive feedback loops reinforce the assembly, as AIM2 and ASC filaments accelerate the assembly of nascent AIM2 with or without dsDNA. Together with a quantitative model of the assembly, our results indicate that an ultrasensitive digital circuit drives the assembly of the AIM2-ASC inflammasome.
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http://dx.doi.org/10.1073/pnas.1712860115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834677PMC
February 2018

The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex.

Elife 2015 Dec 16;4:e11721. Epub 2015 Dec 16.

Johns Hopkins University School of Medicine, Baltimore, United States.

The ability to recognize foreign double-stranded (ds)DNA of pathogenic origin in the intracellular environment is an essential defense mechanism of the human innate immune system. However, the molecular mechanisms underlying distinction between foreign DNA and host genomic material inside the nucleus are not understood. By combining biochemical assays and single-molecule techniques, we show that the nuclear innate immune sensor IFI16 one-dimensionally tracks long stretches of exposed foreign dsDNA to assemble into supramolecular signaling platforms. We also demonstrate that nucleosomes represent barriers that prevent IFI16 from targeting host DNA by directly interfering with these one-dimensional movements. This unique scanning-assisted assembly mechanism allows IFI16 to distinguish friend from foe and assemble into oligomers efficiently and selectively on foreign DNA.
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http://dx.doi.org/10.7554/eLife.11721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829420PMC
December 2015

Assembly-driven activation of the AIM2 foreign-dsDNA sensor provides a polymerization template for downstream ASC.

Nat Commun 2015 Jul 22;6:7827. Epub 2015 Jul 22.

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine Baltimore, Maryland 21205, USA.

AIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2(PYD)) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2(PYD) oligomers define the filamentous structure, and the helical symmetry of the AIM2(PYD) filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2(PYD) is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.
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http://dx.doi.org/10.1038/ncomms8827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525163PMC
July 2015

Single-molecule imaging at high fluorophore concentrations by local activation of dye.

Biophys J 2015 Feb;108(4):949-956

Zernike Institute for Advanced Materials, Groningen, The Netherlands. Electronic address:

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.
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http://dx.doi.org/10.1016/j.bpj.2014.12.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336379PMC
February 2015

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy.

Proc Natl Acad Sci U S A 2014 Jan 23;111(1):E62-71. Epub 2013 Dec 23.

Departments of Biophysics and Biophysical Chemistry and Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Whether host DNA receptors have any capacity to distinguish self from nonself at the molecular level is an outstanding question in the innate immunity of mammals. Here, by using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve as an integral mechanism by which human IFN-inducible protein-16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders, such as Sjögren syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16⋅dsDNA oligomers are clearly different from the conventional noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA-binding pyrin domain of IFI16 that drives the cooperative filament assembly. The surface residues on the pyrin domain that mediate the cooperative DNA binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling foci in a switch-like manner and that it is capable of using the size of naked dsDNA as a molecular ruler to distinguish self from nonself.
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http://dx.doi.org/10.1073/pnas.1313577111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890864PMC
January 2014
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