Biology subjects

Manna, P. T.

Publications and source records attributed to Manna, P. T..

5 recordsLinked to original sources

bioRxiv2025

Exosome tethering requires tetherin homodimerisation

Exosomes are small extracellular vesicles that originate as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Upon fusion of MVBs with the plasma membrane, ILVs are released into the extracellular environment as exosomes. While exosomes can diffuse away from their cells of origin, the expression of the antiviral restriction factor tetherin promotes their retention at the cell surface, thereby limiting their release into the extracellular milieu. Tetherin plays an analogous role in retaining other extracellular particles, including many enveloped viruses and midbody remnants. In this study, we examine the molecular basis of exosome tethering and define the mechanisms underlying tetherin-mediated retention. Using a combination of biochemical, live-cell and ultrastructural imaging approaches, we show that tetherin homodimerisation is essential for exosome tethering. Mutations in regions or motifs of tetherin predicted to impact tetherin traffic to ILVs have only minor impacts on exosome tethering, suggesting redundancy in the mechanisms of tetherin traffic to ILVs, and subsequently, to exosomes. Collectively, these findings provide the first molecular insights into the mechanism that govern exosome tethering.

cell biology↗

bioRxiv2024

Targeting DNA-PK is a highly conserved poxvirus innate immune evasion mechanism

The sensing of viral nucleic acid by pattern recognition receptors (PRRs) is essential for initiation of a type-I interferon response against infection. Intracellular DNA sensing PRRs are responsible for initiating innate immune responses to poxviruses and other double-stranded DNA viruses. Poxviruses, in turn, encode an armoury of immunomodulators that inhibit this host defence mechanism. DNA-dependent protein kinase (DNA-PK) is an essential component of the cGAS/STING-dependent viral DNA sensing machinery that leads to the initiation of a type-I interferon response during poxvirus infection. Poxviruses counter this host sensing mechanism using the C4/C10 family of proteins that target DNA-PK, interfering with its ability to bind viral DNA. Although the DNA-PK complex, known also for its role in double strand break repair, is conserved across multiple taxa, its function in innate immunity outside mammals is unexplored. Here we analysed the contribution of DNA-PK to poxvirus DNA sensing in chickens, a species that is evolutionarily distant from mammals, but that is also infected by poxviruses. We found that DNA-PK functions as a DNA sensor in chickens, and this process is countered by C4/C10 family members found in fowlpox virus. This host/pathogen interaction is conserved across a broader range of species than other mechanisms of poxvirus antagonism of innate immune sensing, which may reflect the difficulty of the host in evolving escape mechanisms that interfere with a protein that is essential for maintenance of genomic stability.

immunology↗

bioRxiv2024

The evolutionary origins of the lysosome-related organelle sorting machinery reveal fundamental homology in post-endosome trafficking pathways

The major organelles and pathways of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) ([~]1.5 billion years ago) and their acquisition were defining milestones during the process of eukaryogenesis itself. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct inter-organelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of new cellular compartments, infer ancestral states pre-dating LECA, and can even provide insight into the process of eukaryogenesis itself. Here we undertake a molecular evolutionary analysis, including providing a transcriptome of the jakobid flagellate Reclinomonas americana, exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles, the so-called Biogenesis of Lysosome-related Organelle Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery, and by inference the corresponding sorting pathway, was likely in place prior to the divergence of eukaryotes and is found in a much more diverse array of eukaryotes than is currently assumed. As such, this sorting pathway is likely an underappreciated facet of broader eukaryotic cellular function. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1/MON1, and a newly identified relationship with HOPS/CORVET) offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LRO biogenesis machinery from prokaryotic origins to current eukaryotic diversity, Asgard archaea to animals, integrating it into the larger mechanistic framework describing endomembrane evolution.

cell biology↗

bioRxiv2023

Mechanisms of type I interferon production by chicken TLR21

The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-{kappa}B and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-{kappa}B and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species.

immunology↗

bioRxiv2019

Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells

CHoP-In (CRISPR/Cas9-mediated, Homology-independent, PCR-product Integration) is a fast and cloning-free strategy for genomic editing of mammalian cells. The desired integration fragment is produced as a PCR product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9/guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of key membrane trafficking proteins. The lack of any donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

molecular biology↗