Biology subjects

Engel, D. A.

Publications and source records attributed to Engel, D. A..

3 recordsLinked to original sources

bioRxiv2024

Host PIK3C3 promotes Shigella flexneri spread from cell to cell through vacuole formation

Shigella flexneri is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). S. flexneri invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell- to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which S. flexneri escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of S. flexneri cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by S. flexneri in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, Listeria monocytogenes did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for L. monocytogenes dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased S. flexneri dissemination in the infant rabbit model of shigellosis. We propose a model of Shigella dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2. Author summaryShigella flexneri is an intracellular pathogen causing bacillary dysentery, a disease responsible for more than 200,000 deaths each year in the world. With the lack of efficient vaccines and the dramatic increase in multi-drug-resistant clinical isolates, it is critical to better understand Shigella pathogenesis to suggest new therapeutic treatments. Previous studies demonstrated that invasion of epithelial cells in the human colon and subsequent spread from cell to cell are critical determinants of pathogenesis. Cell-to-cell spread relies on manipulation of the host cell actin cytoskeleton supporting bacterial movement in the cytosol of infected cells. At cell-cell contacts, bacteria project into adjacent cells through formation of membrane protrusions that resolve into vacuoles from which the bacteria escape to gain access to the cytosol of adjacent cells. Here, we show that the host cell kinase PIK3C3 is critical for efficient cell-to-cell spread through resolution of protrusions into vacuoles. Our work suggests that inhibitors of PIK3C3 may represent novel avenues of therapeutic intervention in shigellosis.

microbiology↗

bioRxiv2024

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

microbiology↗

bioRxiv2020

Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is absolutely required for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles and retention of viral RNA within these particles. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able to overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified domain of NP, the "central domain" (CD). ImportanceInclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

microbiology↗