Phosphatidylserine within the Viral Membrane Enhances Chikungunya Virus Infectivity in a Cell-type Dependent Manner

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.

The role of phosphatidylserine on the membrane in immunity and blood coagulation

The negatively charged aminophospholipid, phosphatidylserine (PtdSer), is located in the inner leaflet of the plasma membrane in normal cells, and may be exposed to the outer leaflet under some immune and blood coagulation processes. Meanwhile, Ptdser exposed to apoptotic cells can be recognized and eliminated by various immune cells, whereas on the surface of activated platelets Ptdser interacts with coagulation factors prompting enhanced production of thrombin which significantly facilitates blood coagulation. In the case where PtdSer fails in exposure or mistakenly occurs, there are occurrences of certain immunological and haematological diseases, such as the Scott syndrome and Systemic lupus erythematosus. Besides, viruses (e.g., Human Immunodeficiency Virus (HIV), Ebola virus (EBOV)) can invade host cells through binding the exposed PtdSer. Most recently, the Corona Virus Disease 2019 (COVID-19) has been similarly linked to PtdSer or its receptors. Therefore, it is essential to comprehensively understand PtdSer and its functional characteristics. Therefore, this review summarizes Ptdser, its eversion mechanism; interaction mechanism, particularly with its immune receptors and coagulation factors; recognition sites; and its function in immune and blood processes. This review illustrates the potential aspects for the underlying pathogenic mechanism of PtdSer-related diseases, and the discovery of new therapeutic strategies as well.

Visualization of SpoVAEa protein dynamics in dormant spores of Bacillus cereus and dynamic changes in their germinosomes and SpoVAEa during germination

Bacillus cereus spores, like most Bacillus spores, can survive for years depending on their specific structure, and germinate when their surroundings become suitable. Spore germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of the dormant state, more information related to the function of germination proteins could be useful to develop new strategies to control B. cereus spores. Prior work has shown that: i) the channel protein SpoVAEa exhibits high frequency movement in the outer leaflet of the inner membrane (IM) in dormant spores of B. subtilis; ii) the dynamics of germinosome formation in developing spores of B. cereus indicate that the formation of germinosome foci is slower than foci formation of germinant receptor GerR and scaffold protein GerD. However, the dynamics of movement of SpoVAEa in B. cereus spores, and the complete behavior of the germinosome in germinated spores of B. cereus are still unclear. In this study, we found that the SpoVAEa fluorescent foci in dormant spores of B. cereus redistribute at a lower frequency than in B. subtilis, and likely colocalize with GerD in dormant spores. Our results further indicate that: i) overexpression of GerR(A-C-B)-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus; ii), germinosome foci composed of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I were lost prior to the phase transition in germination; and iii) GerD-mScarlet-I foci spread out but continued to exist beyond the phase transition of B. cereus spores.

Recognition and Chaperoning by Pex19, Followed by Trafficking and Membrane Insertion of the Peroxisome Proliferation Protein, Pex11

Pex11, an abundant peroxisomal membrane protein (PMP), is required for division of peroxisomes and is robustly imported to peroxisomal membranes. We present a comprehensive analysis of how the Pichia pastoris Pex11 is recognized and chaperoned by Pex19, targeted to peroxisome membranes and inserted therein. We demonstrate that Pex11 contains one Pex19-binding site (Pex19-BS) that is required for Pex11 insertion into peroxisomal membranes by Pex19, but is non-essential for peroxisomal trafficking. We provide extensive mutational analyses regarding the recognition of Pex19-BS in Pex11 by Pex19. Pex11 also has a second, Pex19-independent membrane peroxisome-targeting signal (mPTS) that is preserved among Pex11-family proteins and anchors the human HsPex11γ to the outer leaflet of the peroxisomal membrane. Thus, unlike most PMPs, Pex11 can use two mechanisms of transport to peroxisomes, where only one of them depends on its direct interaction with Pex19, but the other does not. However, Pex19 is necessary for membrane insertion of Pex11. We show that Pex11 can self-interact, using both homo- and/or heterotypic interactions involving its N-terminal helical domains. We demonstrate that Pex19 acts as a chaperone by interacting with the Pex19-BS in Pex11, thereby protecting Pex11 from spontaneous oligomerization that would otherwise cause its aggregation and subsequent degradation.

Absence of Cardiolipin From the Outer Leaflet of a Mitochondrial Inner Membrane Mimic Restricts Opa1-Mediated Fusion

Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.

ABCA1 causes an asymmetric cholesterol distribution to regulate intracellular cholesterol homeostasis

Cholesterol is a major and essential component of the mammalian cell plasma membrane (PM) and the loss of cholesterol homeostasis leads to various pathologies. Cellular cholesterol uptake and synthesis are regulated by a cholesterol sensor in the endoplasmic reticulum (ER). However, it remains unclear how the PM cholesterol level is sensed. Here we show that the sensing depends on ATP-binding cassette A1 (ABCA1) and Aster-A, which cooperatively maintain the asymmetric transbilayer cholesterol distribution in the PM. ABCA1 translocates (flops) cholesterol from the inner to the outer leaflet of the PM to maintain a low inner cholesterol level. When the inner cholesterol level exceeds a threshold, Aster-A is recruited to the PM-ER contact site to transfer cholesterol to the ER. These results show unknown synergy between ABCA1 and Aster-A in intracellular cholesterol homeostasis.

Phosphatidylserine receptors enhance SARS-CoV-2 infection

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

Ultrastructure of organohalide-respiring Dehalococcoidia revealed by cryo-electron tomography

Dehalococcoides mccartyi ( Dhc ) and Dehalogenimonas spp. ( Dhgm ) are members of the class Dehalococcoidia , phylum Chloroflexi, characterized by streamlined genomes and a strict requirement for organohalogens as electron acceptors. Here, we used cryo-electron tomography to reveal morphological and ultrastructural features of Dhc strain BAV1 and ‘ Candidatus Dehalogenimonas etheniformans’ strain GP cells at unprecedented resolution. Dhc cells were irregularly shaped discs (890 ± 110 nm long, 630 ± 110 nm wide and 130 ± 15 nm thick) with curved and straight sides that intersected at acute angles, whereas Dhgm cells appeared as slightly flattened cocci (760 ± 85 nm). The cell envelopes were composed of a cytoplasmic membrane (CM), a paracrystalline surface layer (S-layer) with hexagonal symmetry and ∼22 nm spacing between repeating units, and a layer of unknown composition separating the CM and the S-layer. Cell surface appendages were only detected in Dhc cells, whereas both cell types had bundled cytoskeletal filaments. Repetitive globular structures, ∼5 nm in diameter and ∼9 nm apart, were observed associated with the outer leaflet of the CM. We hypothesized that those represent organohalide respiration (OHR) complexes and estimated ∼30,000 copies per cell. In Dhgm cultures, extracellular lipid vesicles (20 - 110 nm in diameter) decorated with putative OHR complexes but lacking an S-layer were observed. The new findings expand our understanding of the unique cellular ultrastructure and biology of organohalide-respiring Dehalococcoidia . Importance: Dehalococcoidia respire organohalogen compounds and play relevant roles in bioremediation of groundwater, sediments and soils impacted with toxic chlorinated pollutants. Using advanced imaging tools, we have obtained 3-dimensional images at macromolecular resolution of whole Dehalococcoidia cells revealing their unique structural components. Our data detail the overall cellular shape, cell envelope architecture, cytoskeletal filaments, the likely localization of enzymatic complexes involved in reductive dehalogenation, and the structure of extracellular vesicles. The new findings expand our understanding of the cell structure-function relationship in Dehalococcoidia with implications for Dehalococcoidia biology and bioremediation.

Free energies of membrane stalk formation from a lipidomics perspective

Many biological membranes are asymmetric and exhibit complex lipid composition, comprising hundreds of distinct chemical species. Identifying the biological function and advantage of this complexity is a central goal of membrane biology. Here, we study how membrane complexity controls the energetics of the first steps of membrane fusions, that is, the formation of a stalk. We first present a computationally efficient method for simulating thermodynamically reversible pathways of stalk formation at coarse-grained resolution. The method reveals that the inner leaflet of a typical plasma membrane is far more fusogenic than the outer leaflet, which is likely an adaptation to evolutionary pressure. To rationalize these findings by the distinct lipid compositions, we computed ~200 free energies of stalk formation in membranes with different lipid head groups, tail lengths, tail unsaturations, and sterol content. In summary, the simulations reveal a drastic influence of the lipid composition on stalk formation and a comprehensive fusogenicity map of many biologically relevant lipid classes.

The Glycosylphosphatidylinositol Anchor: A Linchpin for Cell Surface Versatility of Trypanosomatids

The use of glycosylphosphatidylinositol (GPI) to anchor proteins to the cell surface is widespread among eukaryotes. The GPI-anchor is covalently attached to the C-terminus of a protein and mediates the protein’s attachment to the outer leaflet of the lipid bilayer. GPI-anchored proteins have a wide range of functions, including acting as receptors, transporters, and adhesion molecules. In unicellular eukaryotic parasites, abundantly expressed GPI-anchored proteins are major virulence factors, which support infection and survival within distinct host environments. While, for example, the variant surface glycoprotein (VSG) is the major component of the cell surface of the bloodstream form of African trypanosomes, procyclin is the most abundant protein of the procyclic form which is found in the invertebrate host, the tsetse fly vector. Trypanosoma cruzi, on the other hand, expresses a variety of GPI-anchored molecules on their cell surface, such as mucins, that interact with their hosts. The latter is also true for Leishmania, which use GPI anchors to display, amongst others, lipophosphoglycans on their surface. Clearly, GPI-anchoring is a common feature in trypanosomatids and the fact that it has been maintained throughout eukaryote evolution indicates its adaptive value. Here, we explore and discuss GPI anchors as universal evolutionary building blocks that support the great variety of surface molecules of trypanosomatids.