Mammalian Host Cells Research Articles

Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro

Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.

Leishmanicidal and cytotoxic activity of essential oil from the fruit peel of Myrciaria floribunda (H. West ex Willd.) O. Berg: Molecular docking and molecular dynamics simulations of its major constituent onto Leishmania enzyme targets

Cutaneous Leishmaniasis (CL) is a neglected disease characterized by highest morbidity rates worldwide. The available treatment for CL has several limitations including serious side effects and resistance to the treatment. Herein we aimed to evaluate the activity of essential oil from the peel of Myrciaria floribunda fruits (MfEO) on Leishmania amazonensis. The cytotoxic potential of MfEO on host mammalian cells was evaluated by MTT. The in vitro leishmanicidal effects of MfEO were investigated on the promastigote and intracellular amastigote forms. The ultrastructural changes induced by MfEO were evaluated by Scanning Electron Microscopy (SEM). The molecular docking of the major compounds δ-Cadinene, γ-Cadinene, γ-Muurolene, α-Selinene, α-Muurolene and (E)–Caryophyllene onto the enzymes trypanothione reductase (TreR) and sterol 14-alpha demethylase (C14DM) were performed. Our results showed that MfEO presented moderate cytotoxicity for Vero cells and macrophages. The MfEO inhibited the growth of promastigote and the survival of intracellular amastigotes, in a dose- and time- dependent way. The MfEO presented high selectivity towards amastigote forms, being 44.1 times more toxic for this form than to macrophages. Molecular docking analysis showed that the major compounds of MfEO interact with Leishmania enzymes and that δ-Cadinene (δ-CAD) presented favorable affinity energy values over TreR and C14DM enzymes, when compared with the other major constituents. Molecular dynamics (MD) simulation studies revealed a stable binding of δ-CAD with lowest binding free energy values in MMGBSA assay. Our results suggested that δ-CAD may be a potent inhibitor of TreR and C14DM enzymes. Communicated by Ramaswamy H. Sarma

Visualizing transfer of microbial biomolecules by outer membrane vesicles in microbe-host-communication in vivo.

The intestinal microbiota influences mammalian host physiology in health and disease locally in the gut but also in organs devoid of direct contact with bacteria such as the liver and brain. Extracellular vesicles (EVs) or outer membrane vesicles (OMVs) released by microbes are increasingly recognized for their potential role as biological shuttle systems for inter‐kingdom communication. However, physiologically relevant evidence for the transfer of functional biomolecules from the intestinal microbiota to individual host cells by OMVs in vivo is scarce. By introducing Escherichia coli engineered to express Cre‐recombinase (E. coli Cre) into mice with a Rosa26.tdTomato‐reporter background, we leveraged the Cre‐LoxP system to report the transfer of bacterial OMVs to recipient cells in vivo. Colonizing the intestine of these mice with E. coli Cre, resulted in Cre‐recombinase induced fluorescent reporter gene‐expression in cells along the intestinal epithelium, including intestinal stem cells as well as mucosal immune cells such as macrophages. Furthermore, even far beyond the gut, bacterial‐derived Cre induced extended marker gene expression in a wide range of host tissues, including the heart, liver, kidney, spleen, and brain. Together, our findings provide a method and proof of principle that OMVs can serve as a biological shuttle system for the horizontal transfer of functional biomolecules between bacteria and mammalian host cells.

Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses.

Dengue is a mosquito-borne viral disease (arboviral) caused by the Dengue virus. It is one of the prominent public health problems in tropical and subtropical regions with no effective vaccines. Every year around 400 million people get infected by the Dengue virus, with a mortality rate of about 20% among the patients with severe dengue. The Dengue virus belongs to the Flaviviridae family, and it is an enveloped virus with positive-sense single-stranded RNA as the genetic material. Studies of the infection cycle of this virus revealed potential host targets important for the virus replication cycle. Here in this review article, we will be discussing different stages of the Dengue virus infection cycle inside mammalian host cells and how host proteins are exploited by the virus in the course of infection as well as how the host counteracts the virus by eliciting different antiviral responses.

Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence.

ABSTRACTEpsins play a pivotal role in the formation of endocytic vesicles and potentially provide a linkage between endocytic and other trafficking pathways. We identified a Candida albicans epsin, ENT2, that bears homology to the Saccharomyces cerevisiae early endocytosis genes ENT1 and ENT2 and studied its functions by a reverse genetic approach utilizing CRISPR-Cas9-mediated gene deletion. The C. albicans ent2Δ/Δ null mutant displayed cell wall defects and altered antifungal drug sensitivity. To define the role of C. albicans ENT2 in endocytosis, we performed assays with the lipophilic dye FM4-64 that revealed greatly reduced uptake in the ent2Δ/Δ mutant. Next, we showed that the C. albicans ent2Δ/Δ mutant was unable to form hyphae and biofilms. Assays for virulence properties in an in vitro keratinocyte infection model demonstrated reduced damage of mammalian adhesion zippers and host cell death from the ent2Δ/Δ mutant. We conclude that C. albicans ENT2 has a role in efficient endocytosis, a process that is required for maintaining cell wall integrity, hyphal formation, and virulence-defining traits.IMPORTANCE The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen. In order to support these advancements, a greater understanding of the biology of C. albicans is needed. In these studies, we are focused on the fundamental biological process of endocytosis, of which little is directly known in C. albicans. In addition to studying the function of a key gene in this process, we are examining the role of endocytosis in the virulence-related processes of filamentation, biofilm formation, and tissue invasion. These studies will provide greater insight into the role of endocytosis in causing invasive fungal infections.

Single-cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells.

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen and acquisition of nutrients from host cells is essential for survival and proliferation of intracellular STM. The nutritional environment of intracellular STM is only partially understood. We deploy bacteria harbouring reporter plasmids to interrogate the environmental cues acting on intracellular STM, and flow cytometry allows analyses on level of single STM. Phosphorus is a macro-element for cellular life, and in STM inorganic phosphate (Pi ), homeostasis is mediated by the two-component regulatory system PhoBR, resulting in expression of the high affinity phosphate transporter pstSCAB-phoU. Using fluorescent protein reporters, we investigated Pi availability for intracellular STM at single-cell level over time. We observed that Pi concentration in the Salmonella-containing vacuole (SCV) is limiting and activates the promoter of pstSCAB-phoU encoding a high affinity phosphate uptake system. Correlation between reporter activation by STM in defined media and in host cells indicates Pi concentration less 10μM within the SCV. STM proliferating within the SCV experience increasing Pi limitations. Activity of the Salmonella pathogenicity island 2 (SPI2)-encoded type III secretion system (T3SS) is crucial for efficient intracellular proliferation, and SPI2-T3SS-mediated endosomal remodelling also reliefs Pi limitation. STM that are released from SCV to enter the cytosol of epithelial cells did not indicate Pi limitations. Addition of Pi to culture media of infected cells partially relieved Pi limitations in the SCV, as did inhibition of intracellular proliferation. We conclude that availability of Pi is critical for intracellular lifestyle of STM, and Pi acquisition is maintained by multiple mechanisms. Our work demonstrates the use of bacterial pathogens as sensitive single-cell reporters for their environment in host cell or host organisms. TAKE AWAY: Salmonella strains were engineered to report their intracellular niche and the availability of inorganic phosphate (Pi ) on level of single intracellular bacteria Within the Salmonella-containing vacuole (SCV), Pi is limited and limitation increases with bacterial proliferation Salmonella located in host cell cytosol are not limited in Pi availability Remodelling of the host cell endosomal system mediated by T3SS-2 reliefs Pi limitation in the SCV.

Extracellular Vesicles and Host-Pathogen Interactions: A Review of Inter-Kingdom Signaling by Small Noncoding RNA.

The focus of this brief review is to describe the role of noncoding regulatory RNAs, including short RNAs (sRNA), transfer RNA (tRNA) fragments and microRNAs (miRNA) secreted in extracellular vesicles (EVs), in inter-kingdom communication between bacteria and mammalian (human) host cells. Bacteria secrete vesicles that contain noncoding regulatory RNAs, and recent studies have shown that the bacterial vesicles fuse with and deliver regulatory RNAs to host cells, and similar to eukaryotic miRNAs, regulatory RNAs modulate the host immune response to infection. Recent studies have also demonstrated that mammalian cells secrete EVs containing miRNAs that regulate the gut microbiome, biofilm formation and the bacterial response to antibiotics. Thus, as evidence accumulates it is becoming clear that the secretion of noncoding regulatory RNAs and miRNAs in extracellular vesicles is an important mechanism of bidirectional communication between bacteria and mammalian (human) host cells. However, additional research is necessary to elucidate how noncoding regulatory RNAs and miRNA secreted in extracellular vesicles mediate inter-kingdom communication.

Essential functions of chaperones and adaptors of protein secretion systems in Gram-negative bacteria.

Equipped with a plethora of secreted toxic effectors, protein secretion systems are essential for bacteria to interact with and manipulate their neighboring environment to survive in host microbiota and other highly competitive communities. While effectors have received spotlight attention in secretion system studies, many require accessory chaperone and adaptor proteins for proper folding/unfolding and stability throughout the secretion process. Here, we review the functions of chaperones and adaptors of three protein secretions systems, type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS), which are employed by many Gram-negative bacterial pathogens to deliver toxins to bacterial, plant, and mammalian host cells through direct contact. Since chaperone and adaptor functions of the T3SS and the T4SS are relatively well studied, we discuss in detail the methods of chaperone-facilitated effector secretion by the T6SS and highlight commonalities between the effector chaperone/adaptor proteins of these diverse secretion systems. While the chaperones and adaptors are generally referred to as accessory proteins as they are not directly involved in toxicities to target cells, they are nonetheless vital for the biological functions of the secretion systems. Future research on biochemical and structural properties of these chaperones will not only elucidate the mechanisms of chaperone-effector binding and release process but also facilitate custom design of cargo effectors to be translocated by these widespread secretion systems for biotechnological applications.

Manipulation of Host Cell Organelles by Intracellular Pathogens.

Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.

Mechanisms Associated with Trypanosoma cruzi Host Target Cell Adhesion, Recognition and Internalization.

Chagas disease is caused by the kinetoplastid parasite Trypanosoma cruzi, which is mainly transmitted by hematophagous insect bites. The parasite’s lifecycle has an obligate intracellular phase (amastigotes), while metacyclic and bloodstream-trypomastigotes are its infective forms. Mammalian host cell recognition of the parasite involves the interaction of numerous parasite and host cell plasma membrane molecules and domains (known as lipid rafts), thereby ensuring internalization by activating endocytosis mechanisms triggered by various signaling cascades in both host cells and the parasite. This increases cytoplasmatic Ca2+ and cAMP levels; cytoskeleton remodeling and endosome and lysosome intracellular system association are triggered, leading to parasitophorous vacuole formation. Its membrane becomes modified by containing the parasite’s infectious form within it. Once it has become internalized, the parasite seeks parasitophorous vacuole lysis for continuing its intracellular lifecycle, fragmenting such a vacuole’s membrane. This review covers the cellular and molecular mechanisms involved in T. cruzi adhesion to, recognition of and internalization in host target cells.

Rational Design of an Antifungal Polyacrylamide Library with Reduced Host-Cell Toxicity.

Life-threatening invasive fungal infections represent an urgent threat to human health worldwide. The limited set of antifungal drugs has critical constraints such as resistance development and/or adverse side effects. One approach to overcome these limitations is to mimic naturally occurring antifungal peptides called defensins. Inspired by their advantageous amphiphilic properties, a library of 35 synthetic, linear, ternary polyacrylamides was prepared by controlled/living radical polymerization. The effect of the degree of polymerization (20, 40, and 100) and varying hydrophobic functionalities (branched, linear, cyclic, or aromatic differing in their number of carbons) on their antifungal activity was investigated. Short copolymers with a calculated log P of ∼1.5 revealed optimal activity against the major human fungal pathogen Candida albicans and other pathogenic fungal species with limited toxicity to mammalian host cells (red blood cells and fibroblasts). Remarkably, selected copolymers outperformed the commercial antifungal drug amphotericin B, with respect to the therapeutic index, highlighting their potential as novel antifungal compounds.

Temperate Bacteriophages-The Powerful Indirect Modulators of Eukaryotic Cells and Immune Functions.

Bacteriophages are natural biological entities that limit the growth and amplification of bacteria. They are important stimulators of evolutionary variability in bacteria, and currently are considered a weapon against antibiotic resistance of bacteria. Nevertheless, apart from their antibacterial activity, phages may act as modulators of mammalian immune responses. In this paper, we focus on temperate phages able to execute the lysogenic development, which may shape animal or human immune response by influencing various processes, including phagocytosis of bacterial invaders and immune modulation of mammalian host cells.

The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection.

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3′ end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

Orthobunyaviruses: From Virus Binding to Penetration into Mammalian Host Cells.

With over 80 members worldwide, Orthobunyavirus is the largest genus in the Peribunyaviridae family. Orthobunyaviruses (OBVs) are arthropod-borne viruses that are structurally simple, with a trisegmented, negative-sense RNA genome and only four structural proteins. OBVs are potential agents of emerging and re-emerging diseases and overall represent a global threat to both public and veterinary health. The focus of this review is on the very first steps of OBV infection in mammalian hosts, from virus binding to penetration and release of the viral genome into the cytosol. Here, we address the most current knowledge and advances regarding OBV receptors, endocytosis, and fusion.

A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Current molecular cloning strategies generally lack inter-compatibility, are not strictly modular, or are not applicable to engineer multi-gene expression vectors for transient and stable integration. A standardized molecular cloning platform would advance research, for example, by promoting exchange of vectors between groups. Here, we present a versatile plasmid architecture for mammalian synthetic biology, which we designate VAMSyB, consisting of a three-tier vector family. Tier-1 is designed for easy engineering of fusion constructs, as well as easy swapping of genes and modules to tune the functionality of the vector. Tier-2 is designed for transient multi-gene expression, and is constructed by directly transferring the engineered expression cassettes from tier-1 vectors. Tier-3 enables stable integration into a mammalian host cell through viral transduction, transposons, or homology-directed recombination via CRISPR. This VAMSyB architecture is expected to have broad applicability in the field of mammalian synthetic biology. The VAMSyB collection of plasmids will be available through Addgene.

Cytotoxicity, Intracellular Replication, and Contact-Dependent Pore Formation of Genotyped Environmental Legionella pneumophila Isolates from Hospital Water Systems in the West Bank, Palestine.

Legionella pneumophila is the causative agent of Legionnaires’ disease. Due to the hot climate and intermittent water supply, the West Bank, Palestine, can be considered a high-risk area for this often fatal atypical pneumonia. L. pneumophila occurs in biofilms of natural and man-made freshwater environments, where it infects and replicates intracellularly within protozoa. To correlate the genetic diversity of the bacteria in the environment with their virulence properties for protozoan and mammalian host cells, 60 genotyped isolates from hospital water systems in the West Bank were analyzed. The L. pneumophila isolates were previously genotyped by high resolution Multi Locus Variable Number of Tandem Repeat Analysis (MLVA-8(12)) and sorted according to their relationship in clonal complexes (VACC). Strains of relevant genotypes and VACCs were compared according to their capacity to infect Acanthamoeba castellanii and THP-1 macrophages, and to mediate pore-forming cytotoxicity in sheep red blood cells (sRBCs). Based on a previous detailed analysis of the biogeographic distribution and abundance of the MLVA-8(12)-genotypes, the focus of the study was on the most abundant L. pneumophila- genotypes Gt4(17), Gt6 (18) and Gt10(93) and the four relevant clonal complexes [VACC1, VACC2, VACC5 and VACC11]. The highly abundant genotypes Gt4(17) and Gt6(18) are affiliated with VACC1 and sequence type (ST)1 (comprising L. pneumophila str. Paris), and displayed seroroup (Sg)1. Isolates of these two genotypes exhibited significantly higher virulence potentials compared to other genotypes and clonal complexes in the West Bank. Endemic for the West Bank was the clonal complex VACC11 (affiliated with ST461) represented by three relevant genotypes that all displayed Sg6. These genotypes unique for the West Bank showed a lower infectivity and cytotoxicity compared to all other clonal complexes and their affiliated genotypes. Interestingly, the L. pneumophila serotypes ST1 and ST461 were previously identified by in situ-sequence based typing (SBT) as main causative agents of Legionnaires’ disease (LD) in the West Bank at a comparable level. Overall, this study demonstrates the site-specific regional diversity of L. pneumophila genotypes in the West Bank and suggests that a combination of MLVA, cellular infection assays and hierarchical agglomerative cluster analysis allows an improved genotype-based risk assessment.

On-cell saturation transfer difference NMR for the identification of FimH ligands and inhibitors

We describe the development of an on-cell NMR method for the rapid screening of FimH ligands and the structural identification of ligand binding epitopes. FimH is a mannose-binding bacterial adhesin expressed at the apical end of type 1 pili of uropathogenic bacterial strains and responsible for their d-mannose sensitive adhesion to host mammalian epithelial cells. Because of these properties, FimH is a key virulence factor and an attractive therapeutic target for urinary tract infection.We prepared synthetic d-mannose decorated dendrimers, we tested their ability to prevent the FimH-mediated yeast agglutination, and thus we used the compounds showing the best inhibitory activity as models of FimH multivalent ligands to set up our NMR methodology. Our experimental protocol, based on on-cell STD NMR techniques, is a suitable tool for the screening and the epitope mapping of FimH ligands aimed at the development of new antiadhesive and diagnostic tools against urinary tract infection pathogens. Notably, the study is carried out in a physiological environment, i.e. at the surface of living pathogen cells expressing FimH.

Entry of Phenuiviruses into Mammalian Host Cells.

Phenuiviridae is a large family of arthropod-borne viruses with over 100 species worldwide. Several cause severe diseases in both humans and livestock. Global warming and the apparent geographical expansion of arthropod vectors are good reasons to seriously consider these viruses potential agents of emerging diseases. With an increasing frequency and number of epidemics, some phenuiviruses represent a global threat to public and veterinary health. This review focuses on the early stage of phenuivirus infection in mammalian host cells. We address current knowledge on each step of the cell entry process, from virus binding to penetration into the cytosol. Virus receptors, endocytosis, and fusion mechanisms are discussed in light of the most recent progress on the entry of banda-, phlebo-, and uukuviruses, which together constitute the three prominent genera in the Phenuiviridae family.

Mechanical Competition Triggered by Innate Immune Signaling Drives The Collective Extrusion of Bacterially-Infected Epithelial Cells

Several intracellular bacterial pathogens alter the mechanics of their mammalian host cells to promote their dissemination through tissues. Conversely, host cells may respond to the presence of intracellular pathogens by altering their own mechanical behavior to limit infection spread. Here, we monitored epithelial cell monolayers sparsely infected with the intracellular bacterial pathogens Listeria monocytogenes or Rickettsia parkeri over the course of several days. We found that, under conditions where these pathogens trigger innate immune signaling through the NF-κB pathway and use actin-based motility to spread non-lytically from cell to cell, domains of infected cells form enormous three-dimensional mounds. We show that these infection mounds result from uninfected cells moving toward the site of infection, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the epithelial monolayer. Bacteria in infection mounds are less able to spread laterally in the monolayer, limiting the growth of the focus, while extruded infected cells eventually undergo cell death. This cellular competition between infected and uninfected cells leading to infected cell extrusion en masse is driven by active directional migration in uninfected bystander cells, intact cell-cell junctions and innate immune signaling. in addition, cells in infected monolayers exhibit behavioral and molecular signatures of the epithelial to mesenchymal transition (EMT). in summary our findings showcase that coordinated forceful action by uninfected bystander cells actively eliminates large domains of infected cells, consistent with the hypothesis that this collective cell response represents an innate immunity-driven process that could help to limit the local spread of infection.

Atypical Divergence of SARS-CoV-2 Orf8 from Orf7a within the Coronavirus Lineage Suggests Potential Stealthy Viral Strategies in Immune Evasion.

Orf8, one of the most puzzling genes in the SARS lineage of coronaviruses, marks a unique and striking difference in genome organization between SARS-CoV-2 and SARS-CoV-1. Here, using sequence comparisons, we unequivocally reveal the distant sequence similarities between SARS-CoV-2 Orf8 with its SARS-CoV-1 counterparts and the X4-like genes of coronaviruses, including its highly divergent "paralog" gene Orf7a, whose product is a potential immune antagonist of known structure. Supervised sequence space walks unravel identity levels that drop below 10% and yet exhibit subtle conservation patterns in this novel superfamily, characterized by an immunoglobulin-like beta sandwich topology. We document the high accuracy of the sequence space walk process in detail and characterize the subgroups of the superfamily in sequence space by systematic annotation of gene and taxon groups. While SARS-CoV-1 Orf7a and Orf8 genes are most similar to bat virus sequences, their SARS-CoV-2 counterparts are closer to pangolin virus homologs, reflecting the fine structure of conservation patterns within the SARS-CoV-2 genomes. The divergence between Orf7a and Orf8 is exceptionally idiosyncratic, since Orf7a is more constrained, whereas Orf8 is subject to rampant change, a peculiar feature that may be related to hitherto-unknown viral infection strategies. Despite their common origin, the Orf7a and Orf8 protein families exhibit different modes of evolutionary trajectories within the coronavirus lineage, which might be partly attributable to their complex interactions with the mammalian host cell, reflected by a multitude of functional associations of Orf8 in SARS-CoV-2 compared to a very small number of interactions discovered for Orf7a.IMPORTANCE Orf8 is one of the most puzzling genes in the SARS lineage of coronaviruses, including SARS-CoV-2. Using sophisticated sequence comparisons, we confirm its origins from Orf7a, another gene in the lineage that appears as more conserved, compared to Orf8. Orf7a is a potential immune antagonist of known structure, while a deletion of Orf8 was shown to decrease the severity of the infection in a cohort study. The subtle sequence similarities imply that Orf8 has the same immunoglobulin-like fold as Orf7a, confirmed by structure determination. We characterize the subgroups of this superfamily and demonstrate the highly idiosyncratic divergence patterns during the evolution of the virus.