Host Cell Membrane Research Articles

Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion.

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues.

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by ET. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by pre-hairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow/liver/thymus mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study.IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor-treated tissues of humanized mice. The findings of trapped HIV-1 virions in all tissues examined demonstrate a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that the fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues.

Dissecting the Membrane Association Mechanism of Aerolysin Pores at Femtomolar Concentrations Using Water as a Probe.

Aerolysin is a bacterial toxin that forms transmembrane pores at the host plasma membrane and has a narrow internal diameter and great stability. These assets make it a highly promising nanopore for detecting biopolymers such as nucleic acids and peptides. Although much is known about aerolysin from a microbiological and structural perspective, its membrane association and pore-formation mechanism are not yet fully understood. Here, we used angle-resolved second harmonic scattering (AR-SHS) and single-channel current measurements to investigate how wild-type (wt) aerolysin and its mutants interact with liposomes in aqueous solutions at femtomolar concentrations. Our AR-SHS experiments were sensitive enough to detect changes in the electrostatic properties of membrane-bound aerolysin, which were induced by variations in pH levels. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore and the prepore states show gradually decreasing membrane affinities, incomplete insertion, and a pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. Our study provides a label-free method for efficiently screening biological pores suitable for conducting molecular sensing and sequencing measurements as well as for probing pore-forming processes.

Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryo-electron tomography.

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

In silico investigation of therapeutic potential of phytosteroids against SARS-CoV2 infection via inhibition of viral multiplication and attenuation of host cytokine storm

SARS-CoV2 infection overcomes host cell membrane barrier, followed by utilization of host cellular processes for viral multiplication. Simultaneously, it triggers a cytokine storm within and around infected cells and tissues. Anti-inflammatory agents that can potentially inhibit viral penetration and multiplication within the host cells may be ideal drug candidates against COVID-19. Dietary phytosteroids have significant anti-inflammatory potential. Hence, the present study intends to investigate anti-viral potential of three dietary phytosteroids, namely, brassinolide, 28-homocastasterone and 24-epibrassinolide against SARS-CoV2 proteins, S1 spike protein, nucleocapsid protein and main protease enzyme, in addition to host pro-inflammatory proteins, interleukin-1, tumour necrosis factor-α, cyclooxygenase-2 and prostaglandin synthase, as drug targets. Molecular docking studies by AutoDock version 4.0 was performed. Brassinolide, 28-homocastasterone and 24-epibrassinolide exhibits high docking score against all the seven proteins, as compared to hydroxychloroquine. Brassinolide and 28- homocastasterone has maximum binding affinity for pro-inflammatory proteins and SARS-CoV2 proteins.Dietary phytosteroids may potentially attenuate cytokine storm with simultaneous inhibition of host entry and multiplication of SARS-CoV2. In-vitro and in-vivo anti-viral studies of plant steroids may provide a clear path for the identification and development of novel drug candidates against COVID-19, that also provides evidence for the concept of reverse pharmacognosy.

The Impact of United Kingdom SARS-CoV-2 Spike Mutations on Spike-ACE2 Interactions Investigated Through Molecular Dynamic Simulation

Background: A newly emergent betacoronavirus, SARS-CoV-2, poses a tremendous global threat and causes severe acute respiratory syndrome coronavirus-2, a major pandemic that began worldwide in 2019. The trimeric spike glycoprotein (S) is located on the envelope of the virus and facilitates the fusion of viral and host cell membranes by interacting with a cellular receptor called angiotensin-converting enzyme 2 (ACE2). In the United Kingdom (UK), a variant of SARS-CoV-2 has been detected using sequencing technology. There has been a significant surge in the number of COVID-19 cases in South East England. These lineages are significantly more transmissible than previously circulating variants. Objectives: The aim of this study is to investigate the effects of UK SARS-CoV-2 spike mutations on its interactions with ACE2. Methods: In this study, the structure of the UK spike protein with multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H, was investigated. The research focuses on studying the impact of these mutations on spike function and its interactions with ACE2 through molecular dynamic simulation. Results: The results indicated that hydrophobic interactions, hydrogen bonds, and double/triple bonds are more prevalent in the mutated spike-ACE2 complex. Additionally, the UK spike-ACE2 complex maintained a consistent conformation throughout the simulation, experiencing minimal changes in structure. The mutant spike protein structure is less stable compared to the wild-type spike structure. Conclusions: Multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H in the spike protein of UK SARS-CoV-2, can affect its interaction with the ACE2 receptor and the transmissibility of this variant of SARS-CoV-2.

Novel humanized anti-PcrV monoclonal antibody COT-143 protects mice from lethal Pseudomonas aeruginosa infection via inhibition of toxin translocation by the type III secretion system.

Treatment of Pseudomonas aeruginosa infection is challenging due to its intrinsic and acquired antibiotic resistance. As the number of current therapeutic options for P. aeruginosa infections is limited, developing novel treatments against the pathogen is an urgent clinical priority. The suppression of virulence of P. aeruginosa could be a new therapeutic option, and the type III secretion system (T3SS), which enables the bacteria to translocate various kinds of toxins into host cells and inhibits cellular functions, is considered as one possible target. In this report, we examined T3SS inhibition by COT-143/INFEX702, a humanized monoclonal antibody against PcrV, T3SS component, and present the crystal structure of the antibody-PcrV complex. COT-143 inhibited T3SS-dependent cytotoxicity and protected mice from the mortality caused by P. aeruginosa infection. The inhibition of cytotoxicity coincided with inhibition of translocon formation in a host cell membrane, which is necessary for T3SS intoxication. COT-143 protected murine neutrophils and facilitated phagocytosis of P. aeruginosa. These results suggest that COT-143 facilitates P. aeruginosa clearance by protecting neutrophil via inhibition of T3SS-dependent toxin translocation. This is the first report to show that an anti-PcrV antibody directly interferes with translocon formation to inhibit intoxication of host cells.

A pathogen effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion.

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

Protein mimics of fusion core from SARS-CoV-1 can inhibit SARS-CoV-2 entry

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the genus Betacoronavirus (subgenus Sarbecovirus) and shares significant genomic and phylogenetic similarities with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). SARS-CoV-2 infection occurs through membrane fusion between the virus and host cell membranes, which is facilitated by the spike glycoprotein subunit 2 (S2). The folding of three heptad-repeat regions 1 (HR1) into a central trimeric core structure, along with the binding of three heptad-repeat regions 2 (HR2) in an antiparallel manner within the groove formed between the HR1 regions, which provides the driving force for membrane fusion. In this study, trimeric and monomeric six-helix bundles (6HB) were created by combining various truncations of the sequences from SARS-CoV-2 HR1 and HR2. In addition, monomeric five-helix bundles (5HB) were constructed using a similar method. Finally, we demonstrated a protein mimic, 5HB_V1 (from SARS-CoV-1), that exhibits activity in inhibiting SARS-CoV-2. These findings suggest a strategy to design monomeric 6HB and 5HB based on the SARS-CoV-2 fusion core: maintain the flanking sequences outside the α-helix region in HR2 and introduce point mutations to enhance hydrogen bonding between the helix bundles. The 5HB could be a target for designing new inhibitors against SARS-CoV-1 and SARS-CoV-2.

HIV-1 assembly - when virology meets biophysics.

Cells naturally produce vesicles that bud from different lipid membranes using dedicated molecular machineries. Enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), also generate particles that bud from host cell membranes by hijacking cellular factors and signaling pathways similar to those involved in the budding of extracellular vesicles. HIV-1 buds from the host cell plasma membrane mainly via the self-assembly of Gag, a structural protein. Gag is a polyprotein that forms assembly complexes containing viral genomic RNA (gRNA), host cell lipids and proteins. HIV-1 Gag binds and segregates host cell plasma membrane lipids while self-assembling simultaneously on the gRNA and the plasma membrane. This self-assembly causes membrane bending and formation of a new viral particle with the help of host cell proteins, likely including cortical actin-associated factors. However, it is unclear whether the energy of Gag self-assembly is sufficient to generate new HIV-1 particles. In this Review, we discuss these processes in the light of the past and recent virology literature, incorporating lessons from studies on the quantitative biophysics of viral self-assembly, and explore how Gag might reorganize the plasma membrane and divert host cell membrane curving proteins and cortical actin-related factors to achieve particle assembly and budding.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation.

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell-cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

The Rac1-PAK1-Arp2/3 signaling axis regulates CHIKV nsP1-induced filopodia and optimal viral genome replication.

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex.IMPORTANCEThe alphavirus nsP1 protein dramatically remodels host cellular membranes, resulting in the formation of filopodia-like extensions. Although described decades ago, the molecular mechanisms controlling these membrane deformations and their functional importance remain elusive. Our study provides mechanistic insight, uncovering the critical role of the Rac1 GTPase, along with its downstream effectors PAK1 and the actin nucleator Arp2, in the nsP1-associated phenotype. Furthermore, we demonstrate that the Rac1-PAK1-Arp2 pathway is essential for optimal CHIKV genome replication. Our findings establish a parallel in the cellular mechanisms governing nsP1-induced plasma membrane reshaping and the production of a functional replication complex in infected cells.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

Red Blood Cell‐Derived Small Extracellular Vesicles Inhibit Influenza Virus through Surface‐Displayed Sialic Acids

Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell‐derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface‐displayed SAs to interact with IAV through multivalent HA‐SA interactions. This high‐avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose‐dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti‐IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti‐IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics.

Red Blood Cell-Derived Small Extracellular Vesicles Inhibit Influenza Virus through Surface-Displayed Sialic Acids.

Disrupting the conserved multivalent binding of hemagglutinin (HA) on influenza A virus (IAV) to sialic acids (SAs) on the host cell membrane offers a robust strategy to block viral attachment and infection, irrespective of antigenic evolution or drug resistance. In this study, we exploit red blood cell-derived small extracellular vesicles (RBC sEVs) as nanodecoys by harnessing their high abundance of surface-displayed SAs to interact with IAV through multivalent HA-SA interactions. This high-avidity binding inhibits viral adhesion to the cell surface, effectively preventing both attachment and infection in a dose-dependent manner. Notably, enzymatic removal of SAs from RBC sEVs significantly diminishes their anti-IAV efficacy. Our findings indicate that RBC sEVs possess intrinsic anti-IAV properties due to their native multivalent SAs and hold considerable promise as antiviral therapeutics.

Host restriction factor Rab11a limits Porcine deltacoronavirus invasion of cells via fusion peptide-mediated membrane fusion

Porcine deltacoronavirus (PDCoV) poses a serious threat to pork industry and has the potential for cross-species transmission. Yet, the invasion mechanisms and host factors involved are still unknown. In the present work, using siRNA interference and co-immunoprecipitation, we identified Annexin A2 (ANXA2), Prohibitin-2 (PHB2), or Caveolin-2 (CAV2) as host factors positively regulating the internalization of PDCoV. We further found that Rab11a co-localized with PDCoV S and inhibited PDCoV internalization. Subsequently, a pseudoviral infection model (LV-PDCoV S-GFP) was constructed, and ANXA2 or CAV2 promoted PDCoV invasion by downregulating Rab11a. Our results also indicated that ANXA2, CAV2, and Rab11a interact with the S protein via S-FP, thereby regulating virus-host membrane fusion. Through LV-PDCoV S-GFP infection, we found that Rab11a may act as a host restriction factor, and it could regulate the invasion efficiency of PDCoV by adding of exogenous GTP. These findings revealed that Rab11a was an exciting target to restrict fusion of PDCoV with host cell membranes. Availability of data and materialNot applicable.

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic Escherichia coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse precolonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study suggests that commensal E. coli engineered to deliver payloads that block essential virulence determinants can be developed as a new means to prevent and potentially treat infections including those due to antibiotic resistant microbes.

Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy

Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.

Impedance‐based monitoring of titration and neutralization assays with VSV‐G and SARS‐CoV‐2‐spike pseudoviruses

AbstractSince cell‐based virus neutralization assays are still the gold standard to assess a patient's immune protection against a given virus, they are of utmost importance for serodiagnosis, convalescent plasma therapy, and vaccine development. Monitoring the emergence and characteristics of neutralizing antibodies in an outbreak situation, confirming neutralizing antibodies as correlates of protection from infection and testing vaccine‐induced potency of neutralizing antibody responses, quests for automated, fast, and parallel neutralization assays. We developed an impedance‐based sensor platform (electric cell‐substrate impedance sensing, ECIS) providing time‐resolved monitoring of the host cell response to viral pseudotypes. For validation, the impedance assay was compared with state‐of‐the‐art quantification of virus‐induced reporter protein expression as an independent indicator of virus infection and neutralization. Vesicular stomatitis virus (VSV) derived pseudoviruses encoding the green fluorescent protein (GFP) as reporter and the autologous G protein (VSV‐G) for the initial binding to the host cell membrane were used for monitoring of HEK293T cell infection and neutralization with both, impedance and optical readout. Virus‐induced cytopathic effects (CPE) were detectable for low pseudotype concentrations (multiplicity of infection 1) in time‐resolved impedance profiles as soon as 5–10 h after infection in a concentration‐dependent manner. Neutralization efficacy of α‐VSV‐G antibodies was determined from impedance time courses and IC50 values compared favorably with fluorescence measurements of virus‐borne GFP expression. Sera of convalescent COVID‐19 patients were tested successfully for SARS‐CoV‐2 neutralizing antibodies by incubating VSV, pseudotyped with the SARS‐CoV‐2 spike protein, with different sera before host cell exposure and impedance recordings. In summary: (i) ECIS monitoring was successfully applied to detect virus‐mediated cell infection and neutralization; (ii) Impedance‐based monitoring allows reducing the assay time to 5–10 h; and (iii) the platform is easily adapted to other virus‐based diseases and scalable to high‐throughput.

The host protease KLK5 primes and activates spike proteins to promote human betacoronavirus replication and lung inflammation.

Coronaviruses rely on host proteases to activate the viral spike protein, which facilitates fusion with the host cell membrane and the release of viral genomic RNAs into the host cell cytoplasm. The distribution of specific host proteases in the host determines the host, tissue, and cellular tropism of these viruses. Here, we identified the kallikrein (KLK) family member KLK5 as a major host protease secreted by human airway cells and exploited by multiple human betacoronaviruses. KLK5 cleaved both the priming (S1/S2) and activation (S2') sites of spike proteins from various human betacoronaviruses in vitro. In contrast, KLK12 and KLK13 displayed preferences for either the S2' or S1/S2 site, respectively. Whereas KLK12 and KLK13 worked in concert to activate SARS-CoV-2 and MERS-CoV spike proteins, KLK5 by itself efficiently activated spike proteins from several human betacoronaviruses, including SARS-CoV-2. Infection of differentiated human bronchial epithelial cells (HBECs) with human betacoronaviruses induced an increase in KLK5 that promoted virus replication. Furthermore, ursolic acid and other related plant-derived triterpenoids that inhibit KLK5 effectively suppressed the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2 in HBECs and mitigated lung inflammation in mice infected with MERS-CoV or SARS-CoV-2. We propose that KLK5 is a pancoronavirus host factor and a promising therapeutic target for current and future coronavirus-induced diseases.