Biology subjects

Alto, N. M.

Publications and source records attributed to Alto, N. M..

14 recordsLinked to original sources

bioRxiv2026

Targeting of ELMO2 by the Shigella E3 Ligase IpaH4.5 Reveals Key Differences Between ELMO Paralogs

The IpaH class of effector proteins secreted by Shigella flexneri ubiquitinate specific immune factors, targeting them for proteasomal degradation. ELMO1-3 are key signaling molecules involved in cell motility and phagocytosis, but the distinct roles of these three proteins remain unclear. Here, we identify ELMO2 as a substrate of S. flexneri IpaH4.5 using an unbiased screening approach in human cell lysate. Using in vitro and cellular assays, we show that IpaH4.5 specifically targets ELMO2 for ubiquitination but has no activity against its paralogs ELMO1 and ELMO3. Using chimeric proteins, we show that IpaH4.5 specificity is dictated by divergence within the ELMO2 Armadillo Repeat Region, identifying this domain as a key determinant of ELMO paralog specialization. Consistent with this divergence, we find that commonly used ELMO antibodies lack paralog specificity and that ELMO2 is the predominant paralog expressed across multiple human cell lines. Further, functional assays demonstrate that IpaH4.5-mediated degradation of ELMO2 disrupts RAC activation downstream of the ELMO/DOCK complex, suggesting that this signaling pathway is perturbed during infection. We conclude that ELMO2 plays an as-yet unidentified role in counteracting bacterial infection and is therefore targeted by IpaH4.5.

microbiology↗

bioRxiv2026

Bacterial and human exonucleases mediate interkingdom antiviral immunity

All kingdoms of life have developed strategies to limit viral infection1,2. In humans, interferons induce a suite of antiviral factors that collectively provide immunity3. Some human immune genes are homologous with antiphage defense genes in bacteria, though the extent of this overlap is not known4-12. Here, we screened a panel of human innate immune genes for phage defense in Escherichia coli and found that the RNA exonuclease ISG20 potently restricts the RNA phages MS2 and Q{beta}. Purified ISG20 trims the 3 untranslated regions (UTRs) of RNA phage genomes, explaining its ability to block phage replication in E. coli. Homologs of ISG20 from bacteria function similarly, exhibiting nuclease-dependent antiphage defense in bacteria and UTR-trimming in vitro. When expressed in human cells, these bacterial exonucleases also restrict human RNA viruses with similar potency as the human antiviral protein ISG20. Thus, antiviral genes from both humans and bacteria can function interchangeably. This bidirectional, interkingdom immunity suggests that viral targets overlap, implying that both bacterial and human factors recognize ancient features of RNA viruses.

microbiology↗

bioRxiv2025

Single-cell tracking of genetically minimized Salmonella reveals mechanisms of effector gene cooperation.

Bacterial pathogens encode secretory systems that deliver large repertoires of effector proteins directly into host cells. While many effector proteins have been characterized biochemically, it is unclear where and when they function within complex cellular systems of host tissues. This problem exists because of the extensive genetic and functional redundancies found in large effector protein repertoires. Here, we coupled targeted genome minimization with single cell mass cytometry to track the cellular location where Salmonella Typhimurium (S.Tm) SPI-2 Type Three Secretion System effector proteins function in a time-resolved animal model of infection. This approach revealed the temporal progression of S.Tm transmission within a complex tissue and pinpointed effector genes responsible for pathogen dissemination between host immune cell types. We further show how coupling two effector gene networks acquired during distinct episodes of bacterial evolution has shaped the cellular and tissue tropism of non-Typhoidal Salmonella species. Together, these results illustrate a top-down genetic approach to interrogate host-pathogen interactions hidden by functional redundancies within large virulence factor gene networks.

microbiology↗

bioRxiv2025

Bacterial ubiquitin ligase engineered for small molecule and protein target identification

The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.

molecular biology↗

bioRxiv2024

Salmonella multimutants enable efficient identification of SPI-2 effector protein function in gut inflammation and systemic colonization

Salmonella enterica spp. rely on translocation of effector proteins through the SPI-2 encoded type III secretion system (T3SS) to achieve pathogenesis. More than 30 effectors contribute to manipulation of host cells through diverse mechanisms, but interdependency or redundancy between effectors complicates the discovery of effector phenotypes using single mutant strains. Here, we engineer six mutant strains to be deficient in cohorts of SPI-2 effector proteins, as defined by their reported function. Using various animal models of infection, we show that three principle phenotypes define the functional contribution of the SPI-2 T3SS to infection. Multimutant strains deficient for intracellular replication, for manipulation of host cell defences, or for expression of virulence plasmid effectors all showed strong attenuation in vivo, while mutants representing approximately half of the known effector complement showed phenotypes similar to the wild-type parent strain. By additionally removing the SPI-1 T3SS, we find cohorts of effector proteins that contribute to SPI-2 T3SS-driven enhancement of gut inflammation. Further, we provide an example of how iterative mutation can be used to find a minimal number of effector deletions required for attenuation, and thus establish that the SPI-2 effectors SopD2 and GtgE are critical for the promotion of gut inflammation and mucosal pathology. This strategy provides a powerful toolset for simultaneous parallel screening of all known SPI-2 effectors in a single experimental context, and further facilitates the identification of the responsible effectors, and thereby provides an efficient approach to study how individual effectors contribute to disease.

microbiology↗

bioRxiv2024

Bacterial Effector Screening Reveals RNF214 as a Virus Restriction Factor in Mammals

Arboviruses are a group of arthropod-transmitted viruses that pose a significant threat to public health. Identifying host factors that inhibit arbovirus infection is critical for the development of strategies to prevent or treat these infections. Previously, we showed that bacterial effector proteins can be used as molecular tools to identify host immunity factors in insect cells that restrict arbovirus replication (Embry et al., 2024). Bacteria secrete effectors into the host cell cytoplasm to inhibit various innate immune defenses. Here, we apply our bacterial effector screening system to identify host antiviral immunity factors in two mammalian hosts - bats and humans. By screening a library of 210 effectors encoded by seven distinct bacterial pathogens, we identified three bacterial effectors (IpaH4, SopB, and SidM) that enhance the replication of both togaviruses and rhabdoviruses in bat and human cells. We also discovered several effectors that enhance arbovirus replication in a virus- or host-specific manner. We further characterize the mechanism by which the Shigella flexneri encoded E3 ubiquitin ligase, IpaH4, enhances arbovirus infection in mammalian cells. Using yeast two-hybrid, ubiquitin-activated interaction traps, in vitro ubiquitination assays and cellular approaches, we show the uncharacterized mammalian RING-domain containing protein, RNF214, to be directly targeted by IpaH4 for ubiquitination-mediated degradation. Phylogenetic analyses of RNF214 proteins indicate they are widely conserved among many vertebrate species, suggesting an important evolutionary function. We show that RNF214 overexpression suppresses arbovirus infections in a manner dependent upon its putative E3 ubiquitin ligase activity, while RNF214 depletion enhances these infections in human and bat cells. These data suggest that RNF214 proteins are important innate immune factors involved in combating viral infection. Collectively, our work shows that bacterial effectors can be useful tools for uncovering novel mammalian antiviral machinery. Author SummaryArboviruses are viruses transmitted by arthropod vectors such as mosquitoes or biting flies to both animal and human hosts. Arboviruses cause diseases ranging from mild febrile illnesses to fatal encephalitic infection. Here, we use bacterial effector proteins to identify mammalian factors that block arbovirus replication. Bacterial effectors encoded by pathogenic bacteria are secreted into mammalian host cells to inhibit cellular antimicrobial responses. Like viruses, many pathogenic bacteria replicate inside of mammalian host cells, thus we hypothesized that some bacterial effectors may target and inhibit the host immune response that are normally restrictive to both bacteria and viruses. After screening a library of >200 bacterial effector proteins, we identified three effectors that promote the replication of arboviruses belonging to two distinct families in bat and human cells. We further show that one of our most potent effectors, IpaH4, enhances arbovirus replication by targeting mammalian RNF214 proteins for degradation. RNF214 proteins are poorly characterized, but our phylogenetic analyses suggest these proteins are widely conserved among vertebrate organisms. We show that depletion of RNF214 protein levels in either bat or human cells sensitizes these cells to arbovirus infections, revealing a new role for RNF214 proteins in antiviral defense. Our study demonstrates the utility of bacterial effectors as tools for identifying new host immune machinery in mammalian hosts.

microbiology↗

bioRxiv2024

The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFN{gamma}). The IFN{gamma} receptor is expressed on most human cells, and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFN{gamma}-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFN{gamma}-primed host cells. Linear (M1-) and lysine-linked ubiquitylation of S. flexneri requires RNF213 but is independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as an inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella.

microbiology↗

bioRxiv2024

Exploiting Bacterial Effector Proteins to Uncover Evolutionarily Conserved Antiviral Host Machinery

AbstractArboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify six effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction. Author SummaryMicrobial pathogens such as viruses and bacteria encounter diverse host cell responses during infection. While viruses possess antagonists to counter these responses in natural host species, their replication can be restricted in unnatural host cells where their antagonists are ineffective. Bacteria also employ a diverse repertoire of immune evasion proteins known as "effectors" that can inhibit antimicrobial responses found in invertebrate and vertebrate hosts. In this study, we hypothesized that some bacterial effectors may target host immunity proteins that restrict both bacteria and viruses. To test this hypothesis, we screened a bacterial effector library comprising 210 effectors from seven distinct bacterial pathogens for their ability to rescue the replication of four viruses in insect cells that are normally non-permissive to these viruses. Though numerous effectors were identified to rescue the replication of each virus, the uncharacterized IpaH4 protein encoded by the human pathogen Shigella flexneri was able to rescue all four viruses screened. We discovered that IpaH4 enhances arbovirus replication in both restrictive insect and permissive human cells by directly targeting two novel, evolutionarily conserved antiviral host proteins, SHOC2 and PSMC1, for degradation. Our study establishes bacterial effectors as valuable tools for identifying critical antimicrobial machinery employed by eukaryotic hosts.

microbiology↗

bioRxiv2023

A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 Antiviral Response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

microbiology↗

bioRxiv2022

A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection

Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PMs sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, which is sustained for long periods of time through 25HC-mediated suppression of SREBPs. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.

cell biology↗

bioRxiv2021

Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor of Toxoplasma gondii Infection

Toxoplasma gondii is an important human pathogen infecting an estimated 1 in 3 people worldwide. The cytokine interferon gamma (IFN{gamma}) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction interferon stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFN{gamma}, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFN{gamma} induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFN{gamma} induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFN{gamma} mediated immunity to T. gondii infection is a complex, multifactorial process.

microbiology↗

bioRxiv2019

The mechanisms of dynamin-actin interaction

Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamins proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2018

Screening Mycobacterium tuberculosis secreted proteins identifies Mpt64 as eukaryotic membrane-binding virulence factor

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb exported proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 exported proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb exported proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and was necessary for Mtb survival in primary human macrophages. These data highlight the importance of exported proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.\n\nImportanceAdvances have been made to identify exported proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis exported proteins for eukaryotic membrane binding, host subcellular localization and interactions with host vesicular trafficking. In addition, we observed the localization of one exported protein, Mpt64, during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the Type VII Secretion System. Finally, we observed that Mpt64 contributes to the virulence of M. tuberculosis during infection of primary human macrophages.

microbiology↗

bioRxiv2017

Actin assembly-inducing protein ActA promotes FcγRIa-mediated Listeria internalization

Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of human listeriosis. While the ability of L. monocytogenes to enter and survive in professional phagocytes is critical to establish a successful infection, mechanisms of invasion are poorly understood. Our previous investigation into the role of type I interferon-stimulated genes in bacterial infection revealed that the human immunoglobulin receptor Fc{gamma}RIa served as a L. monocytogenes invasion factor. Fc{gamma}RIa-mediated L. monocytogenes entry occurred independently of immunoglobulin interaction or bacterial internalins. However, the bacterial determinants that mediate Fc{gamma}RIa interaction remain unclear. Using a comparative genomics approach, we identify actin assembly-inducing protein ActA as a pathogen specific ligand of Fc{gamma}RIa. Fc{gamma}RIa enhanced entry of pathogenic L. monocytogenes and L. ivanovii strain but not non-pathogenic L. innocua. We found that the major virulence regulator PrfA is required for pathogen entry into Fc{gamma}RIa-expressing cells and identify its gene target actA as the critical Listeria ligand. ActA alone was sufficient to promote entry into Fc{gamma}RIa-expressing cells, and this function is independent of its actin nucleating activity. Together, these studies present an unexpected role of ActA beyond its canonical function in actin-based motility and expand our understanding of Listeria strategies for host cell invasion.\n\nImportanceListeria monocytogenes is a food-borne bacterial pathogen and a causative agent of listeriosis with up to 50% mortality rate in immunocompromised individuals. While the mechanisms of Listeria entry into non-phagocytic cells have been extensively characterized, the details of phagocytic cell invasion are still poorly understood. We have recently discovered that human immunoglobulin receptor Fc{gamma}RIa mediates Listeria uptake by monocytic cells. This process occurred independently of canonical immunoglobulin interactions as well as classic Listeria internalization factors. Importantly, molecular determinants of Listeria-Fc{gamma}RIa interaction leading to bacterial entry, remained unknown. In this study, we demonstrate that Listeria virulence factor actin-assembly inducing protein ActA is required for Fc{gamma}RIa-mediated entry. Further, ActA was found to be sufficient for the internalization, suggesting its role as a bacterial ligand of Fc{gamma}RIa. Together, these findings expand our knowledge of mechanisms that Listeria has evolved to exploit cellular signaling pathways and immune defense of the host.

microbiology↗