Biology subjects

Miller, M. J.

Publications and source records attributed to Miller, M. J..

At least 19 recordsLinked to original sources

bioRxiv2025

Exploiting Endogenous Cas9-Based Genome Editing of Lacticaseibacillus rhamnosus GG (LGG) for Precision Food Fermentation and Live Biotherapeutics

Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas9 genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable precise deletions and insertions across multiple loci. Using this optimized and precise genome engineering method, we generated a {beta}-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex gut microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/634116v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@4c471eorg.highwire.dtl.DTLVardef@1d133f6org.highwire.dtl.DTLVardef@1aa7400org.highwire.dtl.DTLVardef@1a5d4dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Cilia dysfunction in the lateral ventricles after neonatal intraventricular hemorrhage does not lead to functional changes in cilia-based CSF flow networks

Intraventricular hemorrhage (IVH) has long been thought to lead to motile cilia dysfunction whereby intraventricular blood breakdown products damage and slough cilia from the ependymal wall. However, specifically how IVH may affect cilia development, structure, and transcriptional activation is not well-understood. Moreover, the impact of blood breakdown product-mediated cilia damage on the functional organization of cilia-based CSF flow networks is unknown. Here, we show hemoglobin exposure affects the number of ciliated ependymal cells in the lateral ventricle (LV) but does not impact in vitro beat frequency of the remaining cilia. Ultrastructurally, IVH decreases the total number of ciliary tufts without impacting axoneme structure. IVH does not result in changes in the expression of cilia-related genes and instead leads to downregulation of neurogenesis markers in parallel with innate immune upregulation. Functionally, we identify three previously uncharacterized cilia-mediated CSF flow domains in the LV lateral wall and show that IVH does not result in widespread disruption of their functional organization. These data de-emphasize cilia as a major contributor to global CSF dysfunction after IVH, and instead call attention to preserving the neurodevelopmental environment and preventing runaway innate immune system activation, as considerations to developing treatment strategies to prevent posthemorrhagic hydrocephalus and other neurodevelopmental sequelae.

neuroscience↗

bioRxiv2024

HSV-1 Infection Alters MAPT Splicing and Promotes Tau Pathology in Neural Models of Alzheimer's Disease

INTRODUCTIONHerpes simplex virus 1 (HSV-1) infection alters critical markers of Alzheimers Disease (AD) in neurons. One key marker of AD is the hyperphosphorylation of Tau, accompanied by altered levels of Tau isoforms. However, an imbalance in these Tau splice variants, specifically resulting from altered 3R to 4R MAPT splicing of exon 10, has yet to be directly associated with HSV-1 infection METHODSTo this end, we infected 2D and 3D human neural models with HSV-1 and monitored MAPT splicing and Tau phosphorylation. Further, we transduced SH-SY5Y-neurons with HSV-1 ICP27 which alters RNA splicing to analyze if ICP27 alone is sufficient to induce altered MAPT exon 10 splicing. RESULTSWe show that HSV-1 infection induces altered splicing of MAPT exon 10, increasing 4R-Tau protein levels, Tau hyperphosphorylation, and Tau oligomerization. DISCUSSIONOur experiments reveal a novel link between HSV-1 infection and the development of cytopathic phenotypes linked with AD progression. HIGHLIGHTSO_LIHSV-1 infection in forebrain organoids reduces the neurite length of MAP2-positive neurons. C_LIO_LIHSV-1 infection increases Tau hyperphosphorylation in both two-month-old and four-month-old forebrain organoids. C_LIO_LIHSV-1 infection increases Exon 10 containing (4R) MAPT mRNA and 4R-Tau protein expression in both forebrain organoids and human SH-SY5Y-neurons. C_LIO_LIHSV-1 ICP27 is both necessary and sufficient to induce increased 4R MAPT mRNA and 4R-Tau protein expression in SH-SY5Y-neurons. C_LIO_LIHSV-1 infection increases Tau oligomerization in both forebrain organoids and SH-SY5Y-neurons. C_LI

microbiology↗

bioRxiv2024

Targeting the host transcription factor HSF1 prevents human cytomegalovirus replication in vitro and in vivo

FDA-approved antivirals against HCMV have several limitations, including only targeting the later stages of the viral replication cycle, adverse side effects, and the emergence of drug-resistant strains. Antivirals targeting host factors specifically activated within infected cells and necessary for viral replication could address the current drawbacks of anti-HCMV standard-of-care drugs. In this study, we found HCMV infection stimulated the activation of the stress response transcription factor heat shock transcription factor 1 (HSF1). HCMV entry into fibroblasts rapidly increased HSF1 activity and subsequent relocalization from the cytoplasm to the nucleus, which was maintained throughout viral replication and in contrast to the transient burst of activity induced by canonical heat shock. Prophylactic pharmacological inhibition or genetic depletion of HSF1 prior to HCMV infection attenuated the expression of all classes of viral genes, including immediate early (IE) genes, and virus production, suggesting HSF1 promotes the earliest stages of the viral replication cycle. Therapeutic treatment with SISU-102, an HSF1 inhibitor tool compound, after IE expression also reduced the levels of L proteins and progeny production, suggesting HSF1 regulates multiple steps along the HCMV replication cycle. Leveraging a newly developed human skin xenograft transplant murine model, we found prophylactic treatment with SISU-102 significantly attenuated viral replication in transplanted human skin xenografts as well as viral dissemination to distal sites. These data demonstrate HCMV infection rapidly activates and relocalizes HSF1 to the nucleus to promote viral replication, which can be exploited as a host-directed antiviral strategy. One Sentence SummaryInhibiting of HSF1 as a host-directed antiviral therapy attenuates HCMV replication in vitro and in vivo.

microbiology↗

bioRxiv2024

Fully Human Bifunctional Intrabodies Achieve Graded Reduction of Intracellular Tau and Rescue Survival of MAPT Mutation iPSC-derived Neurons

Tau protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimers disease, frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP), spurring development of tau-lowering therapeutic strategies. Here, we report fully human bifunctional anti-tau-PEST intrabodies that bind the mid-domain of tau to block aggregation and degrade tau via the proteasome using the ornithine decarboxylase (ODC) PEST degron. They effectively reduced tau protein in human iPSC-derived cortical neurons in 2D cultures and 3D organoids, including those with the disease-associated tau mutations R5L, N279K, R406W, and V337M. Anti-tau-hPEST intrabodies facilitated efficient ubiquitin-independent proteolysis, in contrast to tau-lowering approaches that rely on the cells ubiquitination system. Importantly, they counteracted the proteasome impairment observed in V337M patient-derived cortical neurons and significantly improved neuronal survival. By serial mutagenesis, we created variants of the PEST degron that achieved graded levels of tau reduction. Moderate reduction was as effective as high reduction against tau V337M-induced neural cell death.

neuroscience↗

bioRxiv2024

Sterile kidney tissue injury induces neutrophil swarming in lung alveolar capillaries

Sterile acute kidney injury (AKI) is common in the clinic and frequently associated with hypoxemia that does not improve with dialysis and remains incompletely understood. AKI induces remote lung inflammation with neutrophil recruitment in mice and humans, but which cellular cues establish neutrophilic inflammation and how it contributes to hypoxemia is not known. Here we report that AKI induces rapid intravascular neutrophil retention in lung alveolar capillaries without any significant extravasation into tissue or alveoli, causing hypoxemia by reducing lung capillary blood flow in the absence of any significant lung interstitial or alveolar edema. In contrast to direct ischemic lung injury, lung neutrophil recruitment during remote lung inflammation did not require cues from intravascular non-classical monocytes or tissue-resident alveolar macrophages. Instead, lung neutrophil retention depended on neutrophil chemoattractant CXCL2 released by activated intravascular classical monocytes. Comparative single-cell RNA-sequencing analysis of direct and remote lung inflammation revealed that alveolar macrophages are highly activated and produce the neutrophil chemoattractant CXCL2 only in direct lung inflammation. Establishing a CXCL2 gradient into the alveolus by intratracheal administration of CXCL2 during AKI-induced remote lung inflammation enabled neutrophils to extravasate. We thus discovered important differences in lung neutrophil recruitment in direct versus remote lung inflammation and identified lung capillary neutrophil retention that negatively affects oxygenation by causing a ventilation-perfusion mismatch as a novel driver of AKI-induced hypoxemia.

physiology↗

bioRxiv2023

Microbial aromatic amino acid metabolism is modifiable in fermented food matrices to promote bioactivity

Ingestion of fermented foods impacts human immune function, yet the bioactive food components underlying these effects are not understood. Here, we interrogated whether fermented food bioactivity could be traced to a class of microbial metabolites derived from aromatic amino acids (ArAA), termed aryl-lactates. Using targeted metabolomics, we established that the aryl-lactates phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (4-HPLA), and indole-3-lactic acid (ILA), are present in varying concentrations across a wide range of commercially available fermented foods, including many vegetable and dairy ferments. After pinpointing fermented food-associated lactic acid bacteria (LAB) that produce high levels of aryl-lactates (e.g., Lactiplantibacillus plantarum), we utilized our knowledge of LAB metabolism to identify fermentation conditions (added cultures [e.g., L. plantarum] and metabolic co-factors [e.g., aryl-pyruvates]) to increase aryl-lactate production in food matrices up to 5x103 fold vs. standard fermentation conditions. Next, using ex vivo reporter assays, we found that a variety of food matrix conditions optimized for aryl-lactate production exhibited enhanced agonist activity for the human aryl-hydrocarbon receptor (AhR) as compared to standard fermentation conditions and/or commercial brands. Moreover, we determined that strategies to enhance aryl-lactates effectively maintained food matrix AhR bioactivity across 4 weeks of storage. Reduced microbial-induced AhR activity has emerged as a hallmark of many chronic inflammatory diseases, thus we envision strategies to enhance microbially produced aryl-lactates and thus AhR bioactivity of fermented foods can be leveraged to improve human health.

microbiology↗

bioRxiv2023

Engineered Secretory Immunoglobulin A provides insights on antibody-based effector mechanisms targeting Clostridiodes difficile

Secretory (S) Immunoglobin (Ig) A is the predominant mucosal antibody, which mediates host interactions with commensal and pathogenic microbes, including Clostridioides difficile. SIgA adopts a polymeric IgA structure that is bound by secretory component (SC). Despite significance, how SIgA supports diverse effector mechanisms is poorly characterized and SIgA-based therapies nonexistent. We engineered chimeric (c) SIgAs, in which we replaced SC domain D2 with a single domain antibody or a monomeric fluorescent protein, allowing us to investigate and enhance SIgA effector mechanisms. cSIgAs exhibited increased neutralization potency against C. difficile toxins, promoted bacterial clumping and cell rupture, and decreased cytotoxicity. cSIgA also allowed us to visualize and/or quantify C. difficile morphological changes and clumping events. Results reveal mechanisms by which SIgA combats C. difficile infection, demonstrate that cSIgA design can modulate these mechanisms, and demonstrate cSIgAs adaptability to modifications that might target a broad range of antigens and effector mechanisms.

immunology↗

bioRxiv2023

Honey varietals differentially impact Bifidobacterium animalis ssp lactis survivability in yogurt through simulated in vitro digestion

BackgroundBifidobacterium animalis ssp. lactis DN-173 010/CNCM I-2494 (B. animalis) is a probiotic strain commonly added to yogurt. Yogurt and honey are a popular culinary pairing. Honey improves bifidobacteria survival in vitro. However, probiotic survival in yogurt with honey during in vitro digestion has not been investigated. ObjectiveThe study aimed to evaluate the effects of different honey varietals and concentrations on B. animalis survivability in yogurt through in vitro digestion. MethodsYogurt with honey or control-treated samples underwent in vitro simulated oral, gastric, and intestinal digestion. B. animalis cells were enumerated on MRS medium followed by an overlay with a modified selective MRS medium; all underwent anaerobic incubation. B. animalis were enumerated pre-digestion and after oral, gastric, and intestinal digestion. There were two study phases: phase 1 tested four honey varietals at 20% w/w per 170g yogurt, and phase 2 tested seven dosages of clover honey (20, 14, 10, 9, 8, 6, and 4% w/w) per 170g yogurt. ResultsSimilar B. animalis counts were observed between all treatments after oral and gastric digestion (<1 Log CFU/g probiotic reduction). Higher B. animalis survivability was observed in yogurt with clover honey after exposure to simulated intestinal fluids ([~]3.5 Log CFU/g reduction; P < 0.05) compared to all control treatments ([~]5.5 Log CFU/g reduction; P < 0.05). Yogurt with 10 to 20% w/w clover honey increased B. animalis survivability after simulated in vitro digestion (up to [~]4.7 Log CFU/g survival; P < 0.05). ConclusionYogurt with added honey improves probiotic survivability during in vitro digestion. The effective dose of clover honey in yogurt was 10 to 20% w/w per serving (1 - 2 tablespoons per 170g yogurt) for increased probiotic survivability during in vitro digestion.

microbiology↗

bioRxiv2023

An enhancer RNA recruits MLL1 to regulate transcription of Myb

The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81 kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR/Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expression were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the MLL1 complex. Myrlin CRISPRi compromised MLL1 occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting MLL1.

cell biology↗

bioRxiv2023

Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

Establishing a non-productive quiescent/silent infection within monocytes is essential for spread of human cytomegalovirus (HCMV). Yet, how HCMV establishes a quiescent infection in monocytes remains unclear. US28 is a viral G protein-coupled receptor (GPCR) essential for silent infections within cells of the myeloid lineage. We found virion-associated US28 was rapidly delivered to monocytes, while de novo synthesized US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28{Delta}) was unable to establish a quiescent infection, resulting in a fully productive lytic replication cycle. Mechanistically, viral entry of US28{Delta} phosphorylated Akt at both serine 473 (S473) and threonine 308 (T308), which contrasted with the site-specific phosphorylation of Akt at S473 following WT infection. Preventing Akt bi-phosphorylation prevented lytic replication of US28{Delta}, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of WT infection. Our data demonstrate that virion-delivered US28 fine-tunes Akt activity to permit HCMV infection to enter a quiescent state following primary infection of monocytes.

microbiology↗

bioRxiv2023

Comparative phylogeography reveals widespread cryptic diversity driven by ecology in Panamanian birds

Widespread species often harbor unrecognized genetic diversity, and investigating the factors associated with such cryptic variation can help us better understand the forces driving diversification. Here, we identify potential cryptic species based on a comprehensive dataset of COI mitochondrial DNA barcodes from 2,333 individual Panamanian birds across 429 species, representing 391 (59%) of the 659 resident landbird species of the country, as well as opportunistically sampled waterbirds. We complement this dataset with additional publicly available mitochondrial loci, such as ND2 and cytochrome b, obtained from whole mitochondrial genomes from 20 taxa. Using barcode identification numbers (BINs), we find putative cryptic species in 19% of landbird species, highlighting hidden diversity in the relatively well-described avifauna of Panama. Whereas some of these mitochondrial divergence events corresponded with recognized geographic features that likely isolated populations, such as the Cordillera Central highlands, the majority (74%) of lowland splits were between eastern and western populations. The timing of these splits are not temporally coincident across taxa, suggesting that historical events, such as the formation of the Isthmus of Panama and Pleistocene climatic cycles, were not the primary drivers of cryptic diversification. Rather, we observed that forest species, understory species, insectivores, and strongly territorial species--all traits associated with lower dispersal ability--were all more likely to have multiple BINs in Panama, suggesting strong ecological associations with cryptic divergence. Additionally, hand-wing index, a proxy for dispersal capability, was significantly lower in species with multiple BINs, indicating that dispersal ability plays an important role in generating diversity in Neotropical birds. Together, these results underscore the need for evolutionary studies of tropical bird communities to consider ecological factors along with geographic explanations, and that even in areas with well-known avifauna, avian diversity may be substantially underestimated. LAY SUMMARY- What factors are common among bird species with cryptic diversity in Panama? What role do geography, ecology, phylogeographic history, and other factors play in generating bird diversity? - 19% of widely-sampled bird species form two or more distinct DNA barcode clades, suggesting widespread unrecognized diversity. - Traits associated with reduced dispersal ability, such as use of forest understory, high territoriality, low hand-wing index, and insectivory, were more common in taxa with cryptic diversity. Filogeografia comparada revela amplia diversidad criptica causada por la ecologia en las aves de Panama RESUMENEspecies extendidas frecuentemente tiene diversidad genetica no reconocida, y investigando los factores asociados con esta variacion criptica puede ayudarnos a entender las fuerzas que impulsan la diversificacion. Aqui, identificamos especies cripticas potenciales basadas en un conjunto de datos de codigos de barras de ADN mitocondrial de 2,333 individuos de aves de Panama en 429 especies, representando 391 (59%) de las 659 especies de aves terrestres residentes del pais, ademas de algunas aves acuaticas muestreada de manera oportunista. Adicionalmente, complementamos estos datos con secuencias mitocondriales disponibles publicamente de otros loci, tal como ND2 o citocroma b, obtenidos de los genomas mitocondriales completos de 20 taxones. Utilizando los numeros de identificacion de codigo de barras (en ingles: BINs), un sistema taxonomico numerico que proporcina una estimacion imparcial de la diversidad potencial a nivel de especie, encontramos especies cripticas putativas en 19% de las especies de aves terrestres, lo que destaca la diversidad oculta en la avifauna bien descrita de Panama. Aunque algunos de estos eventos de divergencia conciden con caracteristicas geograficas que probablemente aislaron las poblaciones, la mayoria (74%) de la divergencia en las tierras bajas se encuentra entre las poblaciones orientales y occidentales. El tiempo de esta divergencia no coincidio entre los taxones, sugiriendo que eventos historicos tales como la formacion del Istmo de Panama y los ciclos climaticos del pleistoceno, no fueron los principales impulsores de la especiacion. En cambio, observamos asociaciones fuertes entre las caracteristicas ecologicas y la divergencia mitocondriale: las especies del bosque, sotobosque, con una dieta insectivora, y con territorialidad fuerte mostraton multiple BINs probables. Adicionalmente, el indice mano-ala, que esta asociado a la capacidad de dispersion, fue significativamente menor en las especies con BINs multiples, sugiriendo que la capacidad de dispersion tiene un rol importamente en la generacion de la diversidad de las aves neotropicales. Estos resultos demonstran la necesidad de que estudios evolutivos de las comunidades de aves tropicales consideren los factores ecologicos en conjunto con las explicaciones geograficos. Palabras clave: biodiversidad tropical, biogeografia, codigos de barras, dispersion, especies cripticas

evolutionary biology↗

bioRxiv2022

Time in allopatry does not predict the outcome of secondary contact in lowland Panamanian birds

Geographic speciation models assume that time in allopatry should result in greater reproductive isolation between populations. Here we test the prediction that greater time in allopatry results in greater reproductive isolation using comparative ultraconserved element (UCE) data from ten bird lineages in secondary contact in Panama, measuring both genome-wide divergence and the geographic extent of hybridization. The best-fit models for the proportion of fixed Z-linked and autosomal loci to our data includes a combination of both time (as measured by mtDNA divergence) and hand-wing index, emphasizing that the role of time is tempered by dispersal capability. Furthermore, time does not predict the extent of genome-wide introgression as measured by the median width of diagnostic loci clines or the degree of variation in cline centers or widths. These metrics of the outcome of secondary contact were best predicted by ecological and genomic factors, including diet, hand-wing index, and genome-wide FST respectively, that are understood to serve as proxies for dispersal, the variability of population size, and overall genomic divergence. We find a primary role for ecological factors instead of isolation time in determining secondary contact outcomes for a lineage, highlighting how ecology shapes the development of reproductive isolation.

evolutionary biology↗

bioRxiv2022

Listeria motility increases the efficiency of goblet cell invasion during intestinal infection

Listeria monocytogenes (Lm) is a food-borne pathogen that causes severe bacterial gastroenteritis, with high rates of hospitalization and mortality. Lm is ubiquitous in soil, water and livestock, and can survive and proliferate at low temperatures. Following oral ingestion of contaminated food, Lm crosses the epithelial through intestinal goblet cells in a mechanism depending on Lm InlA and host E-cadherin. Importantly, human infections typically occur with Lm growing at or below room temperature, which are flagellated and motile. Even though many important human bacterial pathogens are flagellated, little is known regarding the effect of bacterial motility on invasion and immune evasion. Here, we used complementary imaging and computer modeling approaches to test the hypothesis that bacterial motility helps Lm locate and engage target cells permissive for invasion. Imaging explanted mouse and human intestine, we confirmed that Lm grown at room temperature uses motility to scan the epithelial surface and preferentially attach to target cells. Furthermore, we integrated quantitative parameters from our imaging experiments to construct a versatile "layered" cellular Potts model (L-CPM) that simulates host-pathogen dynamics. Simulated data are consistent with the hypothesis that bacterial motility enhances invasion by allowing bacteria to search the epithelial surface for their preferred invasion targets. Indeed, our model consistently predicts that motile bacteria have invaded [~]2-fold more at the 1-hour mark. This invasion advantage persists even in the presence of host phagocytes, with the balance between invasion and phagocytosis governed almost entirely by bacterial motility. In conclusion, our simulations provide insight into host pathogen interactions and challenge fundamental assumptions regarding how phagocytes might limit bacterial invasion early during infection.

microbiology↗

bioRxiv2022

GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa. SignificanceThe ISR kinase GCN2 is critical for maintaining tumor amino acid levels to facilitate growth, suggesting a novel therapeutic strategy for the treatment of prostate cancer by inducing starvation for essential amino acids.

cancer biology↗

bioRxiv2021

Prostaglandin E2 induction by cytosolic Listeria monocytogenes in phagocytes is necessary for optimal T-cell priming

Listeria monocytogenes is an intracellular bacterium that elicits robust CD8+ T-cell responses. Despite the ongoing development of L. monocytogenes-based platforms as cancer vaccines, our understanding of how L. monocytogenes drives robust CD8+ T-cell responses remains incomplete. One overarching hypothesis is that activation of cytosolic innate pathways is critical for immunity, as strains of L. monocytogenes that are unable to access the cytosol fail to elicit robust CD8+ T-cell responses and in fact inhibit optimal T-cell priming. Counterintuitively, however, activation of known cytosolic pathways, such as the inflammasome and type I IFN, lead to impaired immunity. Here, we describe a cytosol-dependent response that is critical for immunity to L. monocytogenes, namely production of prostaglandin E2 (PGE2) downstream of cyclooxygenase-2 (COX-2). Vacuole-constrained L. monocytogenes elicit reduced PGE2 production compared to wild-type strains in macrophages and dendritic cells ex vivo. In vivo, infection with wild-type L. monocytogenes leads to 10-fold increases in PGE2 production early during infection whereas vacuole-constrained strains fail to induce PGE2 over mock-immunized controls. Mice deficient in COX-2 specifically in Lyz2+ or CD11c+ cells produce less PGE2, suggesting these cell subsets contribute to PGE2 levels in vivo, while depletion of phagocytes with clodronate abolishes PGE2 production completely. Taken together, this work identifies the first known cytosol-dependent innate immune response critical for generating CD8+ T-cell responses to L. monocytogenes, suggesting that one reason cytosolic access is required to prime CD8+ T-cell responses may be due to induction of PGE2. Author summaryL. monocytogenes is an intracellular bacterial pathogen that generates robust cell-mediated immune responses. Due to this robust induction, L. monocytogenes is used as both a model to understand how CD8+ T-cells are primed, as well as a platform for cancer immunotherapy vaccines. L. monocytogenes must enter the cytosol of an infected host cell to stimulate robust T-cell responses, however, which cytosolic innate pathway(s) contribute to T-cell priming remains unclear. Here, we define COX-2 dependent PGE2 production as the first cytosol-dependent innate immune response critical for immunity to L. monocytogenes. We found that ex vivo PGE2 production by macrophages and dendritic cells is partially dependent on cytosolic access, as vacuole-constrained strains of L. monocytogenes elicit reduced PGE2. In vivo, cytosolic access is essential for PGE2 production. L. monocytogenes elicits a 10-fold increase in PGE2 production, whereas strains of L. monocytogenes that cannot access the cytosol fail to elicit PGE2 compared to mock immunized mice. Furthermore, CD11c+ and Lyz2+ cells contribute to PGE2 production in vivo, as mice deficient in COX-2 in these cell subsets have impaired PGE2 production. Taken together, our work identifies the first known cytosol-dependent pathway that is critical for generating immunity to L. monocytogenes.

immunology↗

bioRxiv2020

Proteomic fingerprint identification of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used as biomarkers for taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both principal component analysis (PCA) and linear discriminant analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.Author Summary Hard ticks (Ixodidae) are external parasites that feed on the blood of almost every species of terrestrial vertebrate on earth, including humans. Due to a complete dependency on blood, both sexes and even immature stages, are capable of transmitting disease agents to their hosts, causing distress and sometimes death. Despite the public health significance of ixodid ticks, accurate species identification remains problematic. Vector species identification is core to developing effective vector control schemes. Herein, we provide the first report of MALDI identification of several species of field-collected Neotropical tick specimens preserved in ethanol for up to four years. Our methodology shows that identification does not depend on a commercial reference library of lab-reared samples, but with the help of machine learning it can rely on a self-curated reference library. In addition, our approach offers greater accuracy and lower cost per sample than conventional and modern identification approaches such as morphology and molecular barcoding.Competing Interest StatementThe authors have declared no competing interest.List of abbreviationsMALDImatrix-assisted laser desorption/ionizationPCAprincipal component analysisLDAlinear discriminant analysisDNAdeoxyribonucleic acidINDICASATInstitute for Scientific Research and High Technology ServicesSTRISmithsonian Tropical Research InstituteSNINational System of InvestigationUTPTechnological University of PanamaTOFtime-of-flightMiAmbienteMinistry of Environment.View Full Text

zoology↗

bioRxiv2020

Sonogenetics for noninvasive and cellular-level neuromodulation in rodent brain

Sonogenetics, which uses ultrasound to noninvasively control cells genetically modified with ultrasound-sensitive ion channels, can be a powerful tool for investigating intact brain circuits. Here we show that TRPV1 is an ultrasound-sensitive ion channel that can modify the activity of TRPV1-expressing cells in vitro when exposing to focused ultrasound. We also show that focused ultrasound exposure at the mouse brain in vivo can selectively activate neurons that are genetically modified to express TRPV1. We demonstrate that precise manipulation of neural activity via TRPV1-based sonogenetics can be achieved by spatiotemporal control of ultrasound-induced heating. The focused ultrasound exposure is safe based on our inspection of neuronal integrity, apoptosis, and inflammation markers. This sonogenetic tool enables noninvasive, cell-type specific, spatiotemporally controlled modulation of mammalian brain activity.

neuroscience↗