Biology subjects

Tran, T. M.

Publications and source records attributed to Tran, T. M..

17 recordsLinked to original sources

bioRxiv2026

Application of AI-Designed OpenCRISPR-1 for Highly Efficient Gene Editing in Soybean and Nicotiana benthamiana

The widespread application of CRISPR/Cas genome editing for commercial crop improvement is currently hindered by a complex and restrictive intellectual property (IP) landscape. The recent development of OpenCRISPR-1, a fully AI-designed and open-source Cas9-like nuclease, provides a promising, IP-unencumbered alternative; however, its efficacy in dicotyledonous plants remains largely uncharacterized. Here, we report the successful adaptation of the OpenCRISPR-1 system for highly efficient targeted mutagenesis in dicots. We constructed a plant-optimized binary vector (pBSE-OpenCRISPR-1) and validated its editing capability across two species. In soybean (Glycine max), targeting the GmFAD2-1B gene via an Agrobacterium rhizogenes-mediated hairy root transformation system yielded a robust mutation rate of approximately 50%. In Nicotiana benthamiana, stable Agrobacterium-mediated transformation targeting the phytoene desaturase homologs (NbPDSa/b) achieved a 75% editing efficiency in T0 lines, with up to 13.8% of events displaying complete homozygous or biallelic mutations and the corresponding visible albino phenotypes. Deep amplicon and Sanger sequencing revealed a characteristic mutation profile dominated by 1-bp insertions and small deletions occurring two to three nucleotides upstream of the PAM. These results demonstrate that the AI-designed OpenCRISPR-1 system is a highly active and versatile nuclease for dicot genome engineering, offering a powerful, commercially unencumbered tool to accelerate global crop trait improvement.

plant biology↗

bioRxiv2026

Plasmodium blood-stage induces trained immunity in hepatocytes

Plasmodium parasites, the causative agents of malaria, are transmitted at high levels in endemic areas and sequential infections are common. Using a mouse model of infection we discovered liver burden was suppressed in blood-stage experienced, compared to naive, animals, independently of adaptive responses and inflammation. We observed greater chromatin accessibility of a subset of interferon stimulated genes in blood-stage experienced animals, and a rapid increase in transcription of these genes upon sporozoite challenge. Ex vivo stimulation of hepatocytes from blood-stage experienced mice also led to a rapid and elevated response upon treatment with an unrelated antigen, ctDNA. Taken together, these data are consistent with a model in which hepatocytes are reprogramed by blood stage Plasmodium infection to exhibit trained memory akin to what has been described in innate immune cells. The consequences of training of hepatocytes could be wide-reaching and might alter responses to diverse pathogens and other stimuli.

immunology↗

bioRxiv2026

Matched pancreatic cancer liver metastatic model system reveals cancer cell-dependent organotropism and site-specific tumor microenvironment reflective of human disease

Background: Pancreatic ductal adenocarcinoma (PDAC) mortality is driven largely by liver metastatic disease; however, the malignant cell states and tumor microenvironmental features of PDAC liver metastases remain obscure. Methods: We developed a transplant model system of matched pancreatic and liver tumors to study PDAC metastatic progression. Using this model, we identified murine PDAC cell lines with distinct liver metastatic capacities and performed multiomic profiling of matched primary pancreatic and metastatic liver tumors. Transcriptional programs associated with high and low liver tropism were defined and evaluated across tumor models and independent human PDAC datasets. Spatial and tumor-immune interaction analyses were used to characterize microenvironmental niches, immune composition, and cellular relationships within primary and metastatic tumors. Results: A high-liver-tropic transcriptional program was enriched in high liver-tropic cell lines and malignant cells within liver metastases, conserved across human PDAC datasets, and associated with inferior patient survival. High- and low-liver-tropic tumor states occupied distinct liver microenvironmental niches and exhibited different tumor-immune communication networks, accompanied by local and systemic changes in immune composition. Liver metastases also displayed features of enhanced immunosuppression, including increased proximity of CD4+ and CD8+ T cells to tumor cells and greater spatial association between regulatory T cells and exhausted CD8+ T cells. Conclusions: These findings identify conserved PDAC cell states associated with differential liver metastatic capacity and demonstrate that liver metastasis is accompanied by spatial and immunologic remodeling of the tumor microenvironment. Together, the study provides a framework for understanding how tumor-intrinsic metastatic programs interact with site-specific immune ecosystems in PDAC.

cancer biology↗

bioRxiv2026

Single-Cell Atlas of Renal Cell Carcinoma Brain Metastasis Uncovers Mechanisms of Immune Dysfunction and Resistance

Brain metastasis (BM) in renal cell carcinoma (RCC) remains poorly understood and often resistant to immune checkpoint inhibitors. We generated a large single-nucleus RNA-seq data of RCC BM, profiling 14 BM samples alongside matched extracranial metastases and primary tumors. Tumor cells in BM displayed neuronal infiltration, neural-like adaptation, and marked remodeling of the microenvironment, including expansion of immunosuppressive myeloid cells and depletion of antigen-presenting dendritic cells. Tumor, immune, and stromal cells exhibited metabolic rewiring characterized by fatty-acid metabolism, oxidative phosphorylation, and MYC-driven programs. CD8 T cells showed terminal exhaustion and impaired proliferative capacity, and tertiary lymphoid structures were absent. Spatial profiling of 12 BM samples (13,128 cells) validated key cellular interactions, while ligand-receptor analysis revealed immunoregulatory circuits between tumor, stromal, and immune cells. These findings define BM-specific adaptations that promote immune evasion and resistance, revealing therapeutic vulnerabilities in RCC BM. SIGNIFICANCESingle-nucleus RNA-sequencing profiling reveals tumor, immune, and metabolic adaptations in renal cell carcinoma (RCC) brain metastases, including neuroglial remodelling and immunosuppressive niche formation. These findings identify immune evasion mechanisms that could contribute to therapeutic resistance, providing new avenues for site-specific therapeutic interventions to improve treatment efficacy and outcomes in patients with RCC BM.

cancer biology↗

bioRxiv2025

The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta, we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers "biofilm mobility", which allows wildtype RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia. In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics. Significance StatementRalstonia solanacearum species complex (RSSC) pathogens threaten global food security by fatally wilting plants. A soft matter physics lens demystified the cryptic role of a major virulence factor, the EPS-I exopolysaccharide. EPS-I transforms RSSC biofilms into viscoelastic fluids, a mechanical behavior not previously described for other microbial biofilms that are almost always viscoelastic solids. We demonstrate that the development of fluid biofilms was a key evolutionary innovation that enabled pathogenic success of these aggressive pathogens that rapidly wilt plants.

microbiology↗

bioRxiv2024

Metabolic regulation of RNA methylation by the m6A-reader IGF2BP3

The interplay of RNA modifications - deposited by "writers", removed by "erasers" and identified by RNA binding proteins known as "readers" - forms the basis of the epitranscriptomic gene regulation hypothesis. Recent studies have identified the oncofetal RNA-binding protein IGF2BP3 as a "reader" of the N6-methyladenosine (m6A) modification and crucial for regulating gene expression. Yet, how its function as a reader overlaps with its critical oncogenic function in leukemia remains an open question. Here, we report the novel finding that the reader IGF2BP3 reprograms cellular metabolism, resulting in an altered ability of the "writers" to modify the epitranscriptome. In leukemia cells, IGF2BP3 supports increased glycolytic flux and one-carbon metabolism, leading to increased production of S-adenosyl methionine (SAM), a key substrate for methylation reactions within the cell. IGF2BP3 directly regulates the translation of MAT2B, the regulatory subunit of the methionine-adenosyltransferase complex, which is the final enzyme in a pathway leading to SAM production. This, in turn, results in increased m6A modifications on RNA, resulting in positive feedback regulation. This novel mechanism illustrates how metabolism mutually acts with epitranscriptomic modifications, underscoring the pervasive impact of IGF2BP3 in gene regulatory mechanisms governing a broad range of cancer-specific processes.

cancer biology↗

bioRxiv2024

Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate- binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a {Delta}lecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the {Delta}lecF/X ECM could explain the double mutants hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment. Author SummaryBacterial wilt diseases caused by Ralstonia species inflict significant losses on diverse, globally important agricultural plants. The pathogen first colonizes roots and ultimately the water-transporting xylem. There it attaches to host cell walls and other bacterial cells to form biofilms that eventually block xylem vessels and disrupt sap flow. It is not well known how Ralstonia spp. modulate attachment, but precise control of both attachment and dispersal is critical for successful host colonization over the disease cycle. Excessive adhesion could trap bacteria in a toxic or nutrient-depleted environment. Conversely, insufficient adhesion in a flowing environment could displace bacteria from an optimal niche. We provide evidence of dual, environment-specific roles of carbohydrate-binding lectins and exopolysaccharide EPS I in Ralstonia pseudosolanacearum (Rps) attachment. In static conditions, which Rps experiences on a host root, two lectins suppress bacterial aggregation and adhesion to roots. However, in flowing conditions, which Rps experiences in healthy xylem vessels, the same two lectins and EPS I are essential for biofilm development. The lectins increase the biofilm viscosity and support colony structural integrity, likely by interacting with polysaccharides in the biofilm matrix. This novel multifunctionality of bacterial lectins reveals how pathogens adapt to a physically dynamic host environment.

microbiology↗

bioRxiv2023

Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

molecular biology↗

bioRxiv2022

Immune Contexture of Isocitrate Dehydrogenase stratified Human Gliomas

The brain tumor immune microenvironment (TIME) continuously evolves during glioma progression, but only a limited view of a highly complex glioma associated immune contexture across isocitrate dehydrogenase mutation (IDH) classified gliomas is known. Herein, we present an unprecedentedly comprehensive view of myeloid and lymphoid cell type diversity based on our single cell RNA sequencing and spectral cytometry-based interrogation of tumor-associated leukocytes from fifty-five IDH stratified primary and recurrent human gliomas and three non-glioma brains. Our analyses revealed twenty-two myeloid and lymphoid cell types within and across glioma subtypes. Glioma severity correlated with microglial attrition concomitant with a continuum of invading monocyte-derived microglia-like and macrophages amongst other infiltrating conventional T and NK lymphocytes and unconventional mucosa associated invariant T (MAIT) cells. Specifically, certain microglial and monocyte-derived subpopulations were associated with antigen presentation gene modules, akin to cross-presenting dendritic cells (DCs). Furthermore, we identified phagocytosis and antigen presentation gene modules enriched in Triggering receptor expressed on myeloid (TREM)-2+ cells as a putative anti-glioma axis. Accelerated glioma growth was observed in Trem2 deficient mice implanted with CT2A glioma cells affirming the anti-glioma role of TREM2+ myeloid cells. In addition to providing a comprehensive landscape of glioma-specific immune contexture, our investigations discover TREM2 as a novel immunotherapy target for brain malignancies.

immunology↗

bioRxiv2021

Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

Salmonella Outer Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Furthermore, despite there being several published reports now independently describing (1) the marked prevalence of tRFs within secreted vesicle transcriptomes and (2) roles for specific tRFs in facilitating/inhibiting viral replication, there have been no examinations of the effects of vesicle-secreted tRFs on viral infection reported to date. Notably, while specific tRFs have been reported in a number of bacteria, the tRFs expressed by salmonellae have not been previously characterized. As such, we recently screened small RNA-seq datasets for the presence of recurrent, specifically excised tRFs and identified 31 recurrent, relatively abundant tRFs expressed by Salmonella enterica serovar Typhimurium (SL1344). Whats more, we find S. Typhimurium OMVs contain significant levels of tRFs highly complementary to known Salmonella enterica-infecting bacteriophage with 17 of 31 tRFs bearing marked complementarity to at least one known Salmonella enterica-infecting phage (averaging 97.4% complementarity over 22.9 nt). Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% sequence complementary over its entire 30 nt length to 29 distinct, annotated Salmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22 infectivity. Furthermore, we find P22 phage pre-incubation with OMVs isolated from naive, control SL1344 S. Typhimurium, successfully rescues the ability of S. Typhimurium transformed with a specific tRNA-Thr-CGT-1-1, 44-73 tRF inhibitor to defend against P22. Collectively, these experiments confirm tRFs secreted in S. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of known Salmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized innate antiviral defense.

microbiology↗

bioRxiv2021

Loss of cGMP-dependent protein kinase II alters ultrasonic vocalizations in mice, a model for speech impairment in human microdeletion 4q21 syndrome

Chromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, facial dysmorphisms, mental retardation, and absent or delayed speech. One of the key genes in the affected region of the chromosome is PRKG2, which encodes cGMP-dependent protein kinase II (cGKII). Mice lacking cGKII exhibit restricted growth and deficits in learning and memory, as seen in the human syndrome. However, speech/vocalization impairments in these mice have not been determined. Moreover, the molecular pathway underlying speech impairment in humans is not fully understood. Here, we employed cGKII knockout (KO) mice as a model for the human microdeletion syndrome to test whether vocalizations are affected by loss of the PRKG2 gene. Mice emit ultrasonic vocalizations (USVs) to communicate in social situations, stress, and isolation. We thus recorded ultrasonic vocalizations as a model for speech in humans. We isolated postnatal day 5-7 pups from the nest to record and analyze USVs and found significant differences in vocalizations of KO mice relative to wild-type and heterozygous mutant mice. KO mice produced fewer calls that were shorter duration, higher frequency, and lower intensity. Because neuronal activity in the hypothalamus is important for the production of animal USVs following isolation from the nest, we assessed hypothalamic activity in KO pups following isolation. Indeed, we found abnormal hyperactivation of hypothalamic neurons in cGKII KO pups after isolation. Taken together, our studies indicate that cGKII is important for neuronal activation in the hypothalamus, which is required for the production of USVs in neonatal mice. We further suggest cGKII KO mice can be a valuable animal model for human microdeletion 4q21 syndrome. HighlightsO_LIChromosome 4q21 microdeletion leads to a human syndrome that exhibits restricted growth, mental retardation, and absent or delayed speech. C_LIO_LIThe cGMP-dependent protein kinase II (cGKII) gene is one of the genes located in the affected region of the chromosome. C_LIO_LIcGKII knockout mice show restricted growth and deficits in learning and memory. C_LIO_LIAltered ultrasonic vocalizations and abnormal activation in hypothalamic neurons are found when infant cGKII knockout pups are isolated from the nest. C_LIO_LIcGKII knockout mice can be a valuable animal model for human microdeletion 4q21 syndrome. C_LI

neuroscience↗

bioRxiv2020

Post-transcriptional gene regulation by the RNA binding protein IGF2BP3 is critical for MLL-AF4 mediated leukemogenesis

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes and a high risk of relapse. Here, we found that MLL-AF4, the most common MLL fusion protein in patients, transcriptionally induces IGF2BP3 and that IGF2BP3 strongly amplifies MLL-Af4 mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4 driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. eCLIP and transcriptome analysis of MLL-Af4 transformed stem and progenitor cells and MLL-Af4 bulk leukemia cells reveals a complex IGF2BP3-regulated post-transcriptional operon governing leukemia cell survival and proliferation. Regulated mRNA targets include important leukemogenic genes such as those in the Hoxa locus and numerous genes within the Ras signaling pathway. Together, our findings show that IGF2BP3 is an essential positive regulator of MLL-AF4 mediated leukemogenesis and represents an attractive therapeutic target in this disease.

cancer biology↗

bioRxiv2020

Longitudinal analysis of naturally acquired antibodies to PfEMP1 CIDR domain variants and their association with malaria protection

Malaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum infected erythrocytes to the microvasculature mediated via specific interactions between PfEMP1 variant domains to host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria. We evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission. Using longitudinal data, we found that IgG to the pathogenic domain variants CIDR1.7 and CIDR1.8 were acquired the earliest. Furthermore, IgG to CIDR{gamma}3 was associated with reduced prospective risk of febrile malaria and recurrent malaria episodes. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.

immunology↗

bioRxiv2020

The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely under-studied. In this work, we show that the QS molecule DSF (cis-11-methyl-dodecenoic acid) produced by Xanthomonas campestris pv. campestris can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in Arabidopsis thaliana, mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of oligomerization states and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of Arabidopsis, with a particular increase of the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate PAMP-triggered host immunity. SIGNIFICANCE STATEMENTBacteria rely on small signalling molecules called quorum sensing (QS) signals to communicate and coordinate their behaviors. QS is known to regulate gene expression, production of virulence factors, and biofilm formation for pathogenic bacteria to effectively colonize their hosts and cause diseases. In this work, we found a class of QS molecule called diffusible-signal factor (DSF), produced by devastating phytopathogenic bacteria such as Xanthomonas spp. and Xylella fastidiosa, could communicate directly with plant host and subvert plant innate immunity by inducing plant sterol production and thereby, attenuating receptor signalling through hindering the receptor clustering and plant endocytosis. The results significantly enrich our understanding of the mechanisms in the tug-of-war between bacterial pathogenesis and host immunity.

plant biology↗

bioRxiv2019

qKW9 encodes a pentatricopeptide repeat protein affecting photosynthesis and grain filling in maize

Kernel weight is an important yield component in maize that was selected during domestication. Many kernel weight genes have been identified through mutant analysis, and are mostly involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, only a limited number of loci underlying quantitative variation in kernel weight have been cloned. Here we characterize a maize kernel weight QTL, qKW9 and find that it encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of NdhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qKW9 background, C-to-U editing of NdhB was abolished, and photosynthesis was reduced, suggesting that qKW9 regulates kernel weight by controling the maternal source of photosynthate for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis on maize grain yield production.

plant biology↗

bioRxiv2018

Coxiella burnetii blocks intracellular IL-17 signaling in macrophages

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella Type IVB Secretion System (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild type (WT) bacteria, suggesting Coxiella T4BSS effector proteins downregulate expression of these genes. In addition, the IL-17 signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT or mock-infected cells, including the pro-inflammatory cytokines I11a, Il1b and Tnfa, the chemokines Cxcl2 and Ccl5, and the antimicrobial protein Lcn2. We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/MIP-2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

microbiology↗

bioRxiv2018

Robotic automation of droplet microfluidics

Droplet microfluidics enables new reactions, assays, and analytic capabilities, but often requires complex workflows involving numerous steps of macro- and micro-fluidic processing. We demonstrate robotically-automated droplet microfluidics, an approach to automate workflows with commercial fluid-handling robots. These workflows can be performed without human intervention, increasing reliability and convenience.

bioengineering↗