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Chen, X.

Publications and source records attributed to Chen, X..

At least 847 records · Page 47Linked to original sources

Novel Pan-RAS Inhibitor ADT-007 Induces Tumor Regression in Mouse Models of GI Cancer

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RASWT cancer cells with GTP-activated RAS from upstream mutations were equally sensitive. Conversely, RASWT cancer cells harboring downstream BRAF mutations and normal cells were essentially insensitive to ADT-007. Sensitivity of cancer cells to ADT-007 required activated RAS and dependence on RAS for proliferation, while insensitivity was attributed to metabolic deactivation by UDP-glucuronosyltransferases expressed in RASWT and normal cells but repressed in RAS mutant cancer cells. ADT-007 binds nucleotide-free RAS to block GTP activation of effector interactions and MAPK/AKT signaling, resulting in mitotic arrest and apoptosis. ADT-007 displayed unique advantages over mutant-specific KRAS and pan-KRAS inhibitors, as well as other pan-RAS inhibitors that could impact in vivo antitumor efficacy by escaping compensatory mechanisms leading to resistance. Local administration of ADT-007 showed robust antitumor activity in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer. The antitumor activity of ADT-007 was associated with the suppression of MAPK signaling and activation of innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug also inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for RAS-driven cancers. SIGNIFICANCEADT-007 has unique pharmacological properties with distinct advantages over other RAS inhibitors by circumventing resistance and activating antitumor immunity. ADT-007 prodrugs and analogs with oral bioavailability warrant further development for RAS-driven cancers.

cancer biology↗

TREM2 promotes glioma progression and angiogenesis mediated by microglia/brain macrophages

TREM2, a myeloid cell-specific signaling molecule, controls essential functions of microglia and impacts on the pathogenesis of Alzheimers disease and other neurodegenerative disorders. TREM2 is also highly expressed in tumor-associated macrophages and plays detrimental roles in an experimental mouse sarcoma model. Here we studied whether TREM2 influences glioma progression. We found a sex- dependent effect of TREM2: the glioma volume is significantly attenuated in TREM2- deficient male but not female mice injected with GL261-EGFP glioma cells. The accumulation of glioma-associated microglia/macrophages (GAMs) and vascularization is reduced in male TREM2-deficient mice. A transcriptomic analysis of glioma tissue revealed that TREM2 deficiency suppresses angiogenic genes and MHC clusters. In an organotypic slice model devoid of functional vascularization, the tumor size was not affected by TREM2-deficiency. In human resection samples from glioblastoma, TREM2 is upregulated in GAMs. Based on the TCGA and CGGA databases, the TREM2 expression levels are negatively correlated with survival. Thus, the TREM2-dependent crosstalk between GAMs and the vasculature formation promotes glioma growth. Graphic abstractTREM2-dependent crosstalk between glioma-associated microglia/macrophages and the vasculature formation promotes glioma growth in male glioma mouse model. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/540621v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@65bec7org.highwire.dtl.DTLVardef@1d00961org.highwire.dtl.DTLVardef@117ee5eorg.highwire.dtl.DTLVardef@227994_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Living cell mTORC1 inhibition reporter mTIR reveals nutrient-sensing targets of histone deacetylase inhibitor

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a clinically effective therapeutic target for diseases such as cancer, diabetes, aging, and neurodegeneration, yet an efficient tool to monitor mTORC1 inhibition in living cells or tissues is still lacking. Here we devised a genetically encoded mTORC1 inhibition reporter termed mTIR that exhibits a highly contrasted fluorescence puncta pattern in response to mTORC1 inhibition. mTIR specifically senses physiological, pharmacological and genetic inhibition of mTORC1 signaling in living cells and tissues. Importantly, mTIR can be applied as an powerful tool for imaging-based visual screening of mTORC1 inhibitors. By this method, we identified histone deacetylase inhibitors (HDACi) that selectively inhibit mTORC1 by inducing nutrient-sensing gene expression. Thus, mTIR is a unique living cell reporter efficiently detecting the inhibition of mTORC1 activity, and the HDACi Panobinostat transcriptionally target mTORC1 signaling via amino acids sensing.

cell biology↗

Lactylation-driven FTO-mediated m6A modification of CDK2 aggravates diabetic microvascular anomalies

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. FTO is an N6-methyladenosine (m6A) demethylase that participates in various biological events, while its role in DR remains elusive. Herein, we detected elevated FTO expression in retinal proliferative membranes of DR patients. FTO promoted endothelial cell (EC) cell cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC-microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate mediated histone lactylation. FB23-2, an inhibitor to FTOs m6A demethylase activity, suppressed angiogenic phenotypes in vivo and in vitro. Noteworthy, we developed a nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficiencies in mice. Collectively, our study demonstrated that FTO coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR.

cell biology↗

Co-engagement of TIGIT+ immune cells to PD-L1+ tumours by a bispecific antibody potentiates T-cell response and tumour control

Co-targeting PD-1/PD-L1 and TIGIT/CD226 is being pursued to broaden the efficacy of current immunotherapy. Here we demonstrate that a bispecific antibody (BsAb) targeting PD-L1 and TIGIT, HB0036, shows major advantages over the combination of the two parental monoclonal Abs (mAbs). We demonstrated that HB0036 co-engages PD-L1+ tumour cells and TIGIT+ T cells, and thereby upregulates CD226 on T cells and induces a greater T-cell proliferative response compared to the combination of the parental antibodies in vitro. In vivo, HB0036 recruits greater amounts of TIGIT antibody in PD-L1+ tumours but not in PD-L1- tumours, compared with therapy using the two parental antibodies. We also observed improved tumour control and favourable immunological signatures with HB0036 in syngeneic and xenograft tumour models. Collectively, these findings demonstrate that bispecific antibodies targeting PD-L1 and TIGIT offer superior benefits in cancer immunotherapy compared with therapy using the two parental antibodies. Based on these studies, a phase I clinical trial with HB0036 has been initiated in patients with solid tumours (NCT05417321).

immunology↗

Modulation of infraslow oscillation in the dentate gyrus during Non-REM sleep

Synchronous neuronal activity is organized into neuronal oscillations with various frequency and time domains across different brain areas and brain states. For example, hippocampal theta, gamma and sharp wave oscillations are critical for memory formation and communication between hippocampal subareas and the cortex. In this study, we investigated the neuronal activity of the dentate gyrus (DG) with optical imaging tools during sleep-wake cycles. We found that the activity of major glutamatergic cell populations in the DG is organized into infraslow oscillations (0.01 - 0.03 Hz) during NREM sleep. Although the DG is considered a sparsely active network during wakefulness, we found that 50% of granule cells and about 25% of mossy cells exhibit increased activity during NREM sleep, compared to that during wakefulness. Further experiments revealed that the infraslow oscillation in the DG was correlated with rhythmic serotonin release during sleep, which oscillates at the same frequency but in an opposite phase. Genetic manipulation of 5-HT receptors revealed that this neuromodulatory regulation is mediated by 5-HT1a receptors and the knockdown of these receptors leads to memory impairment. Together, our results provide novel mechanistic insights into how the 5-HT system can influence hippocampal activity patterns during sleep.

neuroscience↗

The soluble guanylyl cyclase pathway is inhibited to evade androgen deprivation-induced senescence and enable progression to castration resistance

Castration-resistant prostate cancer (CRPC) is fatal and therapeutically under-served. We describe a novel CRPC-restraining role for the vasodilatory soluble guanylyl cyclase (sGC) pathway. We discovered that sGC subunits are dysregulated during CRPC progression and its catalytic product, cyclic GMP (cGMP), is lowered in CRPC patients. Abrogating sGC heterodimer formation in castration-sensitive prostate cancer (CSPC) cells inhibited androgen deprivation (AD)-induced senescence, and promoted castration-resistant tumor growth. We found sGC is oxidatively inactivated in CRPC. Paradoxically, AD restored sGC activity in CRPC cells through redox-protective responses evoked to protect against AD-induced oxidative stress. sGC stimulation via its FDA-approved agonist, riociguat, inhibited castration-resistant growth, and the anti-tumor response correlated with elevated cGMP, indicating on-target sGC activity. Consistent with known sGC function, riociguat improved tumor oxygenation, decreasing the PC stem cell marker, CD44, and enhancing radiation-induced tumor suppression. Our studies thus provide the first evidence for therapeutically targeting sGC via riociguat to treat CRPC. Statement of significanceProstate cancer is the second highest cancer-related cause of death for American men. Once patients progress to castration-resistant prostate cancer, the incurable and fatal stage, there are few viable treatment options available. Here we identify and characterize a new and clinically actionable target, the soluble guanylyl cyclase complex, in castration-resistant prostate cancer. Notably we find that repurposing the FDA-approved and safely tolerated sGC agonist, riociguat, decreases castration-resistant tumor growth and re-sensitizes these tumors to radiation therapy. Thus our study provides both new biology regarding the origins of castration resistance as well as a new and viable treatment option.

cancer biology↗

Repeated Omicron infection alleviates SARS-CoV-2 immune imprinting

The continuous emergence of highly immune evasive SARS-CoV-2 variants, like XBB.1.51,2 and XBB.1.163,4, highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by wildtype (WT)-based vaccination would compromise the antibody response to Omicron-based boosters5-9. Vaccination strategies that can counter immune imprinting are critically needed. In this study, we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. Our results show that in mice, the efficacy of single Omicron-boosting is heavily limited by immune imprinting, especially when using variants antigenically distinct from WT, like XBB, while the concerning situation could be largely mitigated by a second Omicron booster. Similarly, in humans, we found that repeated Omicron infections could also alleviate WT-vaccination-induced immune imprinting and generate high neutralizing titers against XBB.1.5 and XBB.1.16 in both plasma and nasal mucosa. By isolating 781 RBD-targeting mAbs from repeated Omicron infection cohorts, we revealed that double Omicron exposure alleviates immune imprinting by generating a large proportion of highly matured and potent Omicron-specific antibodies. Importantly, epitope characterization using deep mutational scanning (DMS) showed that these Omicron-specific antibodies target distinct RBD epitopes compared to WT-induced antibodies, and the bias towards non-neutralizing epitopes observed in single Omicron exposures due to imprinting was largely restored after repeated Omicron stimulation, together leading to a substantial neutralizing epitope shift. Based on the DMS profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated the combinations of these mutations could further boost XBB.1.5s immune-evasion capability while maintaining high ACE2 binding affinity. Our findings suggest the WT component should be abandoned when updating COVID-19 vaccine antigen compositions to XBB lineages, and those who havent been exposed to Omicron yet should receive two updated vaccine boosters.

immunology↗

scEpiTools: a database to comprehensively interrogate analytic tools for single-cell epigenomic data

Single-cell sequencing technology has enabled the characterization of cellular heterogeneity at an unprecedented resolution. To analyze single-cell RNA-sequencing data, numerous tools have been proposed for various analytic tasks, which have been systematically summarized and concluded in a comprehensive database called scRNA-tools. Although single-cell epigenomic data can effectively reveal the chromatin regulatory landscape that governs transcription, the analysis of single-cell epigenomic data presents assay-specific challenges, and an abundance of tools with varying types and functionalities have thus been developed. Nevertheless, these tools have not been well summarized, hindering retrieval, selection, and utilization of appropriate tools for specific analyses. To address the issues, we here proposed scEpiTools database with a multi-functional platform (http://health.tsinghua.edu.cn/scepitools). Specifically, based on the comprehensive collection and detailed annotation of 553 articles, scEpiTools groups articles into 14 major categories and 90 subcategories, provides task-specific recommendation for different emphases, and offers intuitive trend analysis via directed graphs, word clouds, and statistical distributions. For single-cell chromatin accessibility data analysis, we proposed a novel ensemble method named scEpiEnsemble, which, along with multiple methods as built-in kernels, can be used for flexible and efficient online analysis via the scEpiTools platform. We envision that scEpiTools will guide tool usage and development for single-cell epigenomic data and provide valuable resources for understanding regulatory mechanisms and cellular identity. Author summaryCompared to single-cell RNA-sequencing data, single-cell epigenomic data can reflect a set of epigenetic modifications at the cellular level. In general, the analysis of these data is typically divided into several steps: 1) retrieving available tools based on the omics of data and tasks; 2) selecting appropriate tools manually; and 3) utilizing the chosen tools to analyze data. However, due to the rapid development of tools and the unique complexity of the data, each of the above steps is extremely challenging for researchers. To provide researchers with great convenience, we developed scEpiTools (http://health.tsinghua.edu.cn/scepitools), a database with multiple functionalities. For instance, given the omics type and the analytic task, researchers can easily browse all the available tools via the hierarchical categorization of scEpiTools, and get recommendation scores from multiple perspectives. Considering that researchers may encounter difficulties in hardware requirements or environment setup, we also provide online analysis with various commonly used tools, as well as a novel ensemble method named scEpiEnsemble. In summary, scEpiTools represents a valuable resource for the single-cell epigenomics community, facilitating retrieval, selection and utilization of appropriate tools for diverse analyses, and helping to drive future advancements in the field.

bioinformatics↗

Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors.

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

cancer biology↗

The potential of Senicapoc, a KCNN4 inhibitor, for the prevention and treatment of breast cancer

BackgroundGenome-wide association studies have identified a breast cancer risk locus at 19q13.31. The candidate causal variants at this locus are located in the first exon of KCNN4. KCNN4, which regulates membrane potential and Ca2+ signaling, is a good candidate for drug repositioning because its inhibitor, Senicapoc, has been shown to be well tolerated in Phase-II and -III clinical trials for asthma and sickle cell anemia. MethodsWe evaluated public mRNA expression data to determine whether the allele at 19q13.31 associated with increased breast cancer risk was associated with KCNN4 expression. We also used immunohistochemistry to evaluate the relationship between KCNN4 protein expression and breast cancer survival. We then used Senicapoc in two murine mammary tumor models to determine if it would delay tumor development. We also treated mice bearing 4T1 mammary tumors with Senicapoc, by subcutaneous injection and by oral gavage. Finally we used gene editing to make deletions within Kcnn4 in 4T1 to determine whether Senicapoc had off-target effects on tumor growth. ResultsAnalysis of the Genotype-Tissue Expression Project showed that the allele at 19q13.31 associated with increased breast cancer risk is associated with increased KCNN4 expression, suggesting that inhibiting KCNN4 might reduce breast cancer risk. Using immunohistochemistry in a large breast cancer cohort, we found that membrane and cytoplasmic expression is a marker of poor prognosis in triple negative breast cancer. We then tested the efficacy of Senicapoc to prevent and treat breast cancer. This showed that it delays the development of mammary tumors in two murine models, and slows growth of a syngeneic (4T1) model of triple negative breast cancer. Senicapoc monotherapy showed similar efficacy to anthracycline/taxane-based chemotherapy in these studies, with a stronger effect when they were combined. ConclusionsThese results provide a rationale for clinical testing of Senicapoc for treating, and even preventing, breast cancer.

cancer biology↗

Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis

Besides its mitochondria-based anti-apoptotic role, Bcl-xL also travels to the nucleus to promote cancer metastasis by upregulating global histone H3 trimethyl Lys4 (H3K4me3) and TGF{beta} transcription. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates H3K4me3 modification are not understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced cell migration and metastasis in mouse models. Furthermore, knockout of CtBP2 suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGF{beta} mRNA upregulation as well as cell invasion. Moreover, cleavage under targets and release using nuclease (CUT&RUN) coupled with next generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor region of genes encoding TGF{beta} and its signaling pathway in the cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1.

cancer biology↗

The trade-off between individual metabolic specialization and versatility determines the metabolic efficiency of microbial communities

In microbial systems, a metabolic pathway can be either completed by one autonomous population or alternatively be distributed among a consortium performing metabolic division of labor (MDOL), where several specialized populations cooperate to complete the pathway. MDOL facilitates the systems function by reducing the metabolic burden; however, it may also hinder the function by reducing the exchange efficiency of metabolic intermediates among individuals. As a result, the metabolic efficiency of a community is influenced by trade-offs between the metabolic specialization and versatility of individuals, with the latter potentially introducing metabolic redundancy into the community. However, it remains unclear how metabolic specialization and versatility of the individuals involved can be controlled in order to optimize the function of the community. In this study, we deconstructed the metabolic pathway of naphthalene degradation into four specialized steps and introduced them individually or combinatorically into different strains, with varying levels of metabolic specialization. Using these strains, we engineered 1,456 synthetic consortia with varying levels of metabolic redundancy and tested their naphthalene degradation efficiency. We found that 74 consortia possessing metabolic redundancy exhibited higher degradation efficiency than both the autonomous population and the rigorous MDOL community. Quantitative modeling derived from our experiments provides general strategies for identifying the most effective MDOL consortium with functional redundancy (MCFR) from a range of possible MCFRs. Our large-scale genomic analysis suggests that natural communities for hydrocarbon degradation are mostly functionally redundant. In summary, our study provides critical insights into the engineering of high-performance microbial systems and explains why functional redundancy is prevalent in natural microbial communities.

microbiology↗

Identification of allele-specific KIV-2 repeats and impact on Lp(a) measurements for cardiovascular disease risk

The abundance of Lp(a) protein holds significant implications for the risk of cardiovascular disease (CVD), which is directly impacted by the copy number (CN) of KIV-2, a 5.5 kbp sub-region. KIV-2 is highly polymorphic in the population and accurate analysis is challenging. In this study, we present the DRAGEN KIV-2 CN caller, which utilizes short reads. Data across 166 WGS show that the caller has high accuracy, compared to optical mapping and can further phase [~]50% of the samples. We compared KIV-2 CN numbers to 24 previously postulated KIV-2 relevant SNVs, revealing that many are ineffective predictors of KIV-2 copy number. Population studies, including USA-based cohorts, showed distinct KIV-2 CN, distributions for European-, African-, and Hispanic-American populations and further underscored the limitations of SNV predictors. We demonstrate that the CN estimates correlate significantly with the available Lp(a) protein levels and that phasing is highly important.

genomics↗

Context-Aware Deep Learning Enables High-Efficacy Localization of High Concentration Microbubbles for Super-Resolution Ultrasound Localization Microscopy

Ultrasound localization microscopy (ULM) is an emerging super-resolution imaging technique for deep tissue microvascular imaging. However, conventional localization methods are constrained by low microbubble (MB) concentration, as accurate localization requires a strict separation of MB point spread functions (PSFs). Furthermore, deep learning-based localization techniques are often limited in their ability to generalize to in vivo ultrasound data due to challenges in accurately modeling highly variable MB PSF distributions and ultrasound imaging conditions. To address these limitations, we propose a novel deep learning-pipeline, LOcalization with Context Awareness (LOCA)-ULM, which employs simulation that incorporates MB context to generate synthetic data that closely resemble real MB signals, and a loss function that considers both MB count and localization loss. In in silico experiments, LOCA-ULM outperformed conventional localization with superior MB detection accuracy (94.0% vs. 74.9%) and a significantly lower MB missing rate (13.2% vs 74.8%). In vivo, LOCA-ULM achieved up to three-fold increase in MB localization efficiency and a x 9.5 faster vessel saturation rate than conventional ULM.

bioengineering↗

Tuning social interactions' strength drives collective response to light intensity in schooling fish

Schooling fish heavily rely on visual cues to interact with neighbors and avoid obstacles. The availability of sensory information is influenced by environmental conditions and changes in the physical environment that can alter the sensory environment of the fish, which in turn affects individual and group movements. In this study, we combine experiments and data-driven modeling to investigate the impact of varying levels of light intensity on social interactions and collective behavior in rummy-nose tetra fish. The trajectories of single fish and groups of fish swimming in a tank under different lighting conditions were analyzed to quantify their movements and spatial distribution. Interaction functions between two individuals and the fish interaction with the tank wall were reconstructed and modeled for each light condition. Our results demonstrate that light intensity strongly modulates social interactions between fish and their reactions to obstacles, which then impact collective motion patterns that emerge at the group level.

animal behavior and cognition↗

Single cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, ageing and disease

The brain is spatially organized and contains unique cell types, each performing diverse functions, and exhibiting differential susceptibility to neurodegeneration. This is exemplified in Parkinsons disease with the preferential loss of dopaminergic neurons of the substantia nigra pars compacta. Using a Parkinsons transgenic model, we conducted a single-cell spatial transcriptomic and dopaminergic neuron translatomic analysis of young and old mouse brains. Through the high resolving capacity of single-cell spatial transcriptomics, we provide a deep characterization of the expression features of dopaminergic neurons and 27 other cell types within their spatial context, identifying markers of healthy and aging cells, spanning Parkinsons-relevant pathways. We integrate gene enrichment and GWAS data to prioritize putative causative genes for disease investigation, identifying CASR as a novel regulator of dopaminergic calcium handling. These datasets (see: spatialbrain.org) represent the largest public resource for the investigation of spatial gene expression in brain cells in health, aging and disease.

molecular biology↗

Cross regulation in a three-component cell envelope stress signaling system

A multi-layered structure known as the cell envelope separates the controlled interior of bacterial cells from a fluctuating physical and chemical environment. The transcription of genes that determine cell envelope structure and function is commonly regulated by two-component signaling systems (TCS), comprising a sensor histidine kinase and a cognate response regulator. To identify TCS genes that contribute to cell envelope function in the intracellular mammalian pathogen, Brucella ovis, we subjected a collection of non-essential TCS deletion mutants to compounds that disrupt cell membranes and the peptidoglycan cell wall. Our screen led to the discovery of three TCS proteins that coordinately function to confer resistance to cell envelope stressors and to support B. ovis replication in the intracellular niche. This tripartite regulatory system includes the known cell envelope regulator, CenR, and a previously uncharacterized TCS, EssR-EssS, which is widely conserved in Alphaproteobacteria. The CenR and EssR response regulators bind a shared set of sites on the B. ovis chromosomes to control transcription of an overlapping set of genes with cell envelope functions. CenR directly interacts with EssR and functions to stimulate phosphoryl transfer from the EssS kinase to EssR, while CenR and EssR control the cellular levels of each other via a post-transcriptional mechanism. Our data provide evidence for a new mode of TCS cross-regulation in which a non-cognate response regulator affects both the activity and protein levels of a cognate TCS protein pair. ImportanceAs intracellular pathogens, Brucella must contend with a variety of host-derived stressors when infecting a host cell. The inner membrane, cell wall, and outer membrane -- i.e. the cell envelope -- of Brucella provides a critical barrier to host assault. A conserved regulatory mechanism known as two-component signaling (TCS) commonly controls transcription of genes that determine the structure and biochemical composition of the cell envelope during stress. We report the identification of previously uncharacterized TCS genes that determine B. ovis fitness in the presence of cell envelope disruptors and within infected mammalian host cells. Our study reveals a new molecular mechanism of TCS-dependent gene regulation, and thereby advances fundamental understanding of transcriptional regulatory processes in bacteria.

microbiology↗