Intracellular Growth Research Articles

Mutation in the Kinase Domain Alters the VEGFR2 Membrane Dynamics.

Recently, the substitution R1051Q in VEGFR2 has been described as a cancer-associated "gain of function" mutation. VEGFR2R1051Q phosphorylation is ligand-independent and enhances the activation of intracellular pathways and cell growth both in vitro and in vivo. In cancer, this mutation is found in heterozygosity, suggesting that an interaction between VEGFR2R1051Q and VEGFR2WT may occur and could explain, at least in part, how VEGFR2R1051Q acts to promote VEGFR2 signaling. Despite this, the biochemical/biophysical mechanism of the activation of VEGFR2R1051Q remains poorly understood. On these bases, the aim of our study is to address how VEGFR2R1051Q influences the biophysical behavior (dimerization and membrane dynamics) of the co-expressed VEGFR2WT. We employed quantitative FLIM/FRET and FRAP imaging techniques using CHO cells co-transfected with the two forms of VEGFR2 to mimic heterozygosity. Membrane protein biotinylation reveals that VEGFR2WT is more exposed on the cell membrane with respect to VEGFR2R1051Q. The imaging analyses show the ability of VEGFR2WT to form heterodimers with VEGFR2R1051Q and this interaction alters its membrane dynamics. Indeed, when the co-expression of VEGFR2WT/VEGFR2R1051Q occurs, VEGFR2WT shows reduced lateral motility and a minor pool of mobile fraction. This study demonstrates that active VEGFR2R1051Q can affect the membrane behavior of the VEGFR2WT.

Discovery of D8-03 as an Inhibitor of Intracellular Growth of Francisella tularensis.

Francisella tularensis is a Gram-negative facultative intracellular bacterial pathogen that is classified by the Centers for Disease Control and Prevention as a Tier 1 Select Agent. F. tularensis infection causes the disease tularemia, also known as rabbit fever. Treatment of tularemia is limited to few effective antibiotics which are associated with high relapse rates, toxicity, and potential emergence of antibiotic-resistant strains. Consequently, new therapeutic options for tularemia are needed. Through screening a focused chemical library and subsequent structure-activity relationship studies, we have discovered a new and potent inhibitor of intracellular growth of Francisella tularensis, D8-03. Importantly, D8-03 effectively reduces bacterial burden in mice infected with F. tularensis. Preliminary mechanistic investigations suggest that D8-03 works through a potentially novel host-dependent mechanism and serves as a promising lead compound for further development.

Functional profiling of CHAP domain-containing peptidoglycan hydrolases of Staphylococcus aureus USA300 uncovers potential targets for anti-staphylococcal therapies

The bacterial pathogen Staphylococcus aureus employs a thick cell wall for protection against physical and chemical insults. This wall requires continuous maintenance to ensure strength and barrier integrity, but also to permit bacterial growth and division. The main cell wall component is peptidoglycan. Accordingly, the bacteria produce so-called peptidoglycan hydrolases (PGHs) that cleave glycan strands to facilitate growth, cell wall remodelling, separation of divided cells and release of exported proteins into the extracellular milieu. A special class of PGHs contains so-called ‘cysteine, histidine-dependent amidohydrolase/peptidase’ (CHAP) domains. In the present study, we profiled the roles of 11 CHAP PGHs encoded by the core genome of S. aureus USA300 LAC. Mutant strains lacking individual CHAP PGHs were analysed for growth, cell morphology, autolysis, and invasion and replication inside human lung epithelial cells. The results show that several investigated CHAP PGHs contribute to different extents to extracellular and intracellular growth and replication of S. aureus, septation of dividing cells, daughter cell separation once the division process is completed, autolysis and biofilm formation. In particular, the CHAP PGHs Sle1 and SAUSA300_2253 control intracellular staphylococcal replication and the resistance to β-lactam antibiotics like oxacillin. This makes the S. aureus PGHs in general, and the Sle1 and SAUSA300_2253 proteins in particular, attractive targets for future prophylactic or therapeutic anti-staphylococcal interventions. Alternatively, these cell surface-exposed enzymes, or particular domains of these enzymes, could be applied in innovative anti-staphylococcal therapies.

Identifying Rab2 Protein as a Key Interactor of Centrin1 Essential for Leishmania donovani Growth.

Previously, we have demonstrated that deletion of a growth-regulating gene (LdCen1) in the Leishmania donovani parasite (LdCen1-/-) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1-/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1-/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1-/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Physical stimulations and their osteogenesis-inducing mechanisms.

Physical stimulations such as magnetic, electric and mechanical stimulation could enhance cell activity and promote bone formation in bone repair process via activating signal pathways, modulating ion channels, regulating bonerelated gene expressions, etc. In this paper, bioeffects of physical stimulations on cell activity, tissue growth and bone healing were systematically summarized, which especially focused on their osteogenesis-inducing mechanisms. Detailedly, magnetic stimulation could produce Hall effect which improved the permeability of cell membrane and promoted the migration of ions, especially accelerating the extracellular calcium ions to pass through cell membrane. Electric stimulation could induce inverse piezoelectric effect which generated electric signals, accordingly up-regulating intracellular calcium levels and growth factor synthesis. And mechanical stimulation could produce mechanical signals which were converted into corresponding biochemical signals, thus activating various signaling pathways on cell membrane and inducing a series of gene expressions. Besides, bioeffects of physical stimulations combined with bone scaffolds which fabricated using 3D printing technology on bone cells were discussed. The equipments of physical stimulation system were described. The opportunities and challenges of physical stimulations were also presented from the perspective of bone repair.

Distinct cell death pathways induced by granzymes collectively protect against intestinal Salmonella infection

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert.

The Genetic Architecture of the Human Corpus Callosum and its Subregions.

The corpus callosum (CC) is the largest set of white matter fibers connecting the two hemispheres of the brain. In humans, it is essential for coordinating sensorimotor responses, performing associative/executive functions, and representing information in multiple dimensions. Understanding which genetic variants underpin corpus callosum morphometry, and their shared influence on cortical structure and susceptibility to neuropsychiatric disorders, can provide molecular insights into the CC's role in mediating cortical development and its contribution to neuropsychiatric disease. To characterize the morphometry of the midsagittal corpus callosum, we developed a publicly available artificial intelligence based tool to extract, parcellate, and calculate its total and regional area and thickness. Using the UK Biobank (UKB) and the Adolescent Brain Cognitive Development study (ABCD), we extracted measures of midsagittal corpus callosum morphometry and performed a genome-wide association study (GWAS) meta-analysis of European participants (combined N = 46,685). We then examined evidence for generalization to the non-European participants of the UKB and ABCD cohorts (combined N = 7,040). Post-GWAS analyses implicate prenatal intracellular organization and cell growth patterns, and high heritability in regions of open chromatin, suggesting transcriptional activity regulation in early development. Results suggest programmed cell death mediated by the immune system drives the thinning of the posterior body and isthmus. Global and local genetic overlap, along with causal genetic liability, between the corpus callosum, cerebral cortex, and neuropsychiatric disorders such as attention-deficit/hyperactivity and bipolar disorders were identified. These results provide insight into variability of corpus callosum development, its genetic influence on the cerebral cortex, and biological mechanisms related to neuropsychiatric dysfunction.

CD137 expression and signal function drive pleiotropic γδ T-cell effector functions that inhibit intracellular M. tuberculosis growth

Co-activation signal that induces/sustains pleiotropic effector functions of antigen-specific γδ T cells remains unknown. Here, Mycobacteria tuberculosis (Mtb) tuberculin administration during tuberculosis (TB) skin test resulted in rapid expression of co-activation signal molecules CD137 and CD107a by fast-acting Vγ2Vδ2 T cells in TB-resistant subjects (Resisters), but not patients with active TB. And, anti-CD137 agonistic antibody treatment experiments showed that CD137 signaling enabled Vγ2Vδ2 T cells to produce more effector cytokines and inhibit intracellular Mtb growth in macrophages (Mɸ). Consistently, Mtb antigen (Ag) HMBPP stimulation induced sustainable high-level CD137 expression in fresh and activated Vγ2Vδ2 T cells from uninfected subjects, but not TB patients. CD137+Vγ2Vδ2 T-cell subtype predominantly displayed central memory phenotype and mounted better proliferative responses than CD137−Vγ2Vδ2 T-cells. In response to HMBPP, CD137+Vγ2Vδ2 T-cell subtype rapidly differentiated into greater numbers of pleiotropic effector cells producing anti-Mtb cytokines compared to CD137−Vγ2Vδ2 T subtype, with the non-canonical NF-κB pathway involved. CD137 expression in Vγ2Vδ2 T cells appeared to signal anti-Mtb effector functions leading to intracellular Mtb growth inhibition in Mɸ, and active TB disrupted such CD137-driven anti-Mtb effector functions. CD137+Vγ2Vδ2 T-cells subtype exhibited an epigenetic-driven high-level expression of GM-CSF and de novo production of GM-CSF critical for Vγ2Vδ2 T-cell controlling of Mtb growth in Mϕ. Concurrently, exosomes produced by CD137+Vγ2Vδ2 T cells potently inhibited intracellular mycobacterial growth. Furthermore, adoptive transfer of human CD137+Vγ2Vδ2 T cells to Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, our data suggest that CD137 expression/signaling drives pleiotropic γδ T-cell effector functions that inhibit intracellular Mtb growth.

Design of a new class of broad-spectrum therapeutics targeted to drug-resistant bacteria

We hypothesise that antimicrobial resistance (AMR) cannot be overcome by simply ‘drugging’ single biological targets, therefore, our focus is developing broad-spectrum therapeutics. Herein we present the synthesis of two novel spacer-linked, anthraquinone-triphenylphosphonium (AQ-TPP) conjugates (4) and (5) as early lead compounds in a new class of agent designed to penetrate the lipophilic barriers of the membranes of a diverse family of species; and establish bacterial growth inhibitory properties against methicillin-resistant Staphylococcus aureus (NCTC 13616) and Mycobacterium smegmatis [mc2155] (as a surrogate for Mycobacterium tuberculosis). The MIC and MBC values of (4) and (5) were determined against MRSA and found to be equipotent [MIC for each: 1 µg/mL (1.2 µM)]. Whereas the amide-linked conjugate (5) was determined to have 2-fold greater bactericidal potency [MBC 1 µg/mL (1.2 µM] than ester-linked (4). The anthraquinone-TPP conjugates (4) and (5) were active in vitro against Mycobacterium smegmatis (graphical abstract) and were equipotent as determined by their MIC values; in contrast to MRSA, the less hydrophobic (4) had 2-fold greater bactericidal potency than (5) as measured by their MBC values. Notably, both conjugates showed potent intracellular growth inhibitory activity in infected THP-1 macrophages. The conjugates are promising leads for the development of new antibacterial drugs. Key policy highlights Development of a novel class of antimicrobial compounds Drug screen against a WHO ‘high’ priority drug-resistant pathogen that poses the greatest threat to human health (methicillin-resistant Staphylococcus aureus) Drug screen against a surrogate for Mycobacterium tuberculosis, one of the leading causes of mortality worldwide.

Host Cells Upregulate Phosphate Transporter PIT1 to Inhibit Ehrlichia chaffeensis Intracellular Growth.

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

Functional Characterization of Six Eukaryotic Translation Initiation Factors of Toxoplasma gondii Using the CRISPR-Cas9 System.

Eukaryotic translation initiation factors (eIFs) are crucial for initiating protein translation and ensuring the correct assembly of mRNA-ribosomal subunit complexes. In this study, we investigated the effects of deleting six eIFs in the apicomplexan parasite Toxoplasma gondii using the CRISPR-Cas9 system. We determined the subcellular localization of these eIFs using C-terminal endogenous tagging and immunofluorescence analysis. Four eIFs (RH::315150-6HA, RH::286090-6HA, RH::249370-6HA, and RH::211410-6HA) were localized in the cytoplasm, while RH::224235-6HA was localized in the apicoplast. Additionally, RH::272640-6HA was found in both the basal complex and the cytoplasm of T. gondii. Functional characterization of the six RHΔeIFs strains was conducted using plaque assay, cell invasion assay, intracellular growth assay and egress assay in vitro, and virulence assay in mice. Disruption of five eIF genes (RHΔ315150, RHΔ272640, RHΔ249370, RHΔ211410, and RHΔ224235) did not affect the ability of the T. gondii RH strain to invade, replicate, form plaques and egress in vitro, or virulence in Kunming mice (p > 0.05). However, the RHΔ286090 strain showed slightly reduced invasion efficiency and virulence (p < 0.01) compared to the other five RHΔeIFs strains and the wild-type strain. The disruption of the TGGT1_286090 gene significantly impaired the ability of tachyzoites to differentiate into bradyzoites in both type I RH and type II Pru strains. These findings reveal that the eukaryotic translation initiation factor TGGT1_286090 is crucial for T. gondii bradyzoite differentiation and may serve as a potential target for drug development and an attenuated vaccine against T. gondii.

Subinhibitory concentrations of glabridin from Glycyrrhiza glabra L. reduce Listeria monocytogenes motility and hemolytic activity but do not exhibit antimicrobial activity.

Increases in the virulence and survival of some pathogens in the presence of subinhibitory concentrations of antibiotics have been reported. However, research on the effects of subinhibitory concentrations of antimicrobial substances derived from traditional Chinese medicine on pathogens is still insufficient. Glabridin is a well-known active isoflavone found in licorice roots that possesses a wide range of biological activities. Therefore, in this study, Listeria monocytogenes (L. monocytogenes) exposed to subinhibitory concentrations of glabridin was used as the research object. The minimum inhibitory concentration (MIC) was determined for L. monocytogenes. We investigated the impacts of subinhibitory concentrations of glabridin on the morphology, motility, biofilm formation, adherence, and survival of L. monocytogenes. The results indicated that the MIC of glabridin for L. monocytogenes was 31.25 μg/mL. At 1/8, 1/4, or 1/2 of the MIC, glabridin did not affect the growth, morphology, flagellar production, or biofilm formation of L. monocytogenes. However, subinhibitory concentrations of glabridin inhibited bacterial swimming and swarming motility and decreased the hemolytic activity of L. monocytogenes. Glabridin reduced the hemolytic activity of L. monocytogenes culture supernatants. The results also showed that subinhibitory concentrations of glabridin had no toxic effect on RAW264.7 cells but decreased the intracellular growth of L. monocytogenes in RAW264.7 cells. Furthermore, subinhibitory concentrations of glabridin triggered ROS production but did not induce MET formation in macrophages. In addition, glabridin did not enhance the capacity of L. monocytogenes to trigger METs or the extracellular killing of macrophages by METs. Thus, we conclude that subinhibitory concentrations of glabridin reduce L. monocytogenes motility and hemolytic activity but do not exhibit antimicrobial activity. Glabridin could be an interesting food additive as a bacteriostatic agent with anti-Listeria activity.

Cytological and ultrastructural investigation of pathogen infection pathway and host responses in asparagus stem infected by Phomopsis asparagi

Asparagus stem blight, a highly destructive disease in global asparagus cultivation, is caused by the fungus Phomopsis asparagi. However, the underlying mechanisms of the infectious process and pathogenesis of P. asparagi remain poorly understood. This study aims to elucidate the infection event of P. asparagi at the cytological and ultrastructural levels in asparagus stem through a microscopic observation. The host responses were also examined by microscopic observation and fluorescent probe. It revealed that P. asparagi germinated at either the tip or the middle of the conidia to produce short germ tubes on the surfaces of the asparagus stem at 20 h post-inoculation (hpi). The germ tubes penetrated the host cell wall with appressorium-like structures or narrow pegs at 1 day post-inoculation (dpi). At 3 − 5 dpi, a large number of P. asparagi hyphae colonized the epidermal cells. The hyphae were found to grow both intracellularly and intercellularly. The movement of hyphae between cells was facilitated by constricted invasive hyphae pegs. The hyphae exhibited bidirectional intracellular growth, extending and branching along the inner side of the cell wall within the stem cortex and towards the central cylinder. The fungal colonization resulted in cellular damage in plants, which is characterized by plasmolysis, rupture of the cell wall, and disruption of the cytoplasm. At 11 dpi, the fungi penetrated the parenchyma cells, and the fungal pycnidia were formed. At 13 dpi, the fungi penetrated the stem center parenchyma cell, where the conidia were released. In addition, the host defense response was investigated, which revealed a notably reduced germination rate of conidium, the formation of callose analogs, and the reactive oxygen burst. These findings provide unexpected perspectives on the infection process and host response in P. asparagi-plant interaction.

Biofilm Formation, Motility, and Virulence of Listeria monocytogenes Are Reduced by Deletion of the Gene lmo0159, a Novel Listerial LPXTG Surface Protein.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that causes listeriosis in humans and other animals. Surface proteins with the LPXTG motif have important roles in the virulence of L. monocytogenes. Lmo0159 is one such protein, but little is known about its role in L. monocytogenes virulence, motility, and biofilm formation. Here, we constructed and characterized a deletion mutant of lmo0159 (∆lmo0159). We analyzed not only the capacity of biofilm formation, motility, attachment, and intracellular growth in different cell types but also LD50; bacterial load in mice's liver, spleen, and brain; expression of virulence genes; and survival time of mice after challenge. The results showed that the cross-linking density of the biofilm of ∆lmo0159 strain was lower than that of WT by microscopic examination. The expression of biofilm-formation and virulence genes also decreased in the biofilm state. Subsequently, the growth and motility of ∆lmo0159 in the culture medium were enhanced. Conversely, the growth and motility of L. monocytogenes were attenuated by ∆lmo0159 at both the cellular and mouse levels. At the cellular level, ∆lmo0159 reduced plaque size; accelerated scratch healing; and attenuated the efficiency of adhesion, invasion, and intracellular proliferation in swine intestinal epithelial cells (SIEC), RAW264.7, mouse-brain microvascular endothelial cells (mBMEC), and human-brain microvascular endothelial cells (hCMEC/D3). The expression of virulence genes was also inhibited. At the mouse level, the LD50 of the ∆lmo0159 strain was 100.97 times higher than that of the WT strain. The bacterial load of the ∆lmo0159 strain in the liver and spleen was lower than that of the WT strain. In a mouse model of intraperitoneal infection, the deletion of the lmo0159 gene significantly prolonged the survival time of the mice, suggesting that the lmo0159 deletion mutant also exhibited reduced virulence. Thus, our study identified lmo0159 as a novel virulence factor among L. monocytogenes LPXTG proteins.

Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs.

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tristetraprolin mediates immune evasion of mycobacterial infection in macrophages.

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling the stability of targeted mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in intracellular mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by the mTOR inhibitor rapamycin. Rapamycin and AZD8055 specifically blocked 4EBP1 phosphorylation in infected macrophages and suppressed intracellular BCG growth. Rapamycin promoted TTP protein degradation through the ubiquitination pathway, whereas the proteasome inhibitor MG-132 blocked rapamycin function and thus stabilized TTP protein. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened protective immune responses in macrophages, whereas rapamycin enhanced the bactericidal effects through TTP inhibition. Moreover, blocking TTP binding increased the expression of TNF-α and iNOS and suppressed intracellular mycobacterial growth. Overall, our study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against tuberculosis (TB).

The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis modulates the survival of intracellular Mycobacterium tuberculosis and autophagy in macrophages

Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis (ATB) diagnosis. Therefore, we identified significantly dysregulated circRNAs in ATB patients and healthy controls (HC) and explored their molecular mechanism. We found that hsa_circ_0002371 was significantly up-regulated in PBMCs of ATB patients and Mycobacterium tuberculosis H37Rv- or Mycobacterium bovis bacillus Calmette Guerin (BCG)-infected THP-1 cells. Functional experiments demonstrated that hsa_circ_0002371 inhibited autophagy in BCG-infected THP-1 cells and promoted intracellular BCG survival rate. In terms of mechanism, hsa_circ_0002371 facilitated the expression of hsa-miR-502-5p, as shown by bioinformatics and dual-luciferase reporter gene analysis, respectively. Notably, hsa-miR-502-5p inhibited autophagy via suppressing autophagy related 16 like 1 (ATG16L1) in BCG-infected macrophages and thus promoting intracellular BCG growth. In summation, hsa_circ_0002371 increased the suppression of hsa-miR-502-5p on ATG16L1 and inhibited autophagy to promote Mtb growth in macrophages. In Conclusion, our data suggested that hsa_circ_0002371 was significantly up-regulated in the PBMCs of ATB patients compared with HC. The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis promoted the survival of intracellular Mtb and inhibited autophagy in macrophages. Our findings suggested hsa_circ_0002371 could act as a potential diagnostic biomarker and therapeutic target.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Bromocriptine sensitivity in bromocriptine-induced drug-resistant prolactinomas is restored by inhibiting FGF19/FGFR4/PRL.

At present, various treatment strategies are available for pituitary adenomas, including medications, surgery and radiation. The guidelines indicate that pharmacological treatments, such as bromocriptine (BRC) and cabergoline (CAB), are important treatments for prolactinomas, but drug resistance is an urgent problem that needs to be addressed. Therefore, exploring the mechanism of drug resistance in prolactinomas is beneficial for clinical treatment. In our research, BRC-induced drug-resistant cells were established. Previous RNA sequencing data and an online database were used for preliminary screening of resistance-related genes. Cell survival was determined by Cell Counting Kit-8 (CCK-8) assay, colony formation assays and flow cytometry. Quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting, immunohistochemistry, immunofluorescence and Co-immunoprecipitation (Co-IP) were used to assess the molecular changes and regulation. The therapeutic efficacy of BRC and FGFR4 inhibitor fisogatinib (FISO) combination was evaluated in drug-resistant cells and xenograft tumors in nude mice. Consistent with the preliminary results of RNA sequencing and database screening, fibroblast growth factor 19 (FGF19) expression was elevated in drug-resistant cells and tumor samples. With FGF19 silencing, drug-resistant cells exhibited increased sensitivity to BRC and decreased intracellular phosphorylated fibroblast growth factor receptor 4 (FGFR4) levels. After confirming that FGF19 binds to FGFR4 in prolactinoma cells, we found that FGF19/FGFR4 regulated prolactin (PRL) synthesis through the ERK1/2 and JNK signaling pathways. Regarding the effect of targeting FGF19/FGFR4 on BRC efficacy, FISO and BRC synergistically inhibited the growth of tumor cells, promoted apoptosis and reduced PRL levels. Overall, our study revealed FGF19/FGFR4 as a new mechanism involved in the drug resistance of prolactinomas, and combination therapy targeting the pathway could be helpful for the treatment of BRC-induced drug-resistant prolactinomas.

Discovery of piperine derivatives as inhibitors of human dihydroorotate dehydrogenase to induce ferroptosis in cancer cells

Inhibition of human dihydroorotate dehydrogenase (hDHODH) represents a promising strategy for suppressing the proliferation of cancer cells. To identify novel and potent hDHODH inhibitors, a total of 28 piperine derivatives were designed and synthesized. Their cytotoxicities against three human cancer cell lines (NCI-H226, HCT-116, and MDA-MB-231) and hDHODH inhibitory activities were also evaluated. Among them, compound H19, exhibited the strongest inhibitory activities (NCI-H226 IC50 = 0.95 µM, hDHODH IC50 = 0.21 µM). Further pharmacological investigations revealed that H19 exerted anticancer effects by inducing ferroptosis in NCI-H226 cells, with its cytotoxicity being reversed by ferroptosis inhibitors. This was supported by the intracellular growth or decline of ferroptosis markers, including lipid peroxidation, Fe2+, GSH, and 4-HNE. Overall, H19 emerges as a promising hDHODH inhibitor with potential anticancer properties warranting development.