Infection and Immunity最新文献

Streptococcus pneumoniae is a leading cause of pneumonia. Importantly, the extent and impact of changes in the infected airway on bacterial nutrient availability and gene expression are not known. Utilizing untargeted UPLC-ESI-MS/MS metabolomics, we comprehensively characterized the metabolic landscape in the airway across early, mid, and severe stages of pneumococcal pneumonia. This revealed that dynamic shifts in metabolites occurred during pneumonia, with an initial influx of metabolites at the early stage, followed by declines as the disease progressed. Specific host metabolic perturbations were indicative of purine dysregulation, cellular stress, and outright tissue injury. Levels of glucose, a known modulator of pneumococcal capsule production, were highest at the early disease stage and then declined as the disease progressed, overlaying general metabolite trends. Concurrent bacterial transcriptome profiling was performed using a NanoString nCounter custom panel of 66 genes selected for their importance to metabolism, virulence, and stress response; 9% of which had disease-stage significant differences in gene expression. This analysis revealed remarkably high expression of spxB, the gene encoding pyruvate oxidase, at the severe stage of pneumonia compared to the mid-stage pneumonia, consistent with a drop in glucose levels and indicative of a shift toward mixed fermentation and the increased production of hydrogen peroxide. Our study improves our understanding of how pneumococcal infection alters the lung environment, driving profound metabolic shifts that, in turn, influence bacterial phenotypes. This detailed understanding of host-pathogen metabolic interactions offers valuable insights into novel therapeutic strategies.

Toxoplasma gondii is an obligate intracellular parasite capable of subverting host defenses to establish infection. Necroptosis, a lytic pro-inflammatory form of programed cell death, has emerged as a host defense mechanism against intracellular pathogens. However, its relevance in controlling T. gondii replication remains unclear. Here, we investigated the role of necroptosis in limiting T. gondii replication using bone marrow-derived macrophages (BMDMs) deficient in key necroptotic mediators, RIPK3 and MLKL. We demonstrate that under naïve conditions, T. gondii replication proceeds unimpeded in RIPK3^-/- and MLKL^-/- BMDMs. However, co-treatment with TNF-α and the pan-caspase inhibitor Z-VAD-FMK, conditions that promote necroptosis, significantly reduced parasite replication in wild-type BMDMs but not in those lacking RIPK3 or MLKL. This suppression was dependent on RIPK1 activity, as pharmacological inhibition with Necrostatin-1 abrogated the effect. We further confirmed that TNF-α and Z-VAD-FMK treatment induced necroptotic cell death characterized by loss of plasma membrane integrity, both of which were absent in RIPK3^-/- and MLKL^-/- cells. These findings establish that the activation of necroptosis can effectively limit T. gondii replication in BMDMs and underscore the importance of RIPK1-RIPK3-MLKL signaling in mounting a cell-intrinsic immune defense. Our study provides new insight into the functional capacity of necroptosis in restricting intracellular parasites and highlights its potential as a therapeutic target in toxoplasmosis.

Ehrlichia chaffeensis is an obligately intracellular bacterium that manipulates mononuclear phagocytes by hijacking host cell signaling pathways to promote infection. Previous studies from our laboratory have shown that multiple signal transducer and activator of transcription (STAT) family members interact with E. chaffeensis effector proteins. However, the functional role of STATs during infection remains poorly understood. Notably, STAT3, a highly immunomodulatory and pro-survival factor, interacts with the E. chaffeensis effector protein TRP75. In this study, we examined activation of STAT family members and transcription of STAT target genes during E. chaffeensis infection. We observed significant activation of multiple STATs (STAT1, STAT3, STAT5, and STAT6), with STAT3 showing the highest level of activation. Therefore, we further investigated STAT3 activation dynamics and effects of its inhibition on infection. STAT3 phosphorylation and nuclear translocation were detected beginning 48 h post-infection, coinciding with upregulation of STAT3 target genes, including the anti-apoptotic gene MCL-1. Pharmacological inhibition of STAT3 significantly reduced MCL-1 expression and increased caspase cleavage, implicating STAT3 as a regulator of anti-apoptotic signaling during infection. Furthermore, both pharmacological inhibition and genetic knockout of STAT3 significantly reduced bacterial load, highlighting its critical role in supporting infection. Ectopic expression of TRP75 in human embryonic kidney 293 cells induced STAT3 phosphorylation, demonstrating a specific role for TRP75 in STAT3 activation. Collectively, these findings support a model in which E. chaffeensis exploits STAT3 via the TRP75 effector to activate an anti-apoptotic program and other cellular pathways that promote infection.

The intracellular survival and replication of Chlamydia trachomatis rely on the precise manipulation of host signaling pathways. Host kinases are instrumental in the modulation of host signaling during C. trachomatis infection. However, the potential contribution of host phosphatases to chlamydial pathogenesis remains poorly understood. Here, we identified the host tyrosine phosphatase PTP1B as a positive regulator of C. trachomatis intracellular development. Gain-of-function approaches revealed that PTP1B promotes inclusion development and increases the production of infectious elementary bodies, whereas loss-of-function by chemical inhibition or silencing leads to a reduction in both inclusion size and bacterial infectivity. Interestingly, PTP1B inhibition did not affect Chlamydia trachomatis invasion efficiency, suggesting a specific role during the developmental phase of the chlamydial life cycle. To explore the functional relevance of PTP1B and its potential interaction with chlamydial effectors, we focused on the early-secreted effector Tarp, which undergoes tyrosine phosphorylation upon host cell entry. In vitro biochemical assays demonstrated that recombinant PTP1B can dephosphorylate both native and recombinant forms of Tarp. However, PTP1B inhibition during infection did not significantly alter Tarp phosphorylation levels, possibly owing to the overpowering influence of host tyrosine kinases. These findings suggest that while Tarp may not be a major physiological substrate, PTP1B is capable of interacting with phosphorylated chlamydial effectors. Together, these results establish PTP1B as a host factor that supports chlamydial development and underscore the underappreciated role of host phosphatases in bacterial pathogenesis. This study provides a foundation for future work exploring phosphatase-mediated regulation of infection and potential host-directed therapeutic strategies.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a neglected tropical disease with limited therapeutic options and increasing drug resistance. This study investigates the immunological mechanisms and antiparasitic efficacy of imidazoquinoline-based Toll-like receptor 7/8 (TLR7/8) agonists as host-directed agents in an in vitro VL model. Using RAW 264.7 macrophages and L. donovani promastigotes and amastigotes, we examined macrophage activation, nitric oxide (NO) induction, and cell cycle disruption in parasites. The lead compounds (5 and 10) significantly enhanced NO production in macrophages, both in unstimulated and LPS-stimulated conditions, indicating robust innate immune activation. Additionally, parasite-derived reactive oxygen species (ROS) levels were markedly elevated, suggesting oxidative stress as a mechanism of direct leishmanicidal action. Flow cytometric analysis revealed G₀/G₁ arrest in treated promastigotes, further supporting interference with parasite proliferation. Importantly, these compounds exhibited low cytotoxicity toward host cells and favorable selectivity indices. Notably, this is the first in vitro study to comprehensively demonstrate the ability of TLR7/8 agonists to exert direct parasiticidal effects along with immune modulation in the context of VL. The results underscore the potential of TLR-targeted immunomodulation to enhance host defense mechanisms against intracellular protozoan infections and contribute to the development of novel immunopharmacological interventions for VL.

Here, we report the identification of bacteriophage Mu contamination in a commonly used Citrobacter rodentium DBS100 ∆cpxRA mutant strain. After re-constructing a new Mu-free ∆cpxRA strain, we independently replicated the results of a recent study by A. Gilliland, C. Gavino, S. Gruenheid, and T. Raivio (Infect Immun 90:e00314-22, 2022, https://doi.org/10.1128/iai.00314-22). The only result from Gilliland et al. that was impacted by the presence of Mu was the outcome of interbacterial competition assays with the ∆cpxRA strain, as strains carrying Mu consistently outcompeted susceptible Mu-free competitors. These results are important for the field, as the contaminated DBS100 ∆cpxRA mutant strain has been used in six different studies. We believe that the Mu contamination occurred during the construction of the ∆cpxRA allele, during the conjugation of DBS100 with a popular Mu-containing donor strain. Our results highlight the importance of using Mu-free conjugal donor strains and how phage contamination can impact bacterial physiology and experimental results.

As a leading causative agent of pneumonia infection worldwide, Streptococcus pneumoniae (Spn) induces lung injury and presents substantial therapeutic challenges. To elucidate the role of methyltransferase-like 3 (METTL3) in modulating circular RNA_0001239 (circ_0001239), YTH domain containing protein 2 (YTHDC2), and Krüppel-like factor 10 (KLF10) through m6A modification, we established Spn-induced neonatal mouse models. The survival rates, bacterial load in bronchoalveolar lavage fluid, and METTL3 expression in pulmonary tissue were evaluated. After METTL3 downregulation, lung wet-to-dry ratio, myeloperoxidase activity, and inflammatory markers were assessed. Methylated RNA immunoprecipitation detected enriched m6A modification on circ_0001239, while RNA immunoprecipitation validated the bindings of circ_0001239 to YTHDC2 and YTHDC2 to KLF10. The KLF10 mRNA stability was analyzed via actinomycin D treatment. METTL3 and circ_0001239 were upregulated in pneumonic lungs, while KLF10 was downregulated. METTL3 knockdown improved survival, alleviated lung injury, increased superoxide dismutase levels, and suppressed interleukin (IL)-6, IL-1β, and malondialdehyde levels. METTL3 promoted the binding of circ_0001239 to YTHDC2 via m6A modification, destabilizing KLF10 mRNA. Circ_0001239 overexpression or KLF10 knockdown reversed the protective effects of low expression of METTL3 on lung damage in neonatal mice with pneumonia. In conclusion, METTL3 aggravates Spn-induced lung injury via m6A-dependent circ_0001239/YTHDC2/KLF10 axis, thereby providing potential therapeutic targets for severe pneumonia.

Cell death mechanisms play a fundamental role in mycobacterial pathogenesis. We critically reviewed 94 research manuscripts, 44 review articles, and 4 book chapters to analyze important discoveries, background literature, and potential shortcomings in the field. The focus of this review is the pathogen Mycobacterium tuberculosis (Mtb) and other Mtb and Mycobacterium avium complex microorganisms. Virulent strains hijack cell death processes by inhibiting autophagy, apoptosis, and pyroptosis while eliciting necrosis and ferroptosis to multiply intracellularly and spread within and between hosts. In addition, virulent strains may induce apoptosis in epithelial cells or secondary infected macrophages to spread. Autophagy does not control Mtb intracellular replication in vivo but suppresses macrophage and T cell responses in Mtb infections, with a predominant role in preventing neutrophil infiltration. In contrast, attenuated vaccine strains promote apoptosis in macrophages, leading to the activation of innate immunity and, eventually, the acquired immune response. Although Mtb infection activates necroptosis, studies with mutant cell lines have indicated that this process is not essential for cell lysis and that Mtb promotes unprogrammed necrosis. Ferroptosis is discussed in the context of necrotic processes involving lipid peroxidation. Recent research indicated that pyroptosis is more akin to apoptosis as Mtb proteins induce cell membrane repair to prevent inflammasome activation. In the supplementary tables, homologs of mycobacterial cell death pathways and virulence factors were identified using a basic local alignment search tool protein followed by a conserved domain database search to determine the presence of functional domains. Finally, prospects for therapeutic interventions are discussed.

Bacterial vaginosis (BV) is the most prevalent vaginal disorder in women of childbearing age and causes pregnancy complications, including preterm birth, amnionitis, and postpartum endometritis. BV also interferes with sexual health and increases stress. BV is a vaginal dysbiosis that occurs when Lactobacillus species are displaced by facultative and anaerobic bacterial species, including Gardnerella, Prevotella, Fannyhessea, Sneathia, Megasphaera, Mycoplasma, and others. Species of Gardnerella increase just prior to the onset of symptoms and are considered to play major roles in the development and transmission of BV. However, Gardnerella species have remained genetically intractable, limiting investigations of their virulence mechanisms. Here, we describe methods for genetic manipulation of Gardnerella. Through trial and error, we optimized methods for electrotransformation of Gardnerella and created methods for making mutations and complements. We mutated the gene for the toxin vaginolysin (vly) in G. vaginalis and the gene for sialidase nanH3 in G. pickettii. A vly point mutant was tested in human cervix tissue and found to lack lytic activity. The nanH3 mutant lost sialidase and mucus degradation activity. Overall, this genetic toolkit opens a door for molecular characterization of Gardnerella and its mechanisms in BV.

Vaginal colonization by Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a major risk factor for ascending infections, preterm birth, and neonatal sepsis. Current GBS prevention efforts include routine GBS perinatal screening and intrapartum antibiotic prophylaxis, which decrease the rate of early-onset neonatal sepsis, but have drawbacks that include impacting the infant's developing microbiome. Lactobacillus-dominant vaginal microbiomes provide protection against pathogens such as GBS, and using probiotics as an antibiotic-free approach to limit GBS colonization is of increasing interest. In this study, we investigated the ability of Lactobacillus crispatus-loaded electrospun fibers to deliver live L. crispatus cells in an in vitro vaginal epithelial cell model, modulate GBS infection establishment and persistence, and alter vaginal cell inflammatory signaling. Our data demonstrate that electrospun fibers deliver viable L. crispatus to the surface of vaginal epithelial cells and that L. crispatus modulates vaginal cell inflammatory signaling by decreasing inflammatory IL-8 release and increasing anti-inflammatory IL-1RA secretion during established GBS infection. Treatment of pre-established GBS infection with electrospun fibers with or without L. crispatus decreased GBS burden at 24 hours, suggesting L. crispatus-dependent and -independent anti-GBS activity, and L. crispatus elicited an anti-inflammatory response via IL-1RA release. Overall, the data highlight the potential of electrospun fibers as a feasible probiotic delivery platform with antibacterial activity against GBS and which provides commensal lactobacilli capable of modulating host-pathogen interactions and inflammatory signaling of the vaginal epithelium.