biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsSören RobNextSeq 500RNA-seq of total RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15035In order to suppress the host immune system, numerous bacterial pathogens utilize a type 3 secretion system (T3SS) to inject effector proteins into target cells. We investigated Yersinia enterocolitica effectors (Yops) for their individual and combined effects on gene expression, inflammasome formation and Ca ++ signaling in primary human macrophages. The up- or down-regulation of thousands of macrophage genes by Yersinia inflammatory stimuli was effectively suppressed by YopP, and this correlated with suppression of histone-3 serine-10 phosphorylation. Surprisingly, YopM and YopQ counteracted selected YopP effects on gene expression. Inflammasome formation was reduced by a combination of YopP and YopQ, but not by any single effector or combinations of them. Finally, Ca ++ transients in infected macrophages were completely blocked by YopH alone. We conclude that the T3SS effectors of Yersinia antagonistically, synergistically or individually control major immune pathways of human macrophages to jointly suppress the host immune response.biostudies-arrayexpressSample Collection - Bacterial strains Yersinia enterocolitica strains used in this study are derivatives of the serotype O:8 strain WA314 harboring the virulence plasmid pYVO8. Several mutant strains (WA314ΔYopM, WA314ΔYopP, WA314ΔYopMP) were generated and used, as detailed in Table S1. Cell culture Human peripheral blood monocytes were isolated from buffy coats as described in Kopp et al., 2006. Cells were cultured in RPMI1640 with 20% autologous serum at 37°C and 5% CO₂. Medium was changed every three days until differentiation into macrophages by day 7. Infections were typically performed one week after isolation, except for one batch (Table S2), where cells were used after 2 weeks. Infection of cells The day before infection, the medium was changed to antibiotic- and serum-free RPMI1640. Y. enterocolitica strains were grown overnight in LB with antibiotics at 27°C. On the day of infection, cultures were diluted 1:20 in antibiotic-free LB, incubated for 90 min at 37°C to induce T3SS. Bacteria were pelleted, resuspended in PBS with 1 mM MgCl₂ and CaCl₂, and adjusted to OD600 = 3.6. Macrophages were infected at MOI 100, and centrifuged briefly to synchronize infection.Nucleic Acid Extraction - RNA-seq Sample Preparation Total RNA was isolated from 1–2 x 10⁶ infected human macrophages using the RNeasy extraction kit (Qiagen) including on-column DNase treatment, following the manufacturer's instructions. RNA integrity was assessed using RNA 6000 Nano Chip on an Agilent 2100 Bioanalyzer.Library Construction - mRNA enrichment was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina, following the manufacturer’s instructions. Library concentration was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Fragment size distribution was analyzed on an Agilent 2100 Bioanalyzer using the DNA High Sensitivity Chip. Final libraries were normalized to 2 nM and pooled equimolar.Sequencing - Sequencing was performed on an Illumina NextSeq500.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesSören RobIndra BekereMartin AepfelbacherJiabin HuangfalseSuppression of the key immune pathways in primary human macrophages by cooperation of Yersinia effectorsIn order to suppress the host immune system, numerous bacterial pathogens utilize a type 3 secretion system (T3SS) to inject effector proteins into target cells. We investigated Yersinia enterocolitica effectors (Yops) for their individual and combined effects on gene expression, inflammasome formation and Ca ++ signaling in primary human macrophages. The up- or down-regulation of thousands of macrophage genes by Yersinia inflammatory stimuli was effectively suppressed by YopP, and this correlated with suppression of histone-3 serine-10 phosphorylation. Surprisingly, YopM and YopQ counteracted selected YopP effects on gene expression. Inflammasome formation was reduced by a combination of YopP and YopQ, but not by any single effector or combinations of them. Finally, Ca ++ transients in infected macrophages were completely blocked by YopH alone. We conclude that the T3SS effectors of Yersinia antagonistically, synergistically or individually control major immune pathways of human macrophages to jointly suppress the host immune response.2025-04-30T00:00:00Z2025-04-15T13:18:51.134Z2025-04-15T13:18:51.134ZE-MTAB-15035ERP171656E-MTAB-14995EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0004184