(D) Cells were infected at MOI = 10 for 2 hours and incubated in the absence, to measure adherent bacteria, or presence of gentamicin, to measure invading bacteria, and harvested at 3 hpi

(D) Cells were infected at MOI = 10 for 2 hours and incubated in the absence, to measure adherent bacteria, or presence of gentamicin, to measure invading bacteria, and harvested at 3 hpi. with WT PAO1. Number of surviving intracellular bacteria were determined at 24 and 48 hpi. Data shown is representative of three independent experiments.(TIF) ppat.1009534.s002.tif (1.2M) GUID:?C637DE57-36AE-493C-A3B5-BA12675BC946 S3 Fig: strain does not have a general fitness defect. (A, B) Growth of WT and was followed at 37C in either LB (A) or M9 minimal medium (B) by measuring OD600. (C) Antibiotic susceptibility to gentamicin (GM), levofloxacin (LVX), azithromycin (AZM), amikacin (AN), colistin (CL), tobramycin (NN), ciprofloxacin (CIP), imipenem (IMP) and ceftazidime (CAZ) was determined by disc diffusion. Average zone of inhibition for three discs is shown. (D) Survival of WT and after exposure to 25M NaOCl in PBS + 10mM glucose determined by plating for CFU.(TIF) ppat.1009534.s003.tif (1019K) GUID:?0FC213EE-D7CA-4FC6-B686-9917EEED2588 S4 Fig: Expression of and operon during log phase by RNAseq. RNA-seq libraries were made of WT, and D54N and D54E point mutants grown in LB during logarithmic phase. Transcripts per million (TMPs) for (A) gene and (B) the sum of the genes in the operon (and inducers and and two-component regulator controlling quorum sensing and needle component at MOI = 10. At indicated time points the number of intracellular bacteria was determined. (C) Transcripts per million (TMPs) for genes encoding TNF and its receptors, TNFRSF1A and TNFRSF1B, from the indicated sorted populations. Each data point represents a biological replicate.(TIF) ppat.1009534.s007.tif (2.1M) GUID:?47876566-C27C-4B55-B924-5BAD92BADC26 S1 Table: PatH-Cap enrichment enables analysis of invaded cells. Dual RNAseq libraries were made with the scDualseq protocol and were sequenced before and after enrichment of mRNA derived transcripts with PatH-Cap. Percentage of total aligned reads that mapped to the genome and specifically to mRNA are shown. *of total aligned reads (host + bacteria aligned).(DOCX) ppat.1009534.s008.docx (14K) GUID:?B80702A9-7A61-4BEC-AA5F-F509975D1592 S2 Table: Oligonucleotides used. Rabbit Polyclonal to GPR174 Primers used for in-frame nonpolar deletions, complementation and site-directed mutagenesis.(DOCX) ppat.1009534.s009.docx (16K) GUID:?2EB1452F-E7B4-41F2-8378-55103F455A36 S1 Dataset: Bacterial transcriptional analysis of infection. Invaded GFP positive (inv) cells were sorted 2 hours post-infection with WT or PAO1 GFP-expressing bacteria. RNAseq libraries Cilomilast (SB-207499) were made using the scDualseq protocol and enriched for mRNA using PatH-Cap. Libraries from log phase planktonic culture grown in Cilomilast (SB-207499) LB (log) were made using the RNAtag-Seq protocol. Raw counts, and DESeq2 analysis (log2 fold change and pajd) shown for intracellular WT vs intracellular bacteria and intracellular vs log phase bacteria.(XLSX) ppat.1009534.s010.xlsx (720K) GUID:?E608F36C-1F54-4175-B1D3-AB8B6A249A36 S2 Dataset: Host transcriptional analysis of P. aeruginosa infection. Unexposed (unexp), exposed but uninfected GFP negative (exp) and invaded GFP positive (inv) cells were sorted 2 hours post-infection with WT or PAO1 GFP-expressing bacteria. RNAseq libraries were made using the SMARTer protocol. Raw counts and DESeq2 analysis (log2 fold change and pajd) shown for various comparisons.(XLSX) ppat.1009534.s011.xlsx Cilomilast (SB-207499) (2.8M) GUID:?9F0F7838-1B02-4E53-9941-B053BA5B97DA Data Availability StatementAll sequencing files are available from the Sequence Read Archive database (SRA accession numbers PRJNA574611, PRJNA579143, PRJNA580232). Abstract Long-term survival of bacterial pathogens during persistent bacterial infections can be associated with antibiotic treatment failure and poses a serious public health problem. Infections caused by the Gram-negative pathogen into mammalian cells have been described, the subsequent fate of internalized bacteria, as well as host and bacterial molecular pathways facilitating bacterial long-term survival, are not well defined. In particular, long-term survival within bladder epithelial cells has not been demonstrated and this may have important implications for the understanding and treatment of UTIs caused by inside bladder epithelial cells and a mutant with a disruption in the bacterial two-component regulator AlgR that is unable to survive intracellularly. Using simultaneous dual RNA-seq transcriptional profiling, we define the transcriptional response of intracellular bacteria and their corresponding invaded host cells. The bacterial transcriptional response demonstrates that WT bacteria rapidly adapt to the.