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Single cells RNA-Seq of CD45+ cells during Staphylococcus aureus craniotomy infection

ABSTRACT: Single cells RNA-Seq of CD45+ cells during Staphylococcus aureus craniotomy infection

PROVIDER: PRJNA642646 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12103752.fastq.gz	Fastqsanger.gz
	SRR12103753.fastq.gz	Fastqsanger.gz
	SRR12103754.fastq.gz	Fastqsanger.gz
	SRR12103755.fastq.gz	Fastqsanger.gz
	SRR12103756.fastq.gz	Fastqsanger.gz

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Single cells RNA-Seq of CD45+ cells during Staphylococcus aureus craniotomy infection

Project description:Single cells RNA-Seq of CD45+ cells during Staphylococcus aureus craniotomy infection

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Project description:Purpose: The goal of this study was to identify Staphylococcus aureus genes which are important during establishment of metastatic infections in bloodstream infections. Methods: Pooled mariner transposon mutant libraries were generated as previously described (PMID: 27185949). The libraries were sequenced on the Illumina HiSeq 2500 platform with the transposon-specific oligonucleotide primer Himar1-Seq. Illumina adapter sequences were removed via cutadapt version 1.2.1. The reads were filtered for size (>16 bp) and to contain the signature transposon inverted repeat. Reads were mapped to the Staphylococcus aureus 6850 genome (RefSeq accession NC_022222.1) by Bowtie2 v2.1.0. Identification of depleted and enriched mutants was performed via DESeq2 version 1.6.2. Results: S. aureus genes were identified which were upregulated or downregulated upon iduced expression of the LysR-type transcriptional regulator RSAU_000852 in Staphylococcus aureus 6850. Conclusions: Our study identifies a gene for a LysR-type transcription regulator, which is involved in metabolic adaptation of the pathogen during colonization of secondary infection sites in a bloodstream infection model.

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Project description:Young adult fer-15;fem-1 Caenorhabditis elegans were infected with Staphylococcus aureus for 8 h to determine the transcriptional host response to Staphylococcus aureus. Analysis of differential gene expression in C. elegans young adults exposed to two different bacteria: E. coli strain OP50 (control), wild-type Staphylococcus aureus RN6390. Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Keywords: response to pathogen infection, innate immunity, host-pathogen interactions

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Project description:Since infectious diseases caused by Staphylococcus aureus are still a major threat for human health we aimed to gain a detailed understanding of the molecular interplay of pathogen and host upon internalization by proteome analyses. In the present study we infected human alveolar epithelial A549 cells with S. aureus HG001 pMV158GFP and separated intact bacteria from host cell debris or infected from non-infected A549 cells prior to proteome analysis by cell sorting thus facilitating detailed analyses. During the first 6.5 h in the intracellular milieu S. aureus displayed reduced growth rate, induction of the stringent response, and adaptation to microaerobic conditions as well as cell wall stress. Interestingly, both truly infected host cells and those only potentially exposed to secreted S. aureus proteins but not infected displayed differences in the proteome pattern compared to A549 cells which had never been in contact with S. aureus. However, adaptation reactions were more pronounced in infected compared to non-infected A549 cells. Additional cytokine measurements revealed elaborated levels of pro-inflammatory cytokines in supernatants of the infection setting compared to pure host cell cultures which might mediate communication among the host cells and trigger adaptation even in cells not infected with S. aureus.

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CD4+ T cell-innate immune crosstalk is critical during Staphylococcus aureus biofilm infection [CD3+ scRNA-seq]

Project description:Access to the brain for treating neurological sequalae requires a craniotomy, which can be complicated by infection. T cells preferentially home to the brain, but not other tissue sites, during Staphylococcus aureus (S. aureus) craniotomy infection; however, their functional importance is unknown. CD4+ T cells were critical for bacterial containment during craniotomy infection as Rag1-/- mice and WT animals treated with anti-CD4 or VLA-4 and LFA-1 antibodies exhibited elevated bacterial burdens. scRNA-seq revealed transcriptional heterogeneity in brain CD3+ infiltrates, with CD4+ cells most prominent and typified by both Th1 and Th17 signatures, and adoptive transfer of either subset in Rag1-/- mice prevented S. aureus outgrowth. scRNA-seq revealed a profound IFN-γ signature in innate immune cells from Rag1-/- mice during craniotomy infection, supporting extensive T cell-innate immune crosstalk that was validated by immunostaining in the brain parenchyma. A cooperative role for Th1 and Th17 driven responses was demonstrated by treatment of Ifng-/- mice with IL-17A/F neutralizing Ab that recapitulated phenotypes observed in Rag1-/- animals, which were not observed following the loss of either cytokine alone. Collectively, these results implicate a critical role for CD4+ T cells in S. aureus containment during craniotomy infection by shaping the innate immune landscape.

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