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Deciphering Staphylococcus aureus-host dynamics using dual Activity-based protein profiling of ATP-interacting proteins

ABSTRACT: This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during the course of an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus, we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Staphylococcus Aureus

TISSUE(S): Keratinocyte, Macrophage

SUBMITTER: Stephen Dela Ahator

LAB HEAD: Mona Johannessen

PROVIDER: PXD049003 | Pride | 2024-04-22

REPOSITORIES: Pride

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			Action	DRS
	210602_SDAhator__quant_human.msf	Msf
	SDAhator_Bacteria_RPMI.msf	Msf
	SDAhator_Bacteria_RPMI.raw	Raw
	SDAhator_Bacteria_RPMI_2.msf	Msf
	SDAhator_Bacteria_RPMI_2.raw	Raw

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Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression

Project description:Staphylococcus aureus produces the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). A previous study reported that S. aureus mutants not capable of producing these compounds were less virulent in vivo through the deranged regulation of virulence factor genes. These findings, however, have been questioned as an unknown mutation in an operon that regulates virulence was discovered in the mutant strain. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. When administered to human keratinocytes, phevalin had no substantial effect on gene expression. Phevalin had no obvious impact on the extracellular metabolome of S. aureus. However, conditioned medium from S. aureus spiked with phevalin resulted in significant differences in keratinocyte gene expression compared to conditioned medium alone. A role for phevalin in manipulating host responses is apparent. Additionally, phevalin is a potential biomarker and/or therapeutic target for chronic, biofilm-based infections. Secreted factors from S. aureus biofilm and planktonic cultures with equivalent population sizes were placed in contact with human HaCaT keratinocytes for 4 hours. Keratinocytes were grown to ~90% confluency.

2012-10-01 | E-GEOD-32920 | biostudies-arrayexpress

Study of transcriptional effects of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line.

Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks. HaCaT were obtained from Cell Lines Service (Eppelheim, Germany) and grown in DMEM medium (PAN biotech, Aidenbach, Germany) supplemented with 10% FBS (Life Technology, Grand Island, NY, USA), L-glutamine and non-essential amino acid. Before the treatment HaCaT cells were cultured in serum-free medium for 12hrs. HaCaT were treated with MSU (1mg/ml) vs DMEM control for 12hrs then submitted to RNA extration and gene expression profiling. Triplicate experiments were performed: HaCaT control (n=3), MSU-treated (n=3).

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Project description:We employed human HaCaT cells as a model system to identify cellular proteins that accompany SDS-induced toxicity based on a proteomic approach. HaCaT human keratinocyte cell line were treated with a non-cytotoxic dose of SDS (25 µg/ml, as determined by the MTT assay and microscopically examination) for 48 h. The altered abundance of proteins from HaCaT keratinocytes exposed to SDS was analyzed by LC-MS/MS approach and quantified using Progenesis LC software. The abundance of 217 proteins (which were identified by multiple peptides, ≥ 2) was altered in keratinocytes exposed to SDS; in which 131 proteins had increased abundance while 86 proteins was down regulated. The Pathview map of 131 up-regulated proteins was built and enhancement of glycolysis/gluconeogenesis was found.

2017-11-03 | PXD007789 | Pride

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Aberrantly expressed genes in HaCaT keratinocytes chronically exposed to arsenic trioxide

Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5M-BM-5g/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide Two experimental groups: 1. The treatment group was sub-cultured up to passage 22 to establish a chronic exposure state. 2. The passage control group was also sub-cultured up to 22 passages but with no exposure to arsenic trioxide. 4 technical replicates with 3 replicates making a total of 8X3 =24 samples HaCat Cell untreated (passage control): 1. H1_H001, H1_H002, H1_H003 2. H2_ H004, H2_H005, H2_H006 3. H3_ H007, H3_H008, H3_H009 4. H4_ H010, H4_H011, H4_H012 HaCat Cell treated with 0.5M-BM-5g/ml of arsenic trioxide: 5. A1_H013, A1_H014, A1_H015 6. A2_H016, A2_H017, A2_H018 7. A3_H019, A3_H020, A3_H021 8. A4_H022, A4_H023, A4_H024 Cell Type: Human Skin Keratinocyte: 1.5 M-CM-^W105 HaCaT cells were cultured in 7.5 ml of complete DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin, streptomycin in T-25 culture plate. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 M-BM-:C. The treatment groups were exposed to 0.5M-BM-5g/mL As2O3 (equivalent to LC 0.5), and passaged at 90% confluent. Total RNA was extracted from 4 technical replicates of unexposed HaCaT cells and HaCaT cells chronically exposed to arsenic trioxide up to passage 22 using RNA STAT-60 (TEL-TEST, INC, Friendswood, TX, USA).

2012-04-11 | E-GEOD-37192 | biostudies-arrayexpress

Transcriptomic analysis by AmpliSeq Ion Torrent to explore the effects of the long non-coding RNA TGFB2-AS1 on gene expression in human keratinocytes

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