Dataset Information

Proteome-wide identification of in vitro substrates for P. aeruginosa ClpXP

ABSTRACT: we treated the cell lysates of ΔclpX mutant with or without P. aeruginosa ClpXP protease and used Label-free quantitative techniques (LFQ) to identify and quantify the proteolytic targets of ClpXP (p.aeruginosa PAO1). As a result, 3338 proteins were identified and 2493 proteins were quantified. The abundance (LFQ intensity) of 44 proteins were decreased (≥1.5-fold, p < 0.05) by the treatment with ClpXP. Moreover, we observed that there are 31 proteins which have valid LFQ intensity values in all the three biological replicates of the control group but not in the experimental group (i.e., ClpXP-treated). These results suggest that exogenously added ClpXP has a profound effect on the abundance of proteins in our in vitro assays and that those 75 proteins may serve as potential substrates for the P. aeruginosa ClpXP.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Pseudomonas Aeruginosa Pao1

SUBMITTER: Nana Yang

LAB HEAD: Lefu Lan

PROVIDER: PXD014928 | Pride | 2020-04-29

REPOSITORIES: Pride

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Publications

Alteration of protein homeostasis mediates the interaction of Pseudomonas aeruginosa with Staphylococcus aureus.

Yang Nana N Cao Qiao Q Hu Shuyang S Xu Chenchen C Fan Ke K Chen Feifei F Yang Cai-Guang CG Liang Haihua H Wu Min M Bae Taeok T Lan Lefu L

Molecular microbiology 20200508 3

Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co-isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum ...[more]

PMID: 32323346

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Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 ÂµM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.

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