Dataset Information

Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling.

ABSTRACT: Critical illness is characterised by organ failure, dysregulated immune activation and frequent secondary infections. Unbridled complement activation in these patients exposes immune cells to high levels of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of key immune cells, in particular the neutrophil, and this suppression is associated with poorer outcomes amongst critically ill adults. The intracellular signalling pathways which mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor peripheral blood neutrophils exposed to purified C5a demonstrated a prolonged defect in phagocytosis of the common nosocomial pathogen Staphylococcus aureus which persisted for 7 hours after exposure. Phosphoproteomic profiling of 2712 unique phosphoproteins identified persistent C5a signalling at 1 hour, and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A multi-function assay of bacterial ingestion and phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification in a whole blood model of Staphylococcal bacteraemia which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification, late bacterial ingestion and killing of S. aureus in whole blood. This study provides a deep phosphoproteomic assessment of human neutrophil signalling in response to S. aureus, and demonstrates how some of these pathways are disrupted by exposure to C5a, identifying a defect in phagosomal maturation and providing new information on mechanisms of immune failure in critical illness.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Neutrophil

SUBMITTER: Carmen Gonzalez Tejedo

LAB HEAD: Andrew Conway Morris

PROVIDER: PXD017092 | Pride | 2020-08-06

REPOSITORIES: Pride

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			Action	DRS
	010318_PR935_CAG_Set1_FP_RP_1.raw	Raw
	010318_PR935_CAG_Set1_FP_RP_10.raw	Raw
	010318_PR935_CAG_Set1_FP_RP_11.raw	Raw
	010318_PR935_CAG_Set1_FP_RP_12.raw	Raw
	010318_PR935_CAG_Set1_FP_RP_13.raw	Raw

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C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.

Wood Alexander Jt AJ Vassallo Arlette M AM Ruchaud-Sparagano Marie-Hélène MH Scott Jonathan J Zinnato Carmelo C Gonzalez-Tejedo Carmen C Kishore Kamal K D'Santos Clive S CS Simpson A John AJ Menon David K DK Summers Charlotte C Chilvers Edwin R ER Okkenhaug Klaus K Morris Andrew Conway AC

JCI insight 20200806 15

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a s ...[more]

PMID: 32634128

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