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Profiling of the Cellular Proteome and Phosphoproteome of Klebsiella pneumoniae under Zinc Limited and Zinc Replete Conditions

ABSTRACT: Klebsiella pneumoniae is a bacterial pathogen that causes nosocomial infection in humans and is acquiring antibiotic resistance at an alarming rate. This study investigates the effect of zinc limitation on the phosphoproteome of K. pneumoniae using quantitative mass spectrometry to provide insight into the cell signaling methods used to respond to nutrient limited conditions like those experienced when colonizing a host.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Klebsiella Pneumoniae Subsp. Pneumoniae

TISSUE(S): Cell Suspension Culture

SUBMITTER: Chelsea Reitzel

LAB HEAD: Dr. Jennifer Geddes-McAlister

PROVIDER: PXD041015 | Pride | 2023-03-23

REPOSITORIES: Pride

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Project description:Mycoplasma pneumoniae is a significant cause of pneumonia, particularly in children, the elderly and the immunocompromised, and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model pathogen where extensive ‘omics’ studies have been conducted to gain insight into how minimal genome self-replicating organisms function. Here we conducted an N-terminome study to determine which proteins are targets of processing events in M. pneumoniae. We identified 6,973 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Nearly half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events which predominately target negatively charged residues in P1′ (D and E) with lysine or serine/alanine in P2′ and P3′ positions. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Mycoplasma pneumoniae culture conditions The M. pneumoniae (strain M129) cells were cultured in modified Hayflick’s medium at 37 °C in tissue culture flasks using established culture conditions63. Modified Hayflick’s medium contained 21 g PPLO broth base without crystal violet, 5 g of D-glucose, 4 mL of 0.5% phenol red, 100 mL of liquid yeast extract (150 g/L), 200 mL heat-inactivated horse serum (56 °C, 30 min) supplemented with 1 g ampicillin (Sigma, A5354, St. Louis, MO, USA) per litre. M. pneumoniae cells were harvested at mid-log phase by washing adherant cells 3 times with PBS and collected using cell scaping.

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Profiling of the Cellular Proteome and Phosphoproteome of Klebsiella pneumoniae under Zinc Limited and Zinc Replete Conditions

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