Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

The adenylate cyclase toxin (CyaA) of the whooping cough agent Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin (CD11b/CD18, CR3 or Mac-1). CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.

concentration 20 mM) for 30 min at RT in the dark. The excess of IAA was quenched by the addition of DTT to a final concentration of 20 mM and the reaction was left to proceed for 15 min at RT. Proteins were digested by trypsin (Promega) at a ratio 50:1 (w/w) at 37 °C overnight. Digestion was stopped by the addition of TFA to a final concentration of 1% (v/v) to precipitate SDC. Suspensions were mixed with an equal volume of ethyl acetate, vortexed and centrifuged. Upper organic phase was removed and the extraction process was repeated four times to completely extract SDC. Water phase containing peptides was then desalted on Discovery DSC-18 SPE cartridges (500 mg/3 mL; Sigma Aldrich) and the eluate in 80% ACN/0.1% TFA was vacuum dried.

Fractionation of peptides by hydrophilic interaction chromatography (HILIC)
3 to 5 mg of peptide material was injected onto TSKgel Amide-80 HR column (5 μm, 4.6 × 250 mm) with guard column (5 μm, 4.6 × 10 mm; both Tosoh Bioscience) using Alliance 2695 liquid chromatograph (Waters) under conditions of 20% mobile phase A (2% ACN/0.1% TFA) and 80% mobile phase B (98% ACN/0.1% TFA) at a flow rate of 0.5 mL/min. Peptide separation was performed by a linear gradient formed by mobile phase A and mobile phase B, from 80 to 60% of mobile phase B in 40 min and from 60 to 0% of mobile phase B in 5 min. Column temperature was kept constant at 30 °C, and the separation was monitored at 215 nm.

Liquid chromatography-mass spectrometry (LC-MS)
The Ultimate 3000 RSLCnano system connected with a Q Exactive mass spectrometer (Thermo Scientific) were used for instrumental analysis. Approximately one third of the sample material from each phosphopeptide fraction was introduced onto a trap column (PepMap100 C18, 3 µm, 0.075×20 mm) and peptides were separated by running a linear gradient (0.1% FA in water as phase A; 80% acetonitrile, 20% water and 0.1% FA as phase B) from 4 to 34% B in 48 min and from 34 to 55% B in 10 min, at a flow rate of 300 nL/min, using an analytical column (PepMap C18, 2 µm, 0.075×150 mm). The full MS/Top10 setup was used for mass spectra acquisition. The positive ion MS spectra from 350-1750 m/z range were obtained in the Orbitrap at a resolution of 70,000 (at m/z 200). Multiply charged precursor ions with minimal threshold intensity of 5×10 4 counts and not fragmented during previous 30s were admitted for higher energy collisional dissociation (HCD).
Tandem mass spectra were acquired with following settings; resolution at 17,500, AGC target value at 1×10 5 , maximum ion injection time at 100 ms, and normalized collision energy set to 27. Data acquisition was under control of Xcalibur software v3.0. was 20 ppm, taking the 12 most intensive peaks per 100 Da for search (with enabled possibility of co-fragmented peptide identification). Minimal Andromeda score for modified peptides was 40 and minimal delta score for modified peptides was 6. FDR filtering on peptide spectrum match was 0.01 with separate FDR filtering for each modification set to 0.01. For peptide quantitation, Arg+6 [ 13 C 6 ] and Lys+6 [ 13 C 6 ] were set as labels in heavy channel (or in light channel for label-swap experiments to obtain inverted H/L ratios) with re-quantify function enabled. Ratios for individual phosphosites were derived from normalized ratios of the least modified phosphopeptides in a given replicate.

Significance analysis by Global Mean Rank Test (GMRT) -First,
phosphosites classified as contaminants from MaxQuant search (based on the implemented database of contaminants in software) were filtered out. Significantly regulated phosphosites for given experimental condition (time point and toxin-/toxoidtreatment, Figure S1) were then found by GMRT using R package MeanRankTest 

Analysis of GO enrichment by:
o ClueGO plugin 5 -Parameters of enrichment test were as follows: GO Term interval 3 -8, minimum of 3 genes/3 % of all genes from the input. Two-sided hypergeometric test with Benjamini-Hochberg correction was performed, GO Term fusion was allowed and pV for found terms was set to be higher than 0.05.

Western blots
Cell lysis was performed in RIPA buffer (with 1mM EDTA, PhosSTOP and cOmplete Mini, EDTA-free Protease Inhibitor Cocktail (Roche)). Lysates from cells were then separated by SDS-PAGE and transferred to PVDF membrane -5% gel and wet transfer without methanol or SDS were used for mTOR analysis, 7.5% gel and semidry transfer were then used for analyzes of other proteins. Membranes were decorated with appropriate antibodies (dilution 1:5000 for β-Actin, 1:1000 for other antibodies) and visualized using Enhanced chemiluminescence (ECL) with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).

Ratio H/L normalized
Normalized ratios of heavy (control) vs. Light (treated) signal for particular phosphosite. For label-swap experiments (signed with "LS") are these values inverted. Rep1-3 indicates replicate number.

Intensity
Sum of all intensities (all signals from both light and heavy peptides) in particular replicate. Provides information about the strength of the signal.

Intensity log
Logaritmized (log10) Intensity value Localization prob Probability of correctly localized de/phosphorylation in particular peptide. Values > 0.75 are considered as correctly localized.

Ratio H/L nmods
Value indicating how many de/phosphorylations contained peptide, which was used for quantification of particular phosphosite.

Number of Phospho (STY)
Number of de/phosphorylations observed for peptide containing particular phosphosite.

WT-10msignificant
"+" sign indicates significantly regulated phosphosites according to MeanRank test (FDR = 0.05). Only phosphosites which were quantified in all three replicates were used.

WT-10m-q
Q value of particular phosphosite in the sample according to MeanRank test. Used to determine cut-off FDR 0.05, only phosphosites with q < 0.05 were considered significantly regulated.

Proteins
All possible proteins containing particular phosphosite.

Leading proteins
Proteins with the best coverage by identified peptides.

Protein
One chosen protein from the "Leading proteins" group.

Protein names
Name/s of the proteins bearing particular phosphosite.

Gene names
Gene name/s of the proteins bearing particular phosphosite.

Fasta headers
FASTA header/s of the protein/s containing particular phosphosite.

Amino acid
De/phosphorylated amino acid.

Positions within proteins
Phosphosite position/s in protein/s.

Positions
Phosphosite position/s in protein/s restricted only to proteins from "Leading proteins" group.

Position
Phosphosite position in protein from "Protein" column.

Sequence
Sequence containing particular phosphosite, including 15 amino window acids up and downstream.

Phospho (STY) Probabilities
Highlighted position of de/phosphorylation in peptide and its localization probability.
id Unique identifier or every phosphosite.   Relative phosphorylation