Dataset Information

Affinity-purification mass spectrometry on i) streptolysin O or C5a peptidase with human plasma or saliva; ii) human plasminogen with streptococcal proteins

ABSTRACT: In this project, we tried to use affinity-purification mass spectrometry (AP-MS) to study host-pathogen protein interaction, focusing initially on two major virulence factors of group A streptococcus (GAS), streptolysin O (SLO) and C5a peptidase (C5AP) with the immediate host microenvironment (human plasma or saliva); next centering on one major pathogenic host factor of GAS, human plasminogen (PLG) molecule with streptococcal cell-wall associate proteins (CAP) or secreted proteins (SP). Quantitative MS was performed in combination with interaction analysis to pinpoint the high-confidence interactive partner to the corresponding baits.

INSTRUMENT(S):

ORGANISM(S): Streptococcus Pyogenes Mgas315 Streptococcus Pyogenes M1 Gas Homo Sapiens (human)

TISSUE(S): Blood Plasma, Saliva

SUBMITTER: Di Tang

LAB HEAD: Johan Malmström

PROVIDER: PXD069402 | Pride | 2026-03-16

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	CAP_f01_a_DTDNH_P2306_043.raw	Raw
	CAP_f01_b_DTDNH_P2306_044.raw	Raw
	CAP_f02_a_DTDNH_P2306_046.raw	Raw
	CAP_f02_b_DTDNH_P2306_047.raw	Raw
	CAP_f03_a_DTDNH_P2306_049.raw	Raw

Items per page:

1 - 5 of 112

Publications

Rapid Proteome-Wide Discovery of Protein-Protein Interactions With ppIRIS.

Piochi Luiz Felipe LF Tang Di D Malmström Johan J Karami Yasaman Y Khakzad Hamed H

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20260312

Protein-protein interactions (PPIs) are central to cellular processes and host-pathogen dynamics across all domains of life, yet comprehensive interactome mapping remains challenging at the proteome scale. Experimental approaches provide only partial coverage, while existing computational methods often lack generalizability across species or are too resource-intensive for large-scale screening. Here, we introduce ppIRIS (protein-protein Interaction Regression via Iterative Siamese networks), a l ...[more]

PMID: 41816949

Similar Datasets

Project description:The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [spy0874/0875, herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (DeltasptR) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the DeltasptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies. We determined the transcriptome of SptR (spy0874) in human saliva by comparing expression microarray date for parental wild-type strain MGAS5005 and its delta-spy0874 isogenic mutant strain. 4 replicates of each strain were grown to mid-exponential and stationary growth phases in human saliva. RNA was isolated, converted to cDNA, labeled and hybridized to a custom-made Affymetrix GeneChip. Data was analyzed using GCOS.

Project description:We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis - GRaMS (Growth Rate Modeling of Specificity) - that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well characterized C2H2 zinc finger transcription factor on both a 28bp randomized library for the standard B1H method and on 6bp randomized library for the CV-B1H method for which forty-five different experimental conditions were tested: 5 time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media. All selections were performed using the bait transcription factor Zif268. 45 different B1H selections were performed on plates and the selected sites were multiplexed and sequenced in a single lane. A single selection was performed in liquid media and sequenced along with other multiplexed sets of sites in a separate lane. For the experiments performed on plates, the of the selections assays was varied (4, 8, 12, 18, 24 hours). The stringency of the selections was also varied by changing the concentration of IPTG (0, 10, 50 uM) and 3-AT (0.5, 1, 2 mM). The initial randomized library used for all selections was also sequenced in a single lane. The randomized portion of each transcription factor binding site is 6bp long and is immediately 5' of the constant sequence, GCGG, which is the consensus binding site for finger 1 of Zif268. Upstream of the randomized binding site is a 4bp randomized region referred to as the key region. The purpose of the key region is to detect possible PCR 'jackpotting.' The single liquid media experiment was run for 4 hours using 50uM IPTG and 5mM 3-AT. Four selections were performed using a prey library with a 28bp long randomized region and not fixed consensus site region. These selections were performed at either 36 or 48 hours using 10uM IPTG and 0, 2, or 5 mM 3-AT.

Project description:In many metazons, such as humans and Drosophila, homeodomain proteins comprise the second largest family of sequence specific transcription factors. In Drosophila, homeodomains play an important role in development. Many homeodomain proteins display a high level of homology across metazons, presumably due to importance of their functional roles. We comprehensively characterized the DNA binding preferences of all 84 Drosophila homeodomain transcription factors which contain a single DNA binding domain. Previously, we employed a bacterial one hybrid (B1H) assay to select for 20 to 40 high affinity transcription factor binding sites [Noyes et al. (2008). Cell. 133(7):1277-1289]. In this system, E. coli are transfected with two plasmids. One plasmid encodes the DNA binding domain of a homeodomain fused to two zinc finger domains and the omega subunit of RNAP. The other plasmid is drawn from a library of prey plasmids which contain a 10bp randomized transcription factor binding site (TFBS) region in the promoter of the reporter gene His3. The E. coli strain used is a His3 homolog and omega subunit knock out strain. If a transcription factor has high affinity for a TFBS, more His3 will be produced, leading to the production of more histidine and an increase in the growth rate. If the transcription factor does not bind with sufficient affinity to the TFBS, little or no histidine will be produced resulting in little or no growth. The stringency of the B1H system can be tuned using the chemicals IPTG and 3-AT. IPTG induces production of the chimeric transcription factor, and 3-AT is a competitive inhibitor of the enzyme encoded by His3. One of the advantages of the B1H system is that the transcription factor does not have to be purified and that many experiments can be easily conducted in parallel. In this study, in stead of picking 20 to 40 colonies and sequencing their TFBSs, we used high-throughput Illumina sequencing to sequence the selected sites of all of the colonies growing on a plate. This provided quantitative data regarding the growth rate of cells possessing each selected TFBS variant, which is a function of the affinity of the transcription factor for the binding site. With this quantitative data, we can build more accurate models of transcription factor binding. All 84 of the Drosophila homeodomain proteins that contain a single DNA binding domain were analyzed using the B1H assay. The same selection stringency was used for all experiments (10uM IPTG and 5mM 3-AT). All experiments were run for 36 to 48 hours. 10 mutants were also assayed: 3 Caup, 3 Bcd and 4 En mutants. The bait plasmid omegaUV2zf was used in all but 3 cases. In this instances, a slightly different bait plasmid, omegaUV5zf, with a stronger promoter was used. Thirty different replicates were performed in order to insure that sufficient number of reads were obtained for each protein. In total, 126 experiments were performed.