Dataset Information

Phosphorylation of CFP10 modulates Mycobacterium tuberculosis virulence

ABSTRACT: Virulence effectors secreted by Mycobacterium tuberculosis (Mtb) help subvert host immune mechanisms and, therefore, are critical for establishing infection and pathogenesis. However, knowledge in terms of signaling mechanisms that modulate the secretion of virulence factors is sparse. We performed high-throughput secretome, phosphoproteome, and phospho-secretome analysis of Mtb. We combined the analysis with empirical validations to show regulation of mycobacterial secretion through protein phosphorylation. System level PPI network analysis superimposed with the secretome, phosphoproteome, and phospho-secretome profile revealed an intricate relationship between phosphorylation and secretion. At the core of the network was a key virulence factor CFP10. We identified PknA to be the kinase responsible for phosphorylating CFP10. Using genetic tools, we show that phosphomimetic mutation of CFP10 negatively regulates the secretion of virulence mediator ESAT6. Significantly, the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival within macrophages and mice. Together, the results show that the dynamic phosphorylation status of the secretory protein CFP10 regulates the secretion of virulence factors and impacts virulence.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Vinay Kumar Nandicoori

LAB HEAD: Vinay kumar

PROVIDER: PXD045586 | Pride | 2023-09-22

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

Project description:Intracellular small-molecule signaling has drawn increasing attention due to its importance in bacterial physiology and host-pathogen interactions. Here, we show that a new quinone-sensing transcriptional regulator, QsbR (quinone-stress sensing and bleomycin resistance regulator), is important for bacterial stress response and virulence in S. aureus. The mutant strain of qsbR exhibits dramatically reduced staphylococcal virulence in a murine abscess model of infection. Moreover, the qsbR mutant shows an accumulation of riboflavin and an increased resistance to quinone stress and bleomycin compared to the wild-type strain Newman. Microarray analysis confirmed that QsbR impacts the expression of genes that are involved in quinone stress response, bleomycin resistance, riboflavin biosynthesis, and pathogenesis. DNase-I footprinting and gel shift assays suggest that QsbR not only directly regulates transcription of genes involved in quinone detoxification and bleomycin resistance, but is also subject to autoregulation by binding to the palindromic consensus sequence GTATAN(5)TATAC in their promoter regions. This binding could be disrupted by quinone treatment in vitro. Mass spectrometric analysis showed that wild-type QsbR, but not QsbRC5S with Cys5 mutated to Ser, could be alkylated at Cys5 by quinone. Further mutagenesis studies revealed that Cys5 plays a key role in quinone sensing as substitution of Cys5 with Ser renders the bacterium more sensitive to quinone stress. Our results demonstrate that QsbR is a quinone-sensing regulator that dramatically impacts staphylococcal virulence and stress response. Sample preparation for transcriptional analysis. For each strain, 2 clones were inoculated in TSB and collected from mid-log or stationary phase. Appropriate volumes from each of the 2 cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (Qiagen). Cultures were centrifuged and cell pellets were stored at 4 ÂºC until RNA extraction. During the experiment, OD600 and viable cfu per ml were monitored for each of the cultures. This procedure was repeated 2 independent times to generate 2 samples at each time point for each strain.

Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

Dataset Information

Phosphorylation of CFP10 modulates Mycobacterium tuberculosis virulence

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure