The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis*

Fan-Lin Wu,Yin Liu,He-Wei Jiang,Yi-Zhao Luan,Hai-Nan Zhang,Xiang He,Zhao-Wei Xu,Jing-Li Hou,Li-Yun Ji,Zhi Xie,Daniel M Czajkowsky,Wei Yan,Jiao-Yu Deng,Li-Jun Bi,Xian-En Zhang,Sheng-Ce Tao

doi:10.1074/mcp.m116.065771

Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.

Highlights

EXPERIMENTAL PROCEDURESknown STPK-protein interactions (KPIs) Screening on the Mycobacterium tuberculosis (Mtb) Proteome Microarray—M. tuberculosis protein microarrays containing about 4200 full-length GST fusion proteins spotted in duplicate were used in this study [19]
It is known that these Serine/threonine protein kinases (STPKs) play critical roles in adaptation to various environmental conditions [6], cell wall synthesis [7], 1 The abbreviations used are: TB, tuberculosis; Mtb, Mycobacterium tuberculosis; STPK, serine/threonine protein kinase; known STPK-protein interactions (KPIs), STPK protein interaction; BLI, bio-layer interferometry; Y2H, yeast-two-hybrid; KSRs, kinase substrates relationships; PG, peptidoglycan; M. smegmatis (Msm), Mycobacterium smegmatis; ESI-MS, electrospray ionization mass spectrometry
The study was composed of 4 parts: preparation of the Mtb proteome microarray and the functional kinases (Fig. 1B), screening KPIs on the microarray and validation (Fig. 1C), bioinformatics analysis and KPIs map construction (Fig. 1D), and functional studies of select, newly discovered KPIs

Summary

EXPERIMENTAL PROCEDURES

KPI Screening on the Mtb Proteome Microarray—M. tuberculosis protein microarrays containing about 4200 full-length GST fusion proteins spotted in duplicate were used in this study [19]. The arrays were washed 3 times, 10 min each in 25 ml of PBST (1ϫ PBS at pH 7.4, 0.1% Tween-20) buffer at RT with shaking at 60 rpm. Protein Microarray Data Process Analysis—The process of data analysis includes four steps: [1] background correction, [2] normalization, [3] identification of positive hits, and [4] removal of nonspecific interactions. The grouped samples were concentrated to a protein adsorption film (PALL, Port Washington, NY), washed with 50 mM ammonium bicarbonate for 3 times at 4 °C centrifuged at 10,000 ϫ g for 20 min, and reduced with carboxyamidomethyl at 60 °C for 30 min. Mouse anti-flag antibody (Sigma-Aldrich, St. Louis, MO) was added to 2 mg of soluble lysate (1:100 dilution), and the mixture was incubated at 4 °C with shaking for overnight. Biofilm Formation—For biofilm cultures grown on liquid medium, 10 ml of biofilm medium with a modified version of M63 in a 90 ϫ 15 mm PVC Petri dish was inoculated with 10 ␮l of a saturated culture and incubated at 30 °C without disturbance [25]

RESULTS

DISCUSSION

DATA AVALIABILITY