Global Protein-Protein Interaction Network in the Human Pathogen Mycobacterium tuberculosis H37Rv Yi Wang,† Tao Cui,† Cong Zhang, Min Yang, Yuanxia Huang, Weihui Li, Lei Zhang, Chunhui Gao, Yang He, Yuqing Li, Feng Huang, Jumei Zeng, Cheng Huang, Qiong Yang, Yuxi Tian, Chunchao Zhao, Huanchun Chen, Hua Zhang, and Zheng-Guo He* National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Received August 06, 2010
Analysis of the protein-protein interaction network of a pathogen is a powerful approach for dissecting gene function, potential signal transduction, and virulence pathways. This study looks at the construction of a global protein-protein interaction (PPI) network for the human pathogen Mycobacterium tuberculosis H37Rv, based on a high-throughput bacterial two-hybrid method. Almost the entire ORFeome was cloned, and more than 8000 novel interactions were identified. The overall quality of the PPI network was validated through two independent methods, and a high success rate of more than 60% was obtained. The parameters of PPI networks were calculated. The average shortest path length was 4.31. The topological coefficient of the M. tuberculosis B2H network perfectly followed a power law distribution (correlation ) 0.999; R-squared ) 0.999) and represented the best fit in all currently available PPI networks. A cross-species PPI network comparison revealed 94 conserved subnetworks between M. tuberculosis and several prokaryotic organism PPI networks. The global network was linked to the protein secretion pathway. Two WhiB-like regulators were found to be highly connected proteins in the global network. This is the first systematic noncomputational PPI data for the human pathogen, and it provides a useful resource for studies of infection mechanisms, new signaling pathways, and novel antituberculosis drug development. Keywords: Mycobacterium tuberculosis • PPI network • virulence • secretion • WhiB3
Introduction Mycobacterium tuberculosis, one of the most devastating human pathogens, is responsible annually for about 2 million deaths worldwide. One-third of the world’s population is currently infected with the tubercle bacillus.1 The seriousness of tuberculosis infection has become even more significant with the emergence of multidrug-resistant strains of M. tuberculosis, promoting a thorough investigation of this unique pathogen.2 Understanding the virulence and infection mechanisms of M. tuberculosis will require systematic identification of signal transduction systems utilized by this pathogen. One approach to the study of biological regulation at this level is through the construction of a global protein-protein interaction network. Protein-protein interactions (PPIs) are important in many biological processes, such as in the protein secretion or signal transduction. However, interaction data based on highthroughput experimental techniques are currently available for only a very few model organisms.3 Global PPI networks have been investigated with yeast two-hybrid (Y2H) systems in a number of models, including organisms such as Helicobacter * To whom correspondence should be addressed. E-mail:
[email protected] or
[email protected]. Tel.: +86-2787284300. Fax: +86-27-87280670. † These two authors equally contributed to this work. 10.1021/pr100808n
2010 American Chemical Society
pylori,4 Saccharomyces cerevisiae,5-7 Caenorhabditis elegans,8 Drosophila melanogaster,9 humans,10 Synechocystis sp.,11 Campylobacter jejuni,12 and, recently, Treponema pallidum.13 In addition, using a Y2H method, Rajagopala et al.14 studied the motility networks of T. pallidum and C. jejuni. The generation of large Y2H interaction maps has led to comprehensive understanding of the biological processes and functions of many uncharacterized genes. A similar study of protein-protein interactions in genetically intractable pathogens such as M. tuberculosis would be expected to improve the dissection of virulence pathways and to significantly advance our understanding of tuberculosis infection. Computer analysis has been used to model the construction of global PPI networks in M. tuberculosis.15,16 However, the M. tuberculosis genome encodes about 4000 ORFs, with one-third of these ORFs of unknown function. Therefore, previous studies on PPI networks in M. tuberculosis, based on bioinformatics and homology protein mapping, have actually covered only a very small percentage of the genome.17 These studies provide rather limited information, and one-third of the ORFs encoded by the bacterial genome remain uncharacterized. The definition of protein-protein interactions is now recognized as one of the most efficient approaches for mining these types of unknown function. Journal of Proteome Research 2010, 9, 6665–6677 6665 Published on Web 10/25/2010
research articles
Wang et al.
Figure 1. Construction and property of the M. tuberculosis PPI network. (A) The process of constructing large-scale M. tuberculosis protein-protein interaction networks. (B) Schematic representation of bacterial two-hybrid analysis in this study. (C) Global protein-protein interaction network views of M. tuberculosis. A graph of the pathogenic PPI network involving 2907 proteins linked via 8042 interactions. Several major protein families are indicated by different colors. (D) The degrees of top 10 highly connected proteins in the M. tuberculosis B2H PPI network. The numbers of PPIs are indicated on top of the respective columns. The functions of 10 proteins are shown on the right of the panel.
For the study of PPIs, the yeast two-hybrid system has come to the forefront as a powerful tool for PPI identification. However, the development of bacterial two-hybrid systems now allows proteins to be assayed for interactions under conditions that closely match the native environment of M. tuberculosis (Figure 1B).18-20 In the present paper, we show how we successfully constructed a global M. tuberculosis PPI network. We cloned almost the entire ORFeome of the pathogen and identified more than 8000 mostly novel interactions among the 2907 proteins. The overall quality of the PPI network was validated. Two WhiB-like transcriptional factors were also found to be highly connected proteins in the global network, indicating that these genes might be core regulators. The capacity for comprehensive screening of PPI in M. tuberculosis is expected to provide further information regarding infection mechanisms and to aid in identifying novel antitubercular drugs. 6666
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Materials and Methods Cloning of M. tuberculosis ORFeome into the Bacterial Two-Hybrid Plasmids. Primer pairs for a total of 3989 ORFs were designed, and each predicted ORF was amplified with a specific primer pair through PCR. PCR products were digested by corresponding restriction enzyme pairs or were ligated with an EcoRI-SmaI adaptor (TakaRa Biotech. Co.) if this type of enzyme site was lacking within the ORF sequences. The cleaned ORF fragments were then cloned into a pair of BacterioMatch@II vectors of pBT and pTRG vector (Stratagene). We carried out sequencing of the entire clone library to confirm that the ORFs were correctly amplified and cloned. All pTRGORF plasmids were then pooled, and from there, an ORF library for M. tuberculosis was constructed.
Protein-Protein Interaction Network in M. tuberculosis H37Rv @
Bacterial Two-Hybrid Analysis. The BacterioMatch II TwoHybrid System (Stratagene) was used to establish protein-protein interactions between M. tuberculosis proteins. Each pBT recombinant MtbORF was used as a bait plasmid to screen the pTRG-MtbORF library. The selective medium contained 8 mM 3-AT, 8 mg/mL of streptomycin, 15 mg/mL of tetracycline, 34 mg/mL of chloramphenicol, and 50 mg/mL of kanamycin. The plates were incubated at 30 °C for 2-4 days. All resulting positive clones were collected and stored for further use. The ORF in the recombinant plasmid pTRG-MtbORF was sequenced. The unredundant positive cotransformants were spotted onto selective medium to confirm the interactions. The plates were incubated at 30 °C for 2-3 days. The cotransformant containing pBT-LGF2 and pTRG-Gal11P (Stratagene) was used as a positive control for expected growth on the Selective Screening Medium. A cotransformant containing the empty vectors pBT and pTRG was also used as a negative control. SPR Assays. Surface Plasmon Resonance (SPR) analysis on a Biacore 3000 instrument (GE Healthcare) with NTA sensor chips was performed according to our previously published procedures.21 His-tagged M. tuberculosis protein was immobilized onto the nitrilotriacetic acid (NTA) chips. The other purified GST-tagged protein, to be used as the ligand, was diluted in the HBS buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 50 µM EDTA, 5 mM ATP, 0.005% BIAcore surfactant P20] at a concentration of
