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High-throughput screen of essential gene modules in Mycobacterium tuberculosis: a bibliometric approach

BMC Infectious Diseases201313:227

DOI: 10.1186/1471-2334-13-227

Received: 28 April 2012

Accepted: 15 May 2013

Published: 20 May 2013

Abstract

Background

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). The annotation of functional genome and signaling network in M. tuberculosis are still not systematic. Essential gene modules are a collection of functionally related essential genes in the same signaling or metabolic pathway. The determination of essential genes and essential gene modules at genomic level may be important for better understanding of the physiology and pathology of M. tuberculosis, and also helpful for the development of drugs against this pathogen. The establishment of genomic operon database (DOOR) and the annotation of gene pathways have felicitated the genomic analysis of the essential gene modules of M. tuberculosis.

Method

Bibliometric approach has been used to perform a High-throughput screen for essential genes of M. tuberculosis strain H37Rv. Ant colony algorithm were used to identify the essential genes in other M. tuberculosis reference strains. Essential gene modules were analyzed by operon database DOOR. The pathways of essential genes were assessed by Biocarta, KEGG, NCI-PID, HumanCyc and Reactome. The function prediction of essential genes was analyzed by Pfam.

Results

A total approximately 700 essential genes were identified in M. tuberculosis genome. 40% of operons are consisted of two or more essential genes. The essential genes were distributed in 92 pathways in M. tuberculosis. In function prediction, 61.79% of essential genes were categorized into virulence, intermediary metabolism/respiration,cell wall related and lipid metabolism, which are fundamental functions that exist in most bacteria species.

Conclusion

We have identified the essential genes of M. tuberculosis using bibliometric approach at genomic level. The essential gene modules were further identified and analyzed.

Keywords

Mycobacterium tuberculosis Essential gene modules Operon Pathway

Introduction

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) [1, 2]. In recent years, the prevention and treatment of TB have become difficult due to the prevalence of co-infection with HIV, drug resistance and uncertainty of Bacillus Calmette-Guérin (BCG) prevention [35]. Essential genes are those genes required for cell growth and survival [6, 7]. Previous studies on the essential genes of M. tuberculosis pathogenesis primarily using gene knockout or RNA interference [8]. This approach is expensive and inefficient, and due to limitations of experimental techniques, no experimental method can achieve an essential gene screen at a High-throughput level [9, 10]. Essential gene modules are a collection of functionally related essential genes in the same signaling or metabolic pathway [11]. The determination of essential genes and essential gene modules at genomic level may be important for better understanding of the physiology and pathology of M. tuberculosis, and also helpful for the development of drugs against this pathogen.

To date, more than 31 genomes of Mycobacterium spp. have been sequenced including nine M. tuberculosis strains [12]. However, the systematic analysis of functional genomics and metabolic regulation were not established in M. tuberculosis. In this study, we used a bibliometric approach and performed a High-throughput screening of five M. tuberculosis strains to identify the essential genes. We further analyzed the essential operons and pathways, based on early-established genomic operon database and annotation of gene locus [1315].

Material and methods

Bibliometric method

The bibliometric was used as previously described [16]. The keywords “Mycobacterium tuberculosis” “H37Rv” “essential gene” have been used to search the publications from 2002 to 2011 in PubMed, MEDLINE, BiosisPreview, EMbase and SciFinder. Using Epidata3.1, the duplications of literatures and unrelated literatures were deleted by parallel entry and logical error test. A total of 819 literatures were retrieved and 112 literatures were used to analysis the essential gene modules.

Ant colony algorithm

Multiple sequence alignment bases on BLAST algorithm was restricted by the length and number of the sequences. So, in this study, we used ant colony algorithm to optimize it [17, 18]. First, we divided all sequences into several parts and gain an initial population of K as a column, N as a row. K is the number of individuals of the K-substituting groups; N is the number of sequences. dij indicates the ith individual dividing positions on the jth sequence. For the array, the calculation formula of fitness(r) is as follow:
fitness r = i = 1 N - 1 j = i + 1 N SE S i p d ri , S j p d ri + i = 1 N - 1 j = i + 1 N SE S i s d ri , S j s d ri

The following parameters were used for analysis: the Initial = 5, d1 = 2, d2 = 2, d3 = 3, NCmax (The maximum number of iterations) = 100, m(number of ants) = 100; Parameters for information volatile degree are p = 0.05, q = 0.03, q1 = 0.6, q2 = 0.35, q3 = 0.2, a = 5, b = 3, c = 2, T1 = 50 , T2 = 79 , T3 = 99, Q1 = 0.1, Q2 = 0.2.

Results

Operons and pathways of screened essential genes in M. tuberculosis strains

The genome of the highly pathogenic M. tuberculosis strain H37Rv has been sequenced [19]. Using bibliometric analysis, 684 essential genes were identified in H37Rv strain, 617 genes were proved by experiments as well as 67 genes were identified using an in silico approach. (Related literature of these genes listed in Additional file 1: Table S1). These genes were evenly distributed in the genome, consistent with the previous study [20]. The genomes of M. tuberculosis among the five strains (H37Rv, H37Ra, CDC1511, F11, and KZN1435) were highly conserved. Therefore, we searched the Gene Bank (http://www.ncbi.nlm.nih.gov/genbank) using ant colony algorithm to look for essential genes of other reference strains (H37Ra, CDC1511, F11, and KZN1435). The essential genes of these strains were 702, 665, 699 and 697, respectively (Table 1). It was worth noting that the number of essential genes in the different strains are varies, although the genomes of M. tuberculosis are highly conserved. This phenomenon is mainly caused by the total number of genes in each stain are different, and some essential gene will divide into two genes in another genome, which caused the differences of essential genes.
Table 1

Essential genes, operons and pathway in reference strains H37Rv, H37Ra, CDC1511, F11, KZN1435

  

Operons

Pathways

Strains

Essential genes

Contain 1 essential gene

Contain more than 2 essential genes

Contain 1 essential gene

Contain less than 50 essential genes

Contain more than 50 essential genes

H37Rv

684

185(60%)

122(40%)

7(8%)

82(89%)

3(3%)

H37Ra

702

192(59%)

134(41%)

7(8%)

82(89%)

3(3%)

CDC1551

665

178(60%)

119(40%)

7(8%)

82(89%)

3(3%)

F11

699

186(60%)

124(40%)

7(8%)

82(89%)

3(3%)

KZN1435

697

185(59%)

131(41%)

7(8%)

82(89%)

3(3%)

We used operon database (DOOR) to assess the essential gene modules. These essential genes were fallen in 307, 326, 297, 310 and 316 operons in H37Rv, H37Ra, CDC1511, F11 and KZN1435, respectively (Table 1). Statistical analysis showed that there is no relationship between the size of the operon and the number of essential genes identified. In all strains, approximately 40% of operons are consisted of two or more essential genes and some operons controlled more than ten genes, suggesting that these operons may play an important role in physiology or pathogenesis. For example, the operon ID7843 controls eleven genes, nine of which are essential genes (Figure 1A). Of them, five genes are related to cell membrane, two genes encode protein related to PE/PPE family and another two genes encode hypothetical proteins.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-13-227/MediaObjects/12879_2012_Article_2471_Fig1_HTML.jpg
Figure 1

Essential gene modules operons and pathways. (A) An illustration for operon 7843. Non-essential genes are marked in red; PE/PPE family related genes are marked in green; possible conserved membrance genes are marked in yellow; hypothetical protein are marked in blue. (B) An illustration for histidine metabolism pathway. (C) An illustration for petidoglycan biosynthesis pathway.

Pathway is a signal transduction network that involves in multiple gene interaction. We analyzed the essential genes using pathway databases Biocarta, KEGG, NCI-PID, HumanCyc and Reactome. Although the numbers of essential genes in the five strains are slightly different, these essential genes have the same number of pathways (Table 1). The 684 essential genes of the H37Rv strain were distributed in 92 pathways. Of them, seven pathways only have one essential gene; 82 pathways have less than 50 essential genes; three pathways have more than 50 essential genes. It is interesting to note that in a portion of the pathway is entirely constituted by essential genes, which adjacent to each other in the genome. Histidine metabolism pathway, which is related to intermediary metabolism and respiratory, involves in ten essential genes. Seven of them are adjacent to each other and these clustered genes (Rv1599-1606) are required for L-histidine synthesis [21] (Figure 1B). Peptidoglycan synthesis pathway, which is related to cell wall and membrane formation, involves in ten essential genes (Figure 1C). Five essential genes (Rv2152-2157) are clustered together in the genome. Two of them are involved in N-acetyl muramic acid synthesis and the others for uridine monophosphate (UMP) synthesis [22]. We speculated that the linked genes are required for proper function and play crucial roles in pathways.

Function prediction of essential genes in M. tuberculosis

We used Pfam (http://pfam.sanger.ac.uk/) to predict the potential function of the essential genes by analyzing the functional domains of encoded proteins. The functions of the essential genes are categorized into replication, regulatory proteins, virulence, intermediary metabolism and respiration, cell wall related, signal pathways, lipid metabolism, PE/PPE family, insertion sequences/phages and unknown (Table 2). 61.79% of essential genes are fallen into virulence, intermediary metabolism/respiration, cell wall related, signal pathways and lipid metabolism. 0.06% of essential genes are fallen into replication, regulatory proteins, PE/PPE family, and insertion sequences/phages; 38.15% of essential genes into unknown.
Table 2

Function predictions of essential genes for reference strains H37Rv, H37Ra, CDC1511, F11, KZN1435

 

H37Rv

H37Ra

CDC1551

F11

KZN1435

Replication

10

10

10

10

10

(0.01%)

(0.01%)

(0.02%)

(0.01%)

(0.01%)

Regulatory proteins

10

10

10

10

10

(0.01%)

(0.01%)

(0.02%)

(0.01%)

(0.01%)

Virulence

288

293

284

292

291

(42.10%)

(41.74%)

(42.71%)

(41.77%)

(41.75%)

Intermediary metabolism and respiration

265

270

260

269

266

(38.74%)

(38.46%)

(39.10%)

(38.48%)

(38.16%)

Cell wall related

297

302

295

302

299

(43.42%)

(43.021%)

(44.36%)

(43.20%)

(42.90%)

information pathway

251

255

248

256

253

(36.70%)

(36.32%)

(37.29%)

(36.62%)

(36.30%)

Lipid metabolism

257

263

254

263

260

(37.57%)

(37.46%)

(38.20%)

(37.63%)

(37.30%)

PE/PPE family

11

14

8

14

13

(0.02%)

(0.02%)

(0.01%)

(0.02%)

(0.02%)

Insertion seqs and phages

12

13

10

13

12

(0.02%)

(0.02%)

(0.02%)

(0.02%)

(0.02%)

Hypothetical/unknown

261

269

255

269

268

(38.15%)

(38.31%)

(38.35%)

(38.48%)

(38.45%)

Discussion

In the current study, we have done a High-throughput screen for essential genes of M. tuberculosis. A total approximately 700 essential genes are identified in the genome, some genes were proved by experiments as well as some genes were identified using an in silico approach. We further identified the operons and pathways of these essential genes and predicted the functions of these genes.

The numbers of essential genes in the different strains are distinct suggesting that although the genome of M. tuberculosis is highly conserved, variations exist among different strains. The differences lead to the various capacities of virulence, evolution, and immunogenic among M. tuberculosis strains. Therefore, the investigations on the difference among essential genes in different strains probably gain insight the new mechanism of pathogenesis, especially between the virulent stain (H37Rv) and avirulent stain (H37Ra).

In our study, there were about 40% operons having two or more essential genes. Some operons have as much as ten essential genes. In the pathway analysis, some pathways are consisted of as much as 50 essential genes. At present, there is no any experimental methods can perform the scanning of essential genes aspect for M. tuberculosis. In order to further verify whether these identified genes are essential genes, we used pathway analysis to found that if multiple essential genes are adjacent to each other and constitute known essential pathway, we highly suspected these genes identified are essential, which is critical for drug or vaccine development. Histidine metabolism pathway and peptidoglycan synthesis pathway were found in this study base on pathway enrichment analysis,most genes in these two pathways were essential genes and adjacent to each other. In this case, in-depth studies of above two pathways maybe provide more broad perspective for the new drug development.

Function analysis revealed that 61.79% of essential genes were categorized into virulence,intermediary metabolism/respiration,cell wall related and lipid metabolism, which are fundamental functions that exist in most bacteria species [23, 24], however, insertion sequences, phages and horizontal transfer genes (HTG) are also founded. The function of insertion sequence in Mycobacterium tuberculosis are till obscure, and several literatures report that insertion sequences plays a vital role in the growth cycle, which are essential for the bacteria [25, 26]. The PE/PPE family is M. tuberculosis-specific and is involved in M. tuberculosis infection and virulence. PE/PPE genes accounted for 10% of M. tuberculois genome. Several essential genes that are related to PE/PPE family were also identified in this study, which plays an important role in cell wall synthesis [27].

Conclusion

In current study, we have identified the essential genes of M. tuberculosis using bibliometric approach at genomic level. The essential gene modules were further identified and analyzed.

Abbreviations

DOOR: 

Database of prOkaryotic OpeRons

KEGG: 

Kyoto Encyclopedia of Genes and Genome

UMP: 

Uridine monophosphate

Declarations

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81271897 and 81071424), National Basic Research Program of China (973 program, 2011CB512003), Specialized Research Fund for the Doctoral Program of Higher Education of China (20110061120093), China Postdoctoral Science Foundation (20110491311 and 2012 T50285), Foundation of Xinjiang Provincial Science & Technology Department (201091148), Foundation of Jilin Provincial Health Department (2010Z034 and 2011Z049), Norman Bethune Program of Jilin University (2012219), Fundamental of Jilin University Basic Research Program (2012ZKF06).

Authors’ Affiliations

(1)
Key Laboratory of Zoonosis, Ministry of Education, Norman Bethune College of Medicine, Jilin University
(2)
The First Bethune Hospital of Jilin University

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  28. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/227/prepub

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