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Correspondence: Kanglin Wan, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing 102206, China. Tel./fax: +86 10 58900779; e-mail: email@example.com
Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. A previous study showed that human T-cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 173 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Rv2945c and Rv0309, and compared the sequences. The results showed that genetic diversity existed in these two genes among the MTBC strains and two single nucleotide polymorphisms (SNPs) presented higher polymorphisms. Antigen Rv2945c harbored a higher number of amino acid substitutions of its T-cell epitopes, which may reflect ongoing immune evasion. In addition, the high dN/dS value of Rv0309 suggested antigen Rv0309 might be involved in diversifying selection to evade host immunity. Finally, a small group of strains were identified based on the genetic diversity of these two genes, which might indicate that they interact differently with human T cells compared with other strains.
Tuberculosis (TB) is a major infectious disease with a worldwide prevalence. About one-third of the world population is infected with Mycobacterium tuberculosis, 9.3 million people develop active TB, and 1.8 million people die of TB each year (Donald & van Helden, 2009). The current efforts to reduce the global problem have been focused on improved diagnosis and effective vaccines. Biochemical, immunological, and molecular biological characterization of M. tuberculosis has led to the identification of several antigens that may be useful in the development of improved diagnostic methods and/or vaccines (Young et al., 1992).
Lppx (Rv2945c) is a secreted lipoprotein common to Mycobacterium bovis, BCG, M. tuberculosis and Mycobacterium leprae. Secreted proteins like Rv2945c represent a distinct group in mycobacteria and are considered to be important for the development of immune responses following infection (Andersen, 1997; Harboe & Wiker, 1998). Some studies have suggested that the mature protein of Rv2945c is an exported lipoprotein antigen and serves as protective antigen (Mustafa, 2001, 2002). Rv0309 is a hypothetical protein in M. tuberculosis genome and its function is unknown and has been rarely studied. It harbors two T-cell epitopes (Ernst et al., 2008) which suggests it may play some role in the reaction between M. tuberculosis and human T cells.
Materials and methods
A total of 173 clinical isolates were selected from 2346 MTBC strains isolated in Beijing Municipality and 12 provinces and autonomous regions (Table 1) in China, which were genotyped by spoligotyping in a previous study (Dong et al., 2010). Strains belonging to all major and rare spoligotypes in China were included. Considering the predominance of the Beijing family strains in China, about half of the strains we chose were of the Beijing family (86) and half of non-Beijing family strains (87). We randomly selected the 86 Beijing family strains from 1738 Beijing strains among 2346 strains. The remaining 87 strains were selected from 608 non-Beijing family isolates. Furthermore, we attempted to include strains representing different spoligotypes that were isolated from different places. The spoligotype patterns of 173 stains are shown in Fig. 1, which contained 86 Beijing family strains, 12 T family strains, 27 U family strains, 11 MANU strains, five Haarlem strains, two EAI strains, two LAM strains, one H37Rv family strain, two BCG, one S strain, four CAS strains and 20 other strains belonging to 18 different new spoligotypes.
Table 1. Number of strains from different provinces of China
No. of isolates
Guangxi Zhuang Autonomous Region
Xizang (Tibet) Autonomous Region,
Xinjiang Uygur Autonomous Region
Culture and PCR conditions
The strains were cultured using a standard Löwenstein–Jensen medium method, heat-inactivated and then used directly in polymerase chain reactions (PCRs).The nucleotide sequences of the primers (from 5′ to 3′ end) used in this study were designed with dnastar software according to H37Rv genome sequence and were as follows: 5′-ACAGCTACCGGCTCAAAGAC-3′ and 5′-TCGACTCATTAACGGCTGTG-3′ for Rv2945c; 5′-GACCACACGAGGTGATTGTC-3′ and 5′-TGAATCTCACGACGCAACA-3′ for Rv0309. The PCR were performed in a total volume of 20 μL. The PCR mix contained 10 μL PCR buffer, 100 nM of each primer, 200 μM each of the four dNTPs and 0.5 U DNA Taq Polymerase (Takara). An initial denaturation of 5 min at 94 °C was followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 62 °C for 45 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Negative controls (reagents only, no DNA) were included each time when the PCR was performed. The positive control was 500 pg DNA from M. tuberculosis H37Rv. The presence and size of each PCR product were determined by electrophoresis on 2% agarose gel in Tris-boric acid-EDTA buffer followed by staining with ethidium bromide. We performed all of the PCRs at least twice to validate the reproducibility. The sequences of the PCR products were determined with an ABI 3730xl DNA Analyzer. The sequences were compared and sliced by bioedit software. A neighbor-joining phylogenetic tree was constructed by mega5 software. Values of dN and dS were calculated by mega5.
We have amplified the genes encoding Rv2945c and Rv0309 and compared the sequences. All 173 strains yielded PCR products of antigen Rv2945c and Rv0309. There were four nonsynonymous mutations and one synonymous mutation in Rv2945c, of which AA position 152 displayed higher polymorphism (Table 2). In Rv0309, three nonsynonymous mutations and one synonymous mutation were found and AA position 159 presented higher polymorphism. Nine strains with high number of polymorphisms at position 152 in Rv2945c and position 159 in Rv0309 included three T family strains (AH03009, FJ05395 and HuN06026), five U family strains (ShanX05290, XJ06183, GX06043, GX06130, and XJ06116) and one new spoligotype strain (HuN06101). Therefore, the two SNPs that are acting as phylogenetic markers for the strains belong to the same spoligotype. For strains in different spoligotypes, i.e. non-closely related strains, we had one homoplastic SNP (convergent evolution), which usually is a strong indicator of selection.
Table 2. Base changes, mutation type and relative strains in antigen Rv2945c and Rv0309a
The CDS of Rv2945c and Rv0309 of M. tuberculosis H37Rv strain has been used as the reference sequence.
Fifteen T-cell epitopes in the antigen Rv2945c and two in Rv0309 (Table 3) were observed in the Immune Epitopes Database (IEDB; Ernst et al., 2008). Six of 15 epitopes contained in Rv2945c were altered, of which two harbored two amino acid changes and the other four had one amino acid change. One of two T-cell epitopes of Rv0309 had one amino acid change.
Table 3. Amino acid changes of the T-cell epitopes included in antigen Rv2945c and Rv03098*,†
The dN/dS value of Rv2945c was 0.19 (Table 4), lower than 1, suggesting that purifying selection appears to be the driving selective pressure on this gene. The dN/dS value of Rv0309 reached 3.69, suggesting it might be involved in diversifying selection to evade host immunity. For Rv2945c, there were no non-epitope regions in the gene, as the 15 T-cell epitopes covered all of the gene sequence. This also indicates the importance of antigen Lppx for the development of T-cell immune responses following infection. For Rv0309, we calculated the dN/dS of the epitope and non-epitope regions (Table 4). In Rv0309, the dN/dS of the epitope region was higher than in the non-epitope region, which means the former have accumulated significantly more amino acid changes than the latter.
Table 4. dN/dS values of two genes in the epitope and non-epitope region, respectively
There are no non-epitope regions in Rv2945c, as the 15 T-cell epitopes cover all of the gene sequence.
There are no synonymous mutations in the epitope region of Rv0309.
In this study, we chose 173 clinical MTBC strains in China which originated from a very large geographical area and had different spoligotyping patterns; the data provided by them could therefore be considered representative of genetic diversity present within China.
Host–pathogen co-evolution is characterized by reciprocal adaptive changes in interacting species (Andersen, 1997). Host immune pressure and associated parasite immune evasion are key features of this process, often referred to as an ‘evolutionary arms race’ (Dawkins & Krebs, 1979; Brunham et al., 1993). Mycobacterium tuberculosis is the most successful pathogen with multiple mechanisms to subvert the host immune response, resulting in insidious disease. Studies in human pathogenic viruses, bacteria, and protozoa have revealed that genes encoding antigens tend to be highly variable as a consequence of diversifying selection to evade host immunity (Farci et al., 2000; Urwin et al., 2004; Jeffares et al., 2007; Kawashima et al., 2009). Comas et al. (2010) have reported that human T-cell epitopes of M. tuberculosis are evolutionarily hyperconserved and deduced that M. tuberculosis lack antigenic variation and immune evasion. Rv2945c and Rv0309 are both hypothetical proteins in the M. tuberculosis genome and may play some role in the reaction between T cells and M. tuberculosis. Here, we found genetic diversity existed in these two antigens and two SNPs presented higher polymorphisms. The dN/dS value of Rv0309 reached 3.69, suggesting that the gene may undergo antigenic variation in response to host immune pressure and may be involved in diversifying selection to evade host immunity. There are 15 experimentally confirmed T-cell epitopes included in Rv2945c and two in Rv0309. Six of the 15 epitopes contained in Rv2945c were altered, two harboring two amino acid changes, and the other four, one amino acid change. The higher number of amino acid substitutions in T-cell epitopes of Rv2945c may reflect ongoing immune evasion. Further investigation is needed to determine whether the observed changes are due to immune pressure, other selection pressure(s) or merely random genetic drift.
FJ06051 and FJ05406, belonging to EAI strains according to spoligotyping, presented a mutation in position 143 (S-A) of antigen Rv0309, which may represent a special mutation in EAI strains.
Based on the genetic diversity of Rv2945c and Rv0309, 173 strains were clustered into two groups (see Fig. 2), the smallest of which consisted of nine strains. These nine strains included three T family strains (AH03009, FJ05395, and HuN06026), five U family strains (ShanX05290, XJ06183, GX06043, GX06130, and XJ06116) and one new spoligotype strain (HuN06101). These nine strains had the same mutations in Rv2945c and Rv0309 (Table 2). The mutation in Rv2945c did not result in a change in protein function because the mutation was synonymous, whereas in Rv0309, Q was substituted by H, which might indicate a change in function. Moreover, this nonsynonymous mutation was located on a T-cell epitope (IEDB_ID 51117). As the function of Rv0309 is currently unclear, we are not sure whether this change involved a protein function change. However, we can at least assume that these nine strains are different from the others in the interaction between human T cells and the pathogen, and may represent a special type of MTBC strain that merits further investigation.
In conclusion, the gene sequences of Rv2945c and Rv0309 contain genetic diversity, which may reflect that the antigens are involved in diversifying selection to evade host immunity.
We thank the staffs of the respective institutes in Beijing municipality, the 13 provinces and autonomous regions in China for their excellent contribution to this study, especially for the help of Lishui Zhang (Fujian), Yunhong Tan (Hunan), Xiujun Yang (Jilin), Chongxiang Tong (Gansu), Feiying Liu (Guangxi), Yingcheng Qi (Xinjiang), Qing Wang (Anhui), Xiaohui Cao and Ping Zhao (Beijing), Haitao Li (Henan), Jun Yang (Sichuan), Xuanmin Zhang (Shannxi), Li Shi (Xizang), Qing Wang (Anhui) and Xiaomeng Wang (Zhejiang). This work was funded by projects 2013ZX10003006 and 2013ZX10003002-001 of the Chinese National Key Program of Mega Infectious Disease.
Y.J., X.D., W.Z. and H.L. contributed equally to this study.