Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion
- Authors:
- Published online on: November 19, 2015 https://doi.org/10.3892/mmr.2015.4589
- Pages: 947-954
Abstract
Introduction
Comparative genomics has identified numerous genetic regions in Mycobacterium tuberculosis and M. bovis that are deleted in M. bovis Bacillus Calmette-Guérin (BCG), such as region of difference 1 (RD1) and RD2 (1). RD1 was lost during the original derivation of BCG between 1908 and 1921 (2). Proteins encoded in these regions have the potential to form the basis of novel specific T-cell-based blood tests that do not cross-react with BCG. Among these antigens, early secretory antigenic target 6 (ESAT-6; ESXA, Rv3875), ESAT-6-like protein esxB (CFP10; ESXB, Rv3874), Pro-Pro-Glu 68 (PPE68; Rv3873) and Pro-Glu 35 (PE35; Rv3872) are immunodominant (3–5). The former two antigens (ESAT-6 and CFP10) have been investigated in detail in humans and are known to be predominant virulence factors (6,7) and in addition are good candidates for the diagnosis of tuberculosis (TB) (8). The latter two, PE35 and PPE68, are members of the PE/PPE family and exhibit immunodominance (9). The PE/PPE proteins are secreted or associated with the mycobacterial cell envelope, and mediate interactions at the host-pathogen interface (10–12). PE35 and PPE68 have been demonstrated to be associated with cellular immune responses to mycobacterial infections (9).
Numerous previous studies have demonstrated high sequence variation of PE/PPE genes in M. tuberculosis strains (13,14), an observation that suggests involvement in antigenic variation. To improve the understanding of the genetic diversity of PE35 and PPE68 belonging to the PE-PPE genes in the RD1 region, and to explore the effect of immune recognition on the sequence variation of these two genes, the current study selected 161 clinical M. tuberculosis isolates in China, amplified the PE35 and PPE68 genes and compared the sequences. The effect of the polymorphisms in PE35 and PPE68 were investigated at the protein level to identify whether alterations in the function of these proteins occurs as this may potentially affect strain virulence. In addition, the variation in the human T-cell epitopes of PE35 and PPE68 were investigated to explore whether the two antigens are involved in diversifying selection to evade host immunity.
Materials and methods
Ethics statement
The study obtained approval from the Ethics Committee of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (Beijing, China). The patients with TB included in the present study were provided with a subject information sheet and written informed consent was obtained.
Strains and DNA preparation
A total of 161 strains were selected from 2,346 M. tuberculosis complex (MTBC) strains isolated in Beijing Municipality and 12 provinces and autonomous regions in China (Table I), which were genotyped by spoligotyping as described previously (15–18). Strains belonging to all major and rare spoligotypes in China were included. Considering the predominance of the Beijing family strains in China, approximately half of the Beijing family strains (82 strains) and half non-Beijing family strains (79 strains) were selected. The 82 Beijing family strains were randomly selected from the 1,738 Beijing strains among the total 2,346 strains. The remaining 79 strains were selected from 608 non-Beijing family isolates. Furthermore, it was attempted to include strains representing different spoligotypes that were isolated from different locations. Table I presents the numbers of strains used in the present study that were obtained from different provinces in China. The spoligotype patterns of the 161 strains are presented in Table II.
A total of 2,346 M. tuberculosis isolates were randomly collected between 2005 and 2007 from 2,346 patients at 13 different provincial tuberculosis hospitals across China (16). Subsequently, 161 strains were selected from those 2,346 isolates. Sputum specimens were collected from the TB patients and used to inoculate Löwenstein-Jensen slants. The strains were cultured using a standard Löwenstein-Jensen medium (Baso Diagnostics, Inc., Zhuhai, China) method (15), heat inactivated and then used directly in polymerase chain reactions (PCRs).
Primers
The nucleotide sequences of the primers used in the present study were designed with DNASTAR software (version 7.0; DNASTAR, Inc., Madison, WI, USA) according to the M. tuberculosis H37Rv genomic sequence and were as follows: PE35, forward 5′-GTAATCGAGTTCGGGCAATG-3′ and reverse 5′-AGGCTTCTCCCAGAGAGTT-3′; PPE68, forward 5′-GACATTGGCACGCAAGTGAG-3′ and reverse 5′-TAGCGGCATCGGTCTTCATC-3′.
PCR
The PCRs were performed in a total volume of 20 µl. The PCR mix contained 10 µl PCR buffer (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China), 100 nM primer, 200 µM each of the four dNTPs (Tiangen (Beijing) Co., Ltd.) and 0.5 units DNA Taq Polymerase (Takara Bio, Inc., Otsu, Japan). An initial denaturation of 5 min at 94°C was followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 62°C for 45 sec and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min in a Bio-Rad Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA).
Negative controls (reagents only, no DNA) were included each time the PCR was performed. The positive control was 500 pg DNA from the M. tuberculosis reference strain H37Rv. The presence and size of each PCR product was determined by electrophoresis on 2% agarose gel in Tris/boric acid/ethyl-enediaminetetraacetic acid buffer (Tiangen Biotech (Beijing) Co., Ltd.) followed by staining with ethidium bromide (SBS Genetech Co., Ltd., Beijing, China).
The PCRs were conducted a minimum of two times to validate the reproducibility. The variants were confirmed by the sequencing of the new PCR products.
Sequence and data analysis
The sequences of the PCR products were determined using an ABI 3730xl DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The sequences were first aligned using ClustalW software (19) with the PE35 and PPE68 gene sequences from the M. tuberculosis H37Rv genome to determine the PE35 and PPE68 region. Sequence comparisons and translations were conducted using Bioedit software, version 7.1.3.0 (20). The Immune Epitopes Database (IEBD) (http://www.iedb.org/) was used and 1 human T-cell epitope in PE35 and 62 in PPE68 were found (21). In addition, SPSS software, version 14.0 (SPSS, Inc., Chicago, IL, USA) was used to conduct χ2 analysis. P<0.05 was considered to indicate a statistically significant difference.
Results
Mutation and deletion in gene sequences
The genes encoding PE35 and PPE68 were amplified and the sequences compared. All 161 strains yielded PCR products of these two antigens. Among the 161 M. tuberculosis strains, 23 isolates exhibited polymorphisms in the gene sequence of PE35 (Fig. 1) and 8 strains exhibited polymorphisms in PPE68. For PE35, there were 21 strains containing an A deletion and the remaining 2 strains harbored two different nonsynonymous mutations. For PPE68, two isolates had two different deletions and six strains showed five nonsynonymous mutations (Fig. 2).
Changes at the protein level
Figs. 1 and 2 present the amino acid (AA) alterations and their positions in the PE35 and PPE68 antigens. All the alterations resulted in an AA change. A total of 21 strains with an A deletion in PE35 resulted in a frameshift, and therefore the premature termination of the protein, preventing it's production and thereby impacting upon protein function. HeN06041 contained a 5 base pair (bp) deletion located at the fifth AA of PPE68, which additionally resulted in premature termination, and may impact upon protein function due to the deletion abolishing the production of the protein. HuN06004 contained an 18 bp deletion, which resulted in a six AA deletion in PPE68.
Spoligotyping of variant strains
For PE35, 23 variant strains were identified, including 4 Beijing strains, 13 U family strains, 3 T strains, 1 MANU strain and 2 new spoligotype strains. The two strains with nonsynonymous mutations were members of the Beijing family. For PPE68, 8 variant strains including 2 Beijing strains, 2 T strains, 2 EAI strains, 1 U strain and 1 MANU strain were identified. The two EAI strains, FJ06051 and FJ05406, exhibited the same mutation in 229(V-L) in the AA sequence of PPE68, which may represent a unique mutation in EAI strains. HuN06004 exhibited polymorphisms in PE35 and PPE68.
The prevalence of strains containing a PE35 mutation in the non-Beijing family is significantly greater compared with the Beijing family (Table III, P<0.01). The prevalence of strains with the PPE68 mutation in the non-Beijing family is greater compared with the Beijing family, however this was identified to be a significant difference.
 | Table IIIComparison of the Mycobacterium tuberculosis strains containing mutations in the Beijing family and the non-Beijing family. |
Alterations in T-cell epitopes
There is 1 human T-cell epitope in PE35 and 62 in PPE68 according to the IEBD (http://www.iedb.org/) (21). Table IV presents the alterations in the T-cell epitopes of the two antigens. All mutations observed in PE35, except for that in JL06018, affected the T-cell epitopes. For PPE68, there were no nonepitope regions in the gene, as the 62 T-cell epitopes covered the whole gene sequence. This additionally indicates the importance of the PPE68 antigen for the development of T-cell immune responses following infection. Among all of the strains, 58/62 T-cell epitopes in PPE68 (93.5%), exhibited AA alterations resulting from nucleotide alterations (Table IV).
The 5 bp deletion in HeN06041 resulted in a frameshift in the PPE68 protein code, leading to alterations in the corresponding T-cell epitopes including IEDB_ID 191, 65054, 68284, 68285 and further downstream epitopes. The 18 bp deletion in HuN06004 resulted in a 6 AA deletion in IEDB_ID21179.
Discussion
In the present study, 161 clinical M. tuberculosis strains in China were selected which originated from a large geographical area and exhibited different spoligotyping patterns. This strategy was selected so that the data provided would be representative of the genetic diversity that may be present within China, at least to some extent.
In previous studies, genetic approaches coupled with biochemical analyses have indicated that proteins encoded by the RD1 locus are part of a secretion system required for ESAT-6 and CFP-10 export (22–26), hereafter referred to as the ESAT-6 system-1 (ESX-1). PE35 (Rv3872) and PPE68 (Rv3873) are encoded by RD1 and exhibited immunodominance (9). PE35 is an important antigen that stimulates human peripheral blood mononuclear cells in protective Th1 cell assays, demonstrating antigen-induced proliferation and γ-interferon secretion (4). PPE68 is predominantly associated with the cell wall (27) and forms complexes with the RD1 locus proteins Rv3866, Rv3868, CFP-10 and ESAT-6 (28,29). A recent study (9) indicated that PE35 and PPE68 may serve a major role in RD1-associated pathogenesis, and may contribute to the establishment and maintenance of M. tuberculosis infection. Among the 161 strains investigated in the present study, 14.3% of strains with an A deletion in PE35 resulted in premature termination leading to a 16 AA peptide as opposed to the full length protein of 99 AA. This deletion would result in the prevention of protein production, and consequently lead to the complete loss of PE35 function. In addition, the 5 bp deletion in HeN06041 of PPE68 resulted in premature termination, and therefore may exert an effect on protein function via the abolition of protein production. Strains carrying mutations that lead to alterations in the functions of PE35 and PPE68 may be significantly compromised with regards to their virulence. Therefore, polymorphisms in PE35 and PPE68 may result in alterations in the functions of these proteins, which may potentially affect strain virulence. Furthermore, as PE35 has been demonstrated to be essential for ESXA/B secretion and RD1-mediated virulence (30), the null mutant of Rv3872 may influence the release of the ESX-1 antigens, CFP-10 and ESAT-6. PPE68 is a gating protein that regulates the release of ESX-1 antigens (30) therefore, abolition of PPE68 protein may additionally affect the release of CFP-10 and ESTA-6. To investigate this further, virulence comparison of mutant strains and wild strains of PE35 and PPE68 should be conducted.
PE/PPE genes are known to vary and to encode cell surface-exposed proteins, which has led to the hypothesis that they may be involved in antigenic variation (31) and have been suggested to be a 'molecular mantra' to aid in the escape of host immunity. Comas et al (32) reported that the human T-cell epitopes of M. tuberculosis are evolutionarily hyperconserved and suggested that M. tuberculosis lacks antigenic variation and immune evasion ability, however, the study excluded PE/PPE genes. In the current study, there were 63 human T-cell epitopes identified in PE35 and PPE68 according to the IEDB (21). Among the strains, 59/63 T-cell epitopes (93.7%), exhibited AA alterations resulting from nucleotide alterations. The large number of amino acid alterations in these T-cell epitopes may reflect ongoing immune evasion. The data from the current study supports the view that certain PE/PPE genes exhibiting high sequence variation may be involved in antigenic variation induced immune evasion.
The prevalence of strains with PE35 mutations in the non-Beijing family was significantly greater compared with the Beijing family, indicating that the Beijing family strains are less changeable in the T-cell epitopes of PE35 than the non-Beijing family strains. This is supported by a previous study, which demonstrated that Beijing strains from different geographic areas exhibited a high degree of genetic conservation compared with the other M. tuberculosis strains (33). There is evidence that T-cell responses may contribute directly to human-to-human transmission of MTBC (34). The current study indicated that the Beijing strains were more likely to be recognized by host T-cells in PE35 than the non-Beijing strains, which may render them easier to transmit than the non-Beijing strains. Furthermore, analysis of sequences of PE35 and PPE68 in isolates of these various lineages that have been isolated from non-Chinese populations could provide interesting information and insight.
In conclusion, it has been previously reported that PE35 had potential as a serodiagnostic candidate for M. tuberculosis (35). The results of the current study indicate that PE35 harbors a comparatively high number of AA alterations, suggesting that strain diversity should be considered during the further development of novel serodiagnostic candidates that contain PE35.
Acknowledgments
The current study was supported by the projects of the Natural Science Foundation of China (grant no. 81401647) and the Chinese National Key Program of Mega Infectious Disease (grant nos. 2013ZX10003006 and 2013ZX10003002-001).
The authors would like to thank the staff at the institutes in the Beijing municipality, and the 13 provinces and autonomous regions in China for their contribution to the study, and in particular the help of Professor Lishui Zhang (Fuzhou Pulmonary Hospital of Fujian; Fujian, China), Professor Yunhong Tan (Hunan Chest Hospital; Hunan, China), Mr. Xiujun Yang (Jilin Institute for Tuberculosis Control; Jilin, China), Mrs. Chongxiang Tong (Gansu Institute for Tuberculosis Control; Gansu, China), Mrs. Feiying Liu (Guangxi Center for Disease Control and Prevention; Guangxi, China), Mr. Yingcheng Qi (Xinjiang Center for Disease Control and Prevention; Xinjiang, China), Professor Qing Wang (Anhui Chest Hospital; Anhui, China), Professor Xiaohui Cao (Haidian District Center for Disease Control and Prevention; Beijing, China), Professor Ping Zhao (Chaoyang District Center for Disease Control and Prevention; Beijing, China), Mr. Haitao Li (Henan Provincial Chest Hospital; Henan, China), Mrs. Jun Yang (Sichuan Center for Disease Control and Prevention; Sichuan, China), Mr. Xuanmin Zhang (Xi'an Chest Hospital; Xi'an, China), Professor Li Shi (Xizang Center for Disease Control and Prevention; Xizang, China) and Professor Xiaomeng Wang (Zhejiang Center for Disease Control and Prevention; Zhejiang, China).
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