Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion

  • Authors:
    • Yi Jiang
    • Jianhao Wei
    • Haican Liu
    • Guilian Li
    • Qian Guo
    • Yan Qiu
    • Lili Zhao
    • Machao Li
    • Xiuqin Zhao
    • Xiangfeng Dou
    • Kanglin Wan
  • View Affiliations

  • Published online on: November 19, 2015     https://doi.org/10.3892/mmr.2015.4589
  • Pages: 947-954
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Abstract

Previous studies have demonstrated that the Pro‑Glu/Pro‑Pro‑Glu (PE/PPE) genes in strains of Mycobacterium tuberculosis exhibit high sequence variation and may be involved in antigenic variation and immune evasion. Region of Difference 1 (RD1), encoding genes from Rv3871 to Rv3879, was observed to be lost during the original derivation of Bacillus Calmette‑Guérin between 1908 and 1921. It has been previously demonstrated that two PE/PPE proteins, PE35 (Rv3872) and PPE68 (Rv3873), are encoded by RD1 and exhibit immunodominance. To explore the genetic diversity of PE35 and PPE68, and to evaluate the impact of sequence variation on the immune recognition of these proteins, 161 clinical M. tuberculosis strains were selected from China and comparative sequence analysis of PE35 and PPE68 was performed. The results indicated that polymorphisms in PE35 and PPE68 may lead to alterations in the function of these proteins, which may potentially affect strain virulence. In addition, the human T‑cell epitopes of PE35 and PPE68 were highly variable, suggesting that the two antigens may be involved in diversifying selection to evade host immunity. The prevalence of strains with PE35 mutations in the non‑Beijing family was significantly greater compared with the Beijing family, indicating that Beijing strains may be more conservative than non‑Beijing strains in this gene.

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 (35). 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 (1012). 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 (1518). 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.

Table I

Number of strains in different locations in China.

Table I

Number of strains in different locations in China.

LocationNumber of isolates
Anhui11
Shanxi16
Beijing11
Fujian24
Gansu12
Guangxi Zhuang Autonomous Region29
Sichuan1
Henan12
Hunan7
Xizang (Tibet) Autonomous Region,4
Xinjiang Uygur Autonomous Region11
Jilin12
Zhejiang11

Table II

Number of strains of each spoligotype pattern.

Table II

Number of strains of each spoligotype pattern.

SpoligotypeNumber of strains
Beijing82
T12
U27
MANU10
Haarlem4
EAI2
LAM2
S1
CAS3
New18

[i] MANU, a new family from India; LAM, Latin-American-Mediterranean lineage; EAI, East African-Indian lineage; CAS, Central-Asian lineage; S, Sicily and Sardinia family; U, Ural region; T, modern tuberculosis strains.

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 III

Comparison of the Mycobacterium tuberculosis strains containing mutations in the Beijing family and the non-Beijing family.

Table III

Comparison of the Mycobacterium tuberculosis strains containing mutations in the Beijing family and the non-Beijing family.

StrainBeijing familyNon-Beijing family
Strains with PE35 mutation419a
Strains with PPE68 mutation26b
All8279

a P=0.001; Pearson χ2 test, Beijing strains vs. non-Beijing strains.

b P=0.253; Pearson χ2 test, Beijing strains vs. non-Beijing strains. PE35, Pro-Glu 35; PPE68, Pro-Pro-Glu 68.

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).

Table IV

Amino acid alterations of human T-cell epitopes in the antigens, PE35 and PPE68a.

Table IV

Amino acid alterations of human T-cell epitopes in the antigens, PE35 and PPE68a.

Epitope IDEpitope peptide sequenceRv locusGeneAmino acid alteration
144881 EGIQLLASNASAQRv3872PE35GCT(A)-CCT(P); Frameshift
183 AAGSSATGGAAPVGAGAMGQGAQSGRv3873PPE68CCG(P)-TCG(S); Frameshift
191 AAGWQTLSAALDAQAVELTARLNSLRv3873PPE68Frameshift(5 bp deletion)
265 AALAMEVYQAETAVNTLFRv3873PPE68ATG(M)-ATA(I); Frameshift
2434 ALAMEVYQAETAVNTLFEKLEPMASRv3873PPE68ATG(M)-ATA(I); Frameshift
2922 ALTEMDYFIRMWNQAALAMEVYQAERv3873PPE68ATG(M)-ATA(I); Frameshift
3098 AMGQGAQSGGSTRPGLVARv3873PPE68Frameshift
4186 ARLMAGAGPAPMLAAAAGRv3873PPE68No change
4187 ARLMAGAGPAPMLAAAAGWQTLSAARv3873PPE68No change
4727 ASQSTTNPIFGMPSPGSSTPVGQLPRv3873PPE68Frameshift
4969 ATGGAAPVGAGAMGQGAQRv3873PPE68CCG(P)-TCG(S); Frameshift
5063ATNFFGINTIPIALRv3873PPE68Frameshift
11486 EEAAQMGLLGTSPLSNHPRv3873PPE68Frameshift
14339 ETAVNTLFEKLEPMASILRv3873PPE68Frameshift
15812FFGINTIPIARv3873PPE68Frameshift
16010 FGMPSPGSSTPVGQLPPARv3873PPE68Frameshift
18685 GAMGQGAQSGGSTRPGLVAPAPLAQRv3873PPE68Frameshift
18776 GASQSTTNPIFGMPSPGSRv3873PPE68Frameshift
19860 GGGSDKALAAATPMVVWLQTASTQARv3873PPE68Frameshift
20016 GGSGPSAGAGLLRAESLPRv3873PPE68Frameshift
20048 GGTGGGNPADEEAAQMGLRv3873PPE68Frameshift
20997 GLLGTSPLSNHPLAGGSGPSAGAGLRv3873PPE68Frameshift
21179 GLVAPAPLAQEREEDDEDDWDEEDDRv3873PPE6818 bp deletion; Frameshift
21707 GPMQQLTQPLQQVTSLFSRv3873PPE68GTG(V)-CTG(L); Frameshift
22351 GSGPSAGAGLLRAESLPGAGGSLTRRv3873PPE68Frameshift
22531 GSSTPVGQLPPAATQTLGQLGEMSGRv3873PPE68Frameshift
22657 GTGGGNPADEEAAQMGLLGTSPLSNRv3873PPE68Frameshift
29846 KALAAATPMVVWLQTASTRv3873PPE68Frameshift
35251 LDPGASQSTTNPIFGRv3873PPE68GAT(D)-AAT(N); Frameshift
35652 LEPMASILDPGASQSTTNRv3873PPE68GAT(D)-AAT(N); Frameshift
35819 LFEKLEPMASILDPGASQSTTNPIFRv3873PPE68GAT(D)-AAT(N); Frameshift
37727 LLRAESLPGAGGSLTRTPRv3873PPE68Frameshift
38448 LPEIAANHITQAVLTATNRv3873PPE68Frameshift
38492 LPGAGGSLTRTPLMSQLIEKPVAPSRv3873PPE68Frameshift
39817 LTATNFFGINTIPIARv3873PPE68Frameshift
41291 MDYFIRMWNQAALAMEVYRv3873PPE68ATG(M)-ATA(I); Frameshift
42095 MLWHAMPPELNTARLMAGRv3873PPE68No change
46130 NTIPIALTEMDYFIRMWNRv3873PPE68Frameshift
48567 PMLAAAAGWQTLSAALDARv3873PPE68No change
49875 PVGQLPPAATQTLGQLGERv3873PPE68Frameshift
50366 QATAQAAAYTQAMATTPSLPEIAANRv3873PPE68Frameshift
51367 QLIEKPVAPSVMPAAAAGSSATGGARv3873PPE68Frameshift
52167 QQVTSLFSQVGGTGGGNPRv3873PPE68GTG(V)-CTG(L); Frameshift
52556 QTLGQLGEMSGPMQQLTQRv3873PPE68Frameshift
60492 SQLIEKPVAPSVMPAAAARv3873PPE68Frameshift
62250 SVMPAAAAGSSATGGAAPRv3873PPE68CCG(P)-TCG(S); Frameshift
64822 TLGQLGEMSGPMQQLTQPLQQVTSLRv3873PPE68Frameshift
65054 TLSAALDAQAVELTARLNRv3873PPE68Frameshift (5 bp deletion)
65767 TPSLPEIAANHITQAVLTATNFFGIRv3873PPE68Frameshift
65912 TQPLQQVTSLFSQVGGTGGGNPADERv3873PPE68GTG(V)-CTG(L); Frameshift
66074 TRPGLVAPAPLAQEREEDRv3873PPE68Frameshift
66364 TSPLSNHPLAGGSGPSAGRv3873PPE68Frameshift
68284 VELTARLNSLGEAWTGGGRv3873PPE68Frameshift(5 bp deletion)
68285 VELTARLNSLGEAWTGGGSDKALAARv3873PPE68Frameshift (5 bp deletion)
69128 VITMLWHAMPPELNTARLMAGAGPARv3873PPE68Frameshift
69795 VLTATNFFGINTIPIALTRv3873PPE68Frameshift
69796 VLTATNFFGINTIPIALTEMDYFIRRv3873PPE68Frameshift
71944 VWLQTASTQAKTRAMQATRv3873PPE68Frameshift
71945 VWLQTASTQAKTRAMQATAQAAAYTRv3873PPE68Frameshift
73833 YFIRMWNQAALAMEVRv3873PPE68ATG(M)-ATA(I); Frameshift
121011 TPMVVWLQTASTQAKTRRv3873PPE68Frameshift
144907 IALTEMDYFIRMWNQAALAMEVYRv3873PPE68ATG(M)-ATA(I); Frameshift
144964TNFFGINTIPIALTRv3873PPE68Frameshift

a The coding sequence of PE35 and PPE68 of Mycobacterium tuberculosis H37Rv strain has been used as the reference sequence. Bold letters indicate locations of amino acid alterations. PE35, Pro-Glu 35; PPE68, Pro-Pro-Glu 68; ID, identification.

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 (2226), 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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January 2016
Volume 13 Issue 1

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Online ISSN:1791-3004

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APA
Jiang, Y., Wei, J., Liu, H., Li, G., Guo, Q., Qiu, Y. ... Wan, K. (2016). Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion. Molecular Medicine Reports, 13, 947-954. https://doi.org/10.3892/mmr.2015.4589
MLA
Jiang, Y., Wei, J., Liu, H., Li, G., Guo, Q., Qiu, Y., Zhao, L., Li, M., Zhao, X., Dou, X., Wan, K."Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion". Molecular Medicine Reports 13.1 (2016): 947-954.
Chicago
Jiang, Y., Wei, J., Liu, H., Li, G., Guo, Q., Qiu, Y., Zhao, L., Li, M., Zhao, X., Dou, X., Wan, K."Polymorphisms in the PE35 and PPE68 antigens in Mycobacterium tuberculosis strains may affect strain virulence and reflect ongoing immune evasion". Molecular Medicine Reports 13, no. 1 (2016): 947-954. https://doi.org/10.3892/mmr.2015.4589