Abstract
FtsK/SpoIIIE family ATPases are conserved throughout bacteria and are involved in the translocation of DNA and proteins through membrane-spanning pores. Through comparison of 26 whole-genome-sequenced M. tuberculosis complex (MTBC) strains downloaded from NCBI website, we found that FtsK/SpoIIIE protein presented polymorphisms. One M. bovis strain and three BCG strains even showed the two T cell epitopes missing. Then we chose 159 clinical M. tuberculosis isolates from China, amplified gene encoding FtsK/SpoIIIE protein (Rv3871) and compared the sequences. The results showed that there are polymorphisms existed in FtsK/SpoIIIE protein among MTBC, which may affect both protein function and host immune reaction. In addition, position 1497 could be used as a good phylogenetic marker for Beijing strains.
Keywords: Mycobacterium tuberculosis, FtsK/SpoIIIE protein, polymorphisms
Introduction
The majority of protein export systems contain associated ATPases that supply energy for functions including assembly of the secretory apparatus and substrate translocation. FtsK/SpoIIIE family ATPases are conserved throughout bacteria and are involved in the translocation of DNA and proteins through membrane-spanning pores [1]. This translocation is important for cell division, sporulation, DNA conjugation, and other essential cell processes [1,2]. Rv3871 is FtsK/SpoIIIE family ATPases in Mycobacterium tuberculosis [3]. It is a cytosolic ATPase that has been shown to bind to the C-terminal of CFP10 in the ESAT6: CFP10 complex and escort it to Rv3870, the membrane-bound component of the Esx-I system protein, and thereby allow its secretion [4]. The Esx-I system has several other substrates that are co-secreted and are mutually dependent upon each other for secretion, such that the inhibition of secretion of any of these substrates can affect the secretion of the rest of the substrates [5]. Through functional and comparative genomic studies, it is now known that the secretion of ESAT6 and CFP10 is crucial for stimulating host immunogenicity [6] while imparting a fully virulent phenotype to M. tuberculosis [7-9].
Through comparison of 26 whole-genome-sequenced M. tuberculosis complex (MTBC) strains downloaded from NCBI website, we found that FtsK/SpoIIIE protein presented polymorphisms. One M. bovis strain and three BCG strains even showed the two T cell epitopes missing. All these give us a hint that polymorphisms of the FtsK/SpoIIIE protein in the MTBC may be the reason for changes in the antigen produced, which may cause alterations of related functions and host immune reaction.
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. The patients with TB included in the present research protocol were given a Subject information sheet and they all gave written informed consent to participate in the study.
Strains and DNA preparation
Firstly, we used FtsK/SpoIIIE protein sequences of 26 MTBC strains (Table 1) from NCBI genome website to compare and analyze. These 26 strains included one M. africanum strain, one M. bovis strain and 20 M. tuberculosis strains. We can see that FtsK/SpoIIIE protein presented polymorphisms among 26 MTBC strains (Figure 1). Four strains presented a same nonsynonymous mutation of P31A. Mycobacterium tuberculosis F11 had one nonsynonymous mutation of P288S. One M. bovis strain and three BCG strains even showed the two T cell epitopes missing. All these give us a hint that polymorphisms of the FtsK/SpoIIIE protein in the MTBC may be the reason for changes in the antigen produced, which may cause alterations of related functions and host immune reaction.
Table 1.
26 MTBC strains whose data were obtained from NCBI website
| ID No. | Strain name |
|---|---|
| 01 | Mycobacterium tuberculosis H37Rv |
| 02 | Mycobacterium tuberculosis CDC1551 |
| 03 | Mycobacterium bovis AF2122/97 |
| 04 | Mycobacterium bovis BCG str. Pasteur 1173P2 |
| 05 | Mycobacterium tuberculosis H37Ra |
| 06 | Mycobacterium tuberculosis F11 |
| 07 | Mycobacterium bovis BCG str. Tokyo 172 |
| 08 | Mycobacterium tuberculosis KZN 1435 |
| 09 | Mycobacterium tuberculosis KZN 4207 |
| 10 | Mycobacterium bovis BCG str. Mexico |
| 11 | Mycobacterium tuberculosis UT205 |
| 12 | Mycobacterium tuberculosis RGTB327 |
| 13 | Mycobacterium tuberculosis CCDC5180 |
| 14 | Mycobacterium tuberculosis CCDC5079 |
| 15 | Mycobacterium tuberculosis CTRI-2 |
| 16 | Mycobacterium tuberculosis RGTB423 |
| 17 | Mycobacterium tuberculosis KZN 605 |
| 18 | Mycobacterium tuberculosis 7199-99 |
| 19 | Mycobacterium bovis BCG str. Korea |
| 20 | Mycobacterium tuberculosis str. Erdman = ATCC 35801 |
| 21 | Mycobacterium tuberculosis str. Beijing/NITR203 |
| 22 | Mycobacterium tuberculosis str. Haarlem/NITR202 |
| 23 | Mycobacterium tuberculosis CAS/NITR204 |
| 24 | Mycobacterium tuberculosis EAI5/NITR206 |
| 25 | Mycobacterium tuberculosis EAI5 |
| 26 | Mycobacterium tuberculosis str. Haarlem |
Figure 1.
AA sequence alignment for antigen Rv3871 of 26 whole-genome-sequenced MTBC strains. AA changes marked in red color. Shading indicates T cell epitope area. “?” means there were undetermined base(s) in the sequence.
Then we chose 159 clinical M. tuberculosis isolates to clarify this hypothesis. 159 strains were selected from 2346 MTBC strains isolated in Beijing Municipality and 12 provinces and autonomous regions (See Table 2), China, which were genotyped by Spoligotyping in a previous study [10]. Strains belonging to all major and rare spoligotypes in China were included. Considering the predominance of the Beijing family strains in China, we chose about half of the Beijing family strains (81 strains) and half non-Beijing family strains (78 strains). We randomly selected the 81 Beijing family strains from 1738 Beijing strains among 2346 strains. The remaining 78 strains were selected from 608 non-Beijing family isolates. Furthermore, we attempted to include strains representing different spoligotypes that were isolated from different places. Table 2 shows the numbers of strains used in this study that were obtained from different provinces in China. The spoligotype patterns of 159 stains were showed in Table 3.
Table 2.
No. of strains in different provinces of China
| Places | No. of isolates |
|---|---|
| Anhui Province | 11 |
| Shannxi Province | 14 |
| Beijing Municipality | 10 |
| Fujian Province | 25 |
| Gansu Province | 11 |
| Guangxi Zhuang Autonomous Region | 27 |
| Sichuan Province | 1 |
| Henan Province | 12 |
| Hunan Province | 7 |
| Xizang (Tibet) Autonomous Region, | 5 |
| Xinjiang Uygur Autonomous Region | 11 |
| Jilin Province | 13 |
| Zhejiang Province | 12 |
Table 3.
No. of strains of each Spoligotype pattern
| Spoligotypes | No. of strains |
|---|---|
| Beijing | 81 |
| T | 10 |
| U | 25 |
| MANU | 10 |
| Haarlem | 5 |
| EAI | 1 |
| LAM | 2 |
| S | 1 |
| CAS | 4 |
| H37Rv family | 1 |
| New | 19 |
The strains were cultured using a standard Löwenstein-Jensen medium method, heat inactivated and then used directly in polymerase chain reactions (PCRs).
Primers
As the gene sequence of Rv3871 reached to 1775 bp, we split it into two parts and designed two pairs of primers to amplify the gene sequence. The primers (from the 5’ to 3’ end) used in this study were designed with DNASTAR software according to H37Rv genome sequence and were showed in Table 4.
Table 4.
Primers used in the study
| Gene product | Length (bp) | Primers |
|---|---|---|
| Rv3871-1 | 1507 | 5’-CGCGCATTCACAGGTTCAC-3’F |
| 5’-AACCGCTTCTTCAGGTTGAC-3’R | ||
| Rv3871-2 | 909 | 5’-GTATCCACCTGCACGAACTC-3’F |
| 5’-TCCCGTACACAAACCGTTCG-3’R |
PCR
The PCR were performed in a total volume of 20 μL. The PCR mix contained 10 μL PCR buffer, 100 nM 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 variants were confirmed by sequencing of the new PCR products.
Sequence
The sequences of the PCR products were determined by ABI 3730xl DNA Analyzer.
Data analysis
The sequences were first aligned by ClustalW [11] software with the Rv3871 gene sequence from M. tuberculosis H37Rv genome to determine the Rv3871 region, and then this region was split out by a personalized Perl script. The sequence compare and translate were carried out by Bioedit software. The mutated protein structures were predicted by Phyre2 software online (http://www.sbg.bio.ic.ac.uk/phyre2).
Results
Mutation and deletion in gene sequences
All 159 clinical strains we chose presented relative PCR products of antigen Rv3871-1 and Rv3871-2. Table 5 showed the mutations in the gene sequences of Rv3871. Among 159 strains, there were six nonsynonymous mutations, six synonymous mutations and one three-base deletion in Rv3871. Strain JL06035, GX06055, AH03040, FJ05132 and HeN05007 all had one different nonsynonymous mutation. Four Beijing strains, GX06058, GX06047, XZ06007 and GS05121, presented same synonymous mutation. GX06002, GX06046 and FJ05143 owned different one synonymous mutation. There were one nonsynonymous mutation, one synonymous mutation and one three-base deletion in strain HuN06002. Position 1479 presented higher polymorphism, as 87 strains owned an sSNPs (G-A) (Table 7). We counted the frequencies of the synonymous mutation of position 1479. Among the 81 isolates of Beijing phenotype, 97.5% (n=79) isolates presented A; meanwhile, among the 78 non-Beijing isolates, only seven isolates were G in the position 1479 of Rv3871.
Table 5.
Changes in antigen Rv3871 among 159 clinical strains*
| Isolates | Base change | AA change | Spoligotypes |
|---|---|---|---|
| JL06035 | G226A | (GCC) A76T (ACC) | MANU |
| HuN06002 | G394T; T396G Position 397-399 deletion | (GGT) G132W (TGG) Position 133 deletion | Beijing |
| GX06055 | C570A | (GAC) D190E (GAA) | Beijing |
| AH03040 | C631A | (CAG) Q211K (AAG) | Beijing |
| FJ05132 | T685G | (TCC) S229A (GCC) | T |
| GX06058 | T891C | No change | Beijing |
| GX06047 | |||
| XZ06007 | |||
| GS05121 | |||
| HeN05007 | A1564G | (ATG) M522V (GTG) | S |
| GX06002 | G1557A | No change | U |
| GX06046 | C1663A | No change | Beijing |
| FJ05413 | C1761T | No change | U |
| 87 strains | G1479A | No change | -# |
Use the CDS of Rv3871 of M. tuberculosis H37Rv strain as the reference sequence.
Details are showed in Table 7.
Table 7.
Nucleotide in position 1497 of Rv3871 among 159 strains
| Strains | Spoligotyping | Base | Strains | Spoligo typing | Base | Strains | Spoligotyping | Base | Strains | Spoligotyping | Base |
|---|---|---|---|---|---|---|---|---|---|---|---|
| AH03002 | Beijing | A | FJ05349 | T | G | GX06044 | Beijing | A | JL06007 | Beijing | A |
| AH03009 | T | G | FJ05357 | New | G | GX06045 | Beijing | A | JL06009 | Beijing | A |
| AH03019 | Beijing | A | FJ05395 | T | G | GX06046 | Beijing | A | JL06015 | Beijing | A |
| AH03026 | Beijing | A | FJ05406 | EAI | G | GX06047 | Beijing | A | JL06016 | Beijing | A |
| AH03029 | Beijing | A | FJ05413 | U | G | GX06048 | Beijing | A | JL06017 | Beijing | A |
| AH03031 | Beijing | G | FJ05425 | New | G | GX06052 | Beijing | A | JL06018 | Beijing | A |
| AH03032 | Beijing | A | FJ05484 | U | G | GX06055 | Beijing | A | JL06020 | Beijing | A |
| AH03035 | Beijing | A | FJ05490 | U | G | GX06058 | Beijing | A | JL06023 | Beijing | A |
| AH03037 | Beijing | A | FJ05554 | U | G | GX06059 | Beijing | A | JL06035 | MANU | A |
| AH03040 | Beijing | A | FJ06068 | MANU | A | GX06066 | U | G | JL06151 | MANU | G |
| AH04125 | New | G | FJ06025 | T | G | GX06087 | New | G | JL06183 | New | G |
| ShanX05092 | New | G | FJ06038 | Haarlem | G | GX06088 | LAM | G | ZJ06001 | Haarlem | G |
| ShanX05094 | Beijing | A | FJ06051 | New | G | GX06097 | New | G | ZJ06003 | Beijing | A |
| ShanX05096 | Beijing | A | FJ06057 | New | G | GX06112 | U | G | ZJ06006 | Beijing | A |
| ShanX05098 | Beijing | A | FJ07031 | LAM | G | GX06117 | U | G | ZJ06007 | Beijing | A |
| ShanX05106 | Beijing | A | FJ07033 | H37Rv family | G | GX06129 | MANU | G | ZJ06009 | Beijing | A |
| ShanX05105 | Beijing | A | FJ07042 | Haarlem | G | GX06130 | U | G | ZJ06010 | Beijing | A |
| ShanX05111 | Beijing | A | FJ07049 | MANU | G | HeN05007 | S | G | ZJ06015 | Beijing | A |
| ShanX05115 | Beijing | G | FJ07061 | MANU | G | HeN05015 | U | A | ZJ06018 | Beijing | A |
| ShanX05124 | U | G | GS05040 | T | G | HuN06002 | Beijing | A | ZJ06020 | Beijing | A |
| ShanX05177 | New | G | GS05112 | Beijing | A | HuN06004 | MANU | G | ZJ06027 | Beijing | A |
| ShanX05178 | U | G | GS05113 | Beijing | A | HuN06009 | Beijing | A | ZJ06040 | Beijing | A |
| ShanX05260 | U | G | GS05114 | Beijing | A | HuN06022 | Beijing | A | ZJ06098 | U | G |
| ShanX05290 | U | G | GS05116 | Beijing | A | HuN06026 | T | G | HeN06022 | Beijing | A |
| ShanX05296 | MANU | A | GS05121 | Beijing | A | HuN06099 | U | A | HeN06023 | Beijing | A |
| BJ05012 | Beijing | A | GS05125 | Beijing | A | HuN06101 | New | G | HeN06024 | Beijing | A |
| BJ05013 | Beijing | A | GS05127 | Beijing | A | XZ06003 | CAS | G | HeN06035 | Beijing | A |
| BJ05015 | Beijing | A | GS05129 | Beijing | A | XZ06107 | New | G | HeN06039 | Beijing | A |
| BJ05016 | Beijing | A | GS05130 | Beijing | A | XJ06013 | New | G | HeN06040 | Beijing | A |
| BJ05022 | Beijing | A | GS05138 | MANU | G | XJ06018 | CAS | G | HeN06041 | Beijing | A |
| BJ05024 | Beijing | A | GX06002 | U | G | XJ06025 | U | G | HeN06042 | Beijing | A |
| BJ05025 | Beijing | A | GX06027 | U | G | XJ06060 | New | G | HeN06043 | Beijing | A |
| BJ05028 | Beijing | A | GX06037 | Beijing | G | XJ06087 | U | A | HeN06045 | Beijing | A |
| BJ05029 | Beijing | A | GX06040 | U | G | XJ06106 | U | A | XZ06002 | Beijing | A |
| BJ05030 | Beijing | A | GX06137 | T | G | XJ06112 | MANU | A | XZ06006 | Beijing | G |
| FJ05009 | New | G | GX06160 | New | G | XJ06116 | U | G | XZ06007 | Beijing | A |
| FJ05063 | T | G | GX06187 | New | G | XJ06153 | CAS | G | |||
| FJ05086 | Haarlem | A | GX06203 | U | G | XJ06183 | U | G | |||
| FJ05132 | T | G | GX06204 | New | G | XJ06188 | CAS | G | |||
| FJ05159 | T | A | SC06055 | New | G | JL06003 | Beijing | A | |||
| FJ05199 | Haarlem | G | GX06043 | U | G | JL06006 | Beijing | A |
Changes in protein level
Table 5 showed the AA change and position in antigen Rv3871 among 159 strains. For all changes, six synonymous mutations occurred, which resulted in no AA change. Six nonsynonymous mutations led to AA change of the protein. After we predicted protein structures by Phyre2 software online, we found that AA32-309 constitute the domain. Five nonsynonymous mutations and one three-base deletion were located on the domain region (See Figure 2). Also, four of five nonsynonymous mutations were all changed between amino acids with different properties, which may induce alteration of protein function. Mutation in HeN05007 was not located in the region, which resulted in no function change.
Figure 2.
Positions of mutations in the Rv3871 antigen on the tertiary structures which are predicted by Phyre2 software online (http://www.sbg.bio.ic.ac.uk/phyre2).
Changes in T cell epitopes
There are two human T cell epitopes in the antigen Rv3871 (Table 6) according to the Immune Epitopes Database (IEDB) [12]. For 26 MTBC strains from NCBI website, one M. bovis strain and three BCG strains showed two T cell epitopes missing. Among 159 strains, nonsynonymous mutation in HeN05007 affected one of two T cell epitopes in antigen Rv3871.
Table 6.
AA changes of the T cell epitopes included in antigen Rv3871
| IEDB_ID | Peptide sequence | AA changes |
|---|---|---|
| 8651 | DIGLHIIVTCQMSQAYKATMDK | ATG(M)-GTG(V) |
| 144929 | PMAPLAPLL | No change |
Discussion
In this study, we chose 159 clinical M. tuberculosis strains in China which originated from a very large geographical area and had different spoligotyping patterns; the data provided by them could therefore be representative of genetic diversity that might be present within China, at least to some extent.
Rv3871, one of gene products encoded by RD1, is a cytosolic ATPase that has been shown to bind to the C-terminal of CFP10 in the ESAT6: CFP10 complex and escort it to Rv3870, the membrane-bound component of the EsxI system protein, and thereby allow its secretion [5]. It showed that the two T cell epitopes were missing in BCG and M. bovis strains, which at some level explains that BCG vaccine and M. bovis strains are less likely to induce host immune reaction than M. tb strains. Also, one of two T cell epitopes in the protein changed due to a nonsynonymous mutation, which results in alteration of immune reaction between pathogen and host.
In our study, seven strains presented polymorphisms in Rv3871. After we predicted the mutated protein structures by Phyre2 software, we found that five strains with nonsynonymous mutations were located in structure domain and four of them changed to amino acids with different property, which may result in alteration of protein function.
In the gene sequence of Rv3871, position 1479 presented higher polymorphisms, as 87 strains owned an sSNPs (G-A). As 97.5% Beijing strains showed A in the position, it could be used as a good phylogenetic marker for Beijing strains.
In conclusion, there are polymorphisms existed in FtsK/SpoIIIE protein among MTBC, which may affect both protein function and host immune reaction. In addition, position 1497 could be used as a good phylogenetic marker for Beijing strains.
Acknowledgements
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, specially 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), JunYang (Sichuan), Xuanmin Zhang (Shannxi), Li Shi (Xizang), Qing Wang (Anhui) and Xiaomeng Wang (Zhejiang). This work was funded by the project 81401647 of Natural Science Foundation of China, 2013ZX10003006 and 2013ZX10003002-001of Chinese National Key Program of Mega Infectious Diseases.
Disclosure of conflict of interest
None.
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