Diversity of Potential Short Tandem Repeats in Mycobacterium leprae and Application for Molecular Typing
http://www.100md.com
微生物临床杂志 2005年第10期
Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1, Aobacho, Higashimurayama-shi, 189-0002, Tokyo, Japan
Leprosy-TB Program, Provincial Health Service, Jl 17, Agustus, Manado, North Sulawesi, 95117, Indonesia
Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1, Aobacho, Higashimurayama-shi, 189-0002, Tokyo, Japan
ABSTRACT
A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multicase households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.
INTRODUCTION
Mycobacterium leprae is an obligate intracellular parasite with tropism for macrophages and Schwann cells and the only species of mycobacteria to infect peripheral nerves (19). It causes leprosy, a chronic granulomatous infection of the skin and peripheral nerves with characteristic deformities and disability (1). It is generally accepted today that the worldwide implementation of standardized multidrug therapy for leprosy has decreased the number of registered leprosy cases from a peak of 10 to 15 million to a current total of less than 1 million. However, the annual confirmed new cases remain at 500,000 to 700,000. This continuing number suggests that effective multidrug therapy fails to disrupt the chain of leprosy transmission (26).
Even though leprosy is one of the oldest recorded diseases, the source for M. leprae, the portal of its exit/entry, and the mode of transmission are still under investigation. Some regard human beings as a host for the bacteria, while others are still considering more possibilities. It was proposed that the nasal mucosa are the exit/entry pathway of M. leprae (9, 17, 18). In light of the transmission mode, human-to-human direct contact was first generally accepted (6, 16), with time as air-borne (23), as vector-borne (14, 25) and as vehicle-borne (3, 12) routes from evidence that has been obtained. All the progress in leprosy epidemiology was helpful in understanding the chain of transmission, yet at the same time was overwhelming.
In the past few years, studies focusing on leprosy transmission by molecular genotyping have shed new light on it (8, 10, 11, 22, 24, 27, 28). The most recent one was the report from Groathouse et al. (8). By in silico analysis, 44 promising polymorphic short tandem repeat (STR) sites, including both 33 microsatellite loci (repeat units of 1 to 5 bp) and 11 minisatellite (repeat units of >5 bp) were selected from the M. leprae TN genome sequence. Variable-number tandem repeats (VNTRs) at 9 of 11 STR loci from four clinical isolates of M. leprae were found. Clearly, it is urgent to accumulate extensive data for developing powerful typing tools to tracking the transmission of leprosy to finally reach a world free of leprosy.
In this study, we identified 32 polymorphic loci throughout the 44 STRs in a batch of 27 laboratory strains by sequencing and demonstrating the applicability and feasibility of nine potential loci acting as genetic markers to discriminate different M. leprae strains.
MATERIALS AND METHODS
M. leprae strains. A panel of 27 laboratory strains of M. leprae was subjected for genetic analysis. Strains were maintained by inoculation into nude mice footpads annually in our laboratory. All the strains used in this study were recovered from multibacillary cases. Excluding Thai-53, Thai-311 and Thai-237 were from Thailand, Indonesia-1 was from Indonesia, and Korea 3-2 was from Korea, and the others were from Japan.
All patients were from geographically distinct regions. Four strains, Thai-53, Kyoto-1, Zensho-4, and Korea3-2, and 17 samples, namely, the fourth generation of Thai-53 (Thai-53 4th), Thai-53 7th, Thai-53 11th, Kyoto-1 3rd, Kyoto-1 5th, Kyoto-1 7th, Kyoto-1 8th, Zensho-4 (biopsy specimen), Zensho-4 1st, Zensho-4 2nd, Zensho-4 3rd, Zensho-4 4th, Korea3-2 (biopsy specimen), Korea3-2 1st, Korea3-2 2nd, Korea3-2 3rd, and Korea3-2 4th (Table 6) were employed for the stability testing of loci. Partially purified bacterial materials were prepared from the inoculated footpads by deferential centrifuging and suspension in a phosphate buffered saline at concentration of 105-6/ml.
Primer selection. Primer sets for the amplification regions of DNA containing the STR sites were referred to the study of Groathouse et al. (8). The sequences of primer pairs were listed (Table 1).
Slit-skin smears from multicase household. Eleven slit skin smears were collected as the same manner as that for Bacterial Index examination from leprosy patients in a total of three households. Among them, five smears from five patients in household I; four smears from two patients in household II, sample 6 from the left earlobe and sample 7 from the right earlobe within a single individual; sample 8 from the earlobe and sample 9 from the back in another individual; and two smears from two patients in household III (Table 7).
Preparation of M. leprae DNA from strains and slit skin smears and sequencing analysis. M. leprae templates from both strains and slit-skin smears were prepared by treatment with lysis buffer at 60°C overnight as described previously (13). PCR amplification of STR sites as well as sequencing analysis was performed under the same condition as described elsewhere using the listed primer pairs (10, 11, 12, 13). Briefly, target loci were amplified using a G mixture and a FailSafe PCR system (EPICENTRE, Madison, Wis.). DNA samples for sequencing were recovered with a MinElute gel extraction kit (QIAGEN GmbH, Hilden, Germany) after electrophoresis of PCR products. Samples were sequenced with a BigDye terminator cycle sequencing FS Ready Reaction kit (Perkin-Elmer Applied Biosystems, Norwalk, Conn.) and an ABI Prism 310 genetic analyzer (Perkin-Elmer). The nucleotide sequences obtained were analyzed using DNASIS software (Hitachi Software Engineering, Yokohama, Japan).
Multiplication of M. leprae strains in nude mice footpad. Inoculums of each strain were prepared from BALB/c-nu/nu nude mice which were inoculated with isolates of the third to fifth generations about 8 to 10 months before by Nakamura's method as described previously (15). Five-week-old male nude mice were injected with the inoculums containing 104 bacilli/0.05 ml of Hanks' balanced salt solution. Bacillary number in the footpads of six nude mice at 10, 20, 30, 40, and 50 weeks growth were examined by Shepard's method (21).
Ethical approval. Informed consent was obtained from all subjects, and the study was approved by the institutional ethics committee of National Institute of Infectious Diseases, Tokyo, Japan. Bacillary samples of nasal mucus and slit skin smears were collected when informed consent was obtained. Animal experiments were conducted under the approval of the institutional animal experiment committee.
RESULTS
Allelic diversity of 33 microsatellite loci in M. leprae strains. In the 27 M. leprae strains, 8 out of 33 selected microsatellite loci showed the same copy number of allele whereas the other 25 loci, (A)9, (G)9, (C)9, (G)10a, (G)10b, (G)11, (G)12, (C)16(G)8, (C)20, (G)22, (AC)8a, (AC)8b, (AC)9, (CA)6, (TA)8, (TA)9, (TA)10, (TA)13, (AT)10, (AT)15, (AT)17, (TA)18, (GGT)5, (GTA)9, and (AGA)20, presented at least two types of allele. All locus identifications are from Groathouse et al. (8) to maintain integrity. This result validated the comments from Groathouse group on the polymorphic (C)20, (AT)17, (TA)18, (GTA)9, and (AGA)20 and no diversity of (CG)6, albeit (AC)9 was also examined for considerable polymorphism here which was predicted as nonpolymorphic loci by them. However, the last repeat unit of some results was not easily defined for unknown reasons (Table 2).
Allelic diversity of eleven minisatellite loci in M. leprae strains. Throughout the 27 M. leprae strains, four out of 11 selected minisatellite loci showed no diversity while the other seven loci, 6-3a, 6-7, 12-5, 18-8, 21-3, 23-3, and 27-5, exhibited variable characteristics, which also verified Groathouse et al. (8) reports on the polymorphism of loci 6-7, 12-5, 18-8, 21-3, and 27-5 (Table 3).
Variations of allelic diversity at STR loci in M. leprae strains. Forty-four selected STR loci consisted of nine protein coding genes, 19 intergenic regions, and 16 pseudogenes. Polymorphism was revealed in six among nine (67%) protein coding genes, 16 out of 19 (84%) intergenic genes, and 10 of 16 (63%) pseudogenes. Almost all of the STR loci located in intergenic regions or in pseudogenes and were unlikely involved in biological functions. Overall, thirty-two out of 44 STR loci were polymorphic and 12 loci were invariable that might be of limited value as epidemiological markers. The variations were between 2 and 11 alleles (Table 4). Notably, at locus (CA)6, isolate Thai-237 differed from the other 26 strains by one repeat unit (Table 2). Similarly, at locus 23-3, isolate Hoshizuka-1 alone, and at locus 27-5, isolate Airaku-2 alone had one copy difference allele (Table 3). Additionally, at locus 12-5, a five-copy-repeat unit was obtained from M. leprae TN, but all of the strains in this study showed three- and four-copy-repeat units instead of the five-copy-repeat unit (Table 3).
Comparison among the loci fall within protein coding genes with two variations. Locus 6-3a is in the rpoT gene coding RNA polymerase sigma factor in M. leprae contained two variations, three- and four-copy alleles (2, 13). Twelve strains, Zensho-2 to Zensho-12, harbored the three-copy allele whereas 15 strains, Hoshizuka-1 to Kyoto-1, had a four-copy allele in the rpoT gene (Table 3). Geographic distribution of each genotype of M. leprae rpoT revealed the distinguished distribution in several countries in the world (13, 10). Intriguingly, we compared the copy numbers of the other two protein coding genes 12-5 and 18-8 having two alleles with the strains carrying three-copy and four-copy rpoT that were closely associated with geographic distributions and no correlation was exploited.
Discriminatory capacity, stability, and reproducibility of nine potential polymorphic loci detected among serial passage strains by nude mice. Nine polymorphic microsatellite loci, (AC)9, (AC)8b, (AC)8a, (TA)10, (AT)17, (AT)15 (GTA)9, (TA)18, and (AGA)20 (also named TTC) were selected for discriminatory capacity analysis. All 27 strains were divided into two groups, three-copy rpoT and four-copy rpoT, based on the rpoT polymorphism. By adding the conjunction of 9 loci, they were distinguished from each other (Table 5). Then stability and reproducibility testing was carried out among them through serial passage strains by nude mice owing to the nature of susceptibility of replication slippage of microsatellite (28). One generation designated approximately 1 year, these strains have been in passage for 4 to 11 years. Not only the identical profile of allele was shared among the different generations of the strains at each locus, but also the copy number of repeats was in agreement with that in the repeated experiment of the same strains, which ensured these polymorphic microsatellite loci were highly stable and reproducible (Tables 1 and 6). The stability of TTCwas done previously (11)]. It was the rationale for these nine polymorphic microsatellite loci to be a significant source of informative markers for the identification and discrimination of M. leprae strains.
Application of polymorphic microsatellite loci for multicase households. Based on the condition of leprosy patients living in same household and possessing the identical TTC pattern, eleven smears from three household were chosen (Table 7). Five bacterial materials from household I shared identical 13-copy of TTC repeat were also subjected to loci (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15, (AT)17, (TA)18, and (GTA)9 and presented the identical copy number of 12, 7, 10, 10, 13, 15, 14, and 9, respectively. In household II which had 12 copies of the TTC repeat, four smears at the above loci showed equal repeats of 10, 7, 9, 10, 13, 13, 11, and 11. There was an exact match by copy number at each locus between samples 6 and 7 as well as samples 8 and 9 within one individual. In household III, two smears harboring eight copies of the TTC repeat, the copy number of loci (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15 and (TA)18 was 10, 7, 9, 8, 16, and 15. respectively. Strikingly, at locus (AT)17, a difference of 9 and 10 copies and a mismatch of 11 and 12 copies at locus (GTA)9 were found in these two smears, respectively.
Linkage between the growth rate of M. leprae in nude mice footpad and allelic diversity. Shepard et al. (20) reported the hereditary fast-slow growth difference among M. leprae strains in conventional mouse footpads, which was also observed in nude mice footpads in our laboratory. However, in this study, the growth curve of M. leprae in nude mice footpads gave no difference between strains with three-copy rpoT (Zensho-2, Airaku-3, and Thai-53) and those with four-copy rpoT (Izumi and Zensho-4) except Kyoto-2, which revealed the copy number of rpoT was irrelevant to the fastidious growth. In addition, no correlation was found between the VNTR of the other protein coding genes and M. leprae growth.
DISCUSSION
Poor understanding of leprosy transmission has hindered us from eradicating the disease even though M. leprae was identified as the pathogen as early as 1873 by Hansen (19). The unusual biological traits of being especially slow growing as well as the inability to culture M. leprae in vitro have partially accounted for the setbacks of detailed studies on leprosy. However, the determination of the complete M. leprae TN genome sequence (4) is a breakthrough for leprosy research, which has served as a public domain used by the Groathouse study group for the screening of potential VNTR for molecular typing (8). Tandem repeats are usually classified among satellites (spanning megabases of DNA, associated with heterochromatin), minisatellites (repeat units in the range from 6 to 100 bp, spanning hundreds of base pairs) and microsatellites (repeat units in the range from 1 to 5 bp, spanning a few tens of nucleotides) (5, 7). Both microsatellite and minisatellite loci have been selected for this study to substantiate the polymorphic loci as promising candidates used as the molecular typing tools for leprosy epidemiology.
In order to generate comprehensive and reliable data, a battery of 27 laboratory strains was used to develop PCR systems to amplify 44 STR target loci and the PCR products were sequenced. Unexpectedly, the results disclosed that not all the STR loci were polymorphic. Some of the loci were with no polymorphism which may have little or no long-term epidemiological value. In bacteria, loci containing a tandem repeat from the microsatellite class have been called simple sequence contingency loci. Altered number of repeats allows reversible on and off states of expression for the corresponding gene. In such an extreme situation, the microsatellite is not suitable for strain identification, epidemiological, or phylogenetic studies (7). The slight allele shift found in a few loci, (CA)6, 23-3, 27-5, and 12-5, was on one hand supposed to be a result of replication slippage, which can lead to an increase or a decrease in the copy number of the repeat element during cell division and inclined to be attributed to the locus with no polymorphism, and on the other hand, there might be different subpopulations of M. leprae.
As far as we are concerned, the nine combined microsatellite loci with strong discriminatory capacity which have been also tested for its stability and reproducibility in this study were the most likely and reliable sites for genetic diversity for M. leprae. Our previous study showed the 10-copy TTC repeat was the predominant genotype in a leprosy-endemic village (11). Nonetheless, by TTC alone, no conclusion could be drawn that bacteria were from the same population. Meanwhile, the multicase household which was examined the identical genotype for TTC alone does not mean an identical M. leprae isolate at all. As a matter of fact, when eight polymorphic loci rather than TTC, (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15, (AT)17, (TA)18, and (GTA)9, were added to the genotype, M. leprae could be further subdivided into two populations. Moreover, all 27 strains could also be identified individually by these combined nine polymorphic loci. Hence, this group of locus is anticipated to a wide application for clinical samples as a preliminary molecular typing system in leprosy epidemiology.
It is thought that tandem repeats located within the regulatory region of a gene can constitute an on/off switch of gene expression at the transcriptional level (7). In spite of this, the intergenic or interpseudogenic location of the microsatellite loci makes it unlikely that a difference in repeat copy number would itself confer any biological advantage, but it is possible that it acts as a marker for some biologically distinct subpopulation of bacteria and that it is carried along during selective expansion of this subpopulation (28). On the basis of this, the repeat unit which was differed by one copy number at (AT)17 and (GTA)9 loci when multicase household were subjected to the nine combined polymorphic loci indicated the presence of two subpopulations of M. leprae which have different dominant genotypes and presumably have propagated independently of each other. With regard to the fully matching polymorphism profile in each sample from the same dwelling at each polymorphic microsatellite loci, it was possible that the patients were infected by the same strain of M. leprae that survived in some common infectious sources. Taking these into consideration, there might be certain publicly shared infectious sources other than multibacillary cases played a role in the transmission of leprosy.
The unique function of rpoT was precisely evaluated in tracking the possible worldwide spread of leprosy and its contribution to the study of anthropology and archaeology (10, 13). We analyzed the other two loci with characteristics common to locus 6-3a in rpoT and found no regularity and significance such as rpoT had.
Young et al. (28) compared microsatellite profiles for samples taken from different anatomic sites from the same individual and observed that while skin, blood, and nasal cavity samples consistently generated matching profiles, frequent mismatches were found when bacteria in skin and nerves were compared. Mostly, we obtained identical repeats from different skin lesions on the same individual such as samples 6 (left earlobe), 7 (right earlobe) and 8 (earlobe) and 9 (back). Rarely, different skin lesions from the same patient exhibited mismatch profiles at the TTC locus (data was not shown). This probably suggested different bacterial populations coexisted instead of resulting from replication slippage. Nevertheless, the underlying mechanisms are awaiting to be elucidated.
Molecular typing systems are undergoing rapid technical improvements. Advance in the understanding of the biological basis of microbial biodiversity at subspecies levels will improve the conceptual framework required for proper epidemiologic interpretation of disease transmission. Wider application of these systems will surely contribute to the epidemiology of leprosy transmission and, therefore, allow for more effective control and prevention strategies to reach a world free of leprosy.
ACKNOWLEDGMENTS
This study was supported by a Health Research Grant of Emerging and Re-emerging Infectious Diseases, Ministry of Health, Labor and Welfare, Government of Japan, a grant of the Postdoctoral Fellowship Program, Japan Society for the Promotion of Science, a grant of the Foundation for Hansen's diseases in Asia, and a grant of the U.S.-Japan Cooperative Medical Science Program.
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Leprosy-TB Program, Provincial Health Service, Jl 17, Agustus, Manado, North Sulawesi, 95117, Indonesia
Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1, Aobacho, Higashimurayama-shi, 189-0002, Tokyo, Japan
ABSTRACT
A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multicase households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.
INTRODUCTION
Mycobacterium leprae is an obligate intracellular parasite with tropism for macrophages and Schwann cells and the only species of mycobacteria to infect peripheral nerves (19). It causes leprosy, a chronic granulomatous infection of the skin and peripheral nerves with characteristic deformities and disability (1). It is generally accepted today that the worldwide implementation of standardized multidrug therapy for leprosy has decreased the number of registered leprosy cases from a peak of 10 to 15 million to a current total of less than 1 million. However, the annual confirmed new cases remain at 500,000 to 700,000. This continuing number suggests that effective multidrug therapy fails to disrupt the chain of leprosy transmission (26).
Even though leprosy is one of the oldest recorded diseases, the source for M. leprae, the portal of its exit/entry, and the mode of transmission are still under investigation. Some regard human beings as a host for the bacteria, while others are still considering more possibilities. It was proposed that the nasal mucosa are the exit/entry pathway of M. leprae (9, 17, 18). In light of the transmission mode, human-to-human direct contact was first generally accepted (6, 16), with time as air-borne (23), as vector-borne (14, 25) and as vehicle-borne (3, 12) routes from evidence that has been obtained. All the progress in leprosy epidemiology was helpful in understanding the chain of transmission, yet at the same time was overwhelming.
In the past few years, studies focusing on leprosy transmission by molecular genotyping have shed new light on it (8, 10, 11, 22, 24, 27, 28). The most recent one was the report from Groathouse et al. (8). By in silico analysis, 44 promising polymorphic short tandem repeat (STR) sites, including both 33 microsatellite loci (repeat units of 1 to 5 bp) and 11 minisatellite (repeat units of >5 bp) were selected from the M. leprae TN genome sequence. Variable-number tandem repeats (VNTRs) at 9 of 11 STR loci from four clinical isolates of M. leprae were found. Clearly, it is urgent to accumulate extensive data for developing powerful typing tools to tracking the transmission of leprosy to finally reach a world free of leprosy.
In this study, we identified 32 polymorphic loci throughout the 44 STRs in a batch of 27 laboratory strains by sequencing and demonstrating the applicability and feasibility of nine potential loci acting as genetic markers to discriminate different M. leprae strains.
MATERIALS AND METHODS
M. leprae strains. A panel of 27 laboratory strains of M. leprae was subjected for genetic analysis. Strains were maintained by inoculation into nude mice footpads annually in our laboratory. All the strains used in this study were recovered from multibacillary cases. Excluding Thai-53, Thai-311 and Thai-237 were from Thailand, Indonesia-1 was from Indonesia, and Korea 3-2 was from Korea, and the others were from Japan.
All patients were from geographically distinct regions. Four strains, Thai-53, Kyoto-1, Zensho-4, and Korea3-2, and 17 samples, namely, the fourth generation of Thai-53 (Thai-53 4th), Thai-53 7th, Thai-53 11th, Kyoto-1 3rd, Kyoto-1 5th, Kyoto-1 7th, Kyoto-1 8th, Zensho-4 (biopsy specimen), Zensho-4 1st, Zensho-4 2nd, Zensho-4 3rd, Zensho-4 4th, Korea3-2 (biopsy specimen), Korea3-2 1st, Korea3-2 2nd, Korea3-2 3rd, and Korea3-2 4th (Table 6) were employed for the stability testing of loci. Partially purified bacterial materials were prepared from the inoculated footpads by deferential centrifuging and suspension in a phosphate buffered saline at concentration of 105-6/ml.
Primer selection. Primer sets for the amplification regions of DNA containing the STR sites were referred to the study of Groathouse et al. (8). The sequences of primer pairs were listed (Table 1).
Slit-skin smears from multicase household. Eleven slit skin smears were collected as the same manner as that for Bacterial Index examination from leprosy patients in a total of three households. Among them, five smears from five patients in household I; four smears from two patients in household II, sample 6 from the left earlobe and sample 7 from the right earlobe within a single individual; sample 8 from the earlobe and sample 9 from the back in another individual; and two smears from two patients in household III (Table 7).
Preparation of M. leprae DNA from strains and slit skin smears and sequencing analysis. M. leprae templates from both strains and slit-skin smears were prepared by treatment with lysis buffer at 60°C overnight as described previously (13). PCR amplification of STR sites as well as sequencing analysis was performed under the same condition as described elsewhere using the listed primer pairs (10, 11, 12, 13). Briefly, target loci were amplified using a G mixture and a FailSafe PCR system (EPICENTRE, Madison, Wis.). DNA samples for sequencing were recovered with a MinElute gel extraction kit (QIAGEN GmbH, Hilden, Germany) after electrophoresis of PCR products. Samples were sequenced with a BigDye terminator cycle sequencing FS Ready Reaction kit (Perkin-Elmer Applied Biosystems, Norwalk, Conn.) and an ABI Prism 310 genetic analyzer (Perkin-Elmer). The nucleotide sequences obtained were analyzed using DNASIS software (Hitachi Software Engineering, Yokohama, Japan).
Multiplication of M. leprae strains in nude mice footpad. Inoculums of each strain were prepared from BALB/c-nu/nu nude mice which were inoculated with isolates of the third to fifth generations about 8 to 10 months before by Nakamura's method as described previously (15). Five-week-old male nude mice were injected with the inoculums containing 104 bacilli/0.05 ml of Hanks' balanced salt solution. Bacillary number in the footpads of six nude mice at 10, 20, 30, 40, and 50 weeks growth were examined by Shepard's method (21).
Ethical approval. Informed consent was obtained from all subjects, and the study was approved by the institutional ethics committee of National Institute of Infectious Diseases, Tokyo, Japan. Bacillary samples of nasal mucus and slit skin smears were collected when informed consent was obtained. Animal experiments were conducted under the approval of the institutional animal experiment committee.
RESULTS
Allelic diversity of 33 microsatellite loci in M. leprae strains. In the 27 M. leprae strains, 8 out of 33 selected microsatellite loci showed the same copy number of allele whereas the other 25 loci, (A)9, (G)9, (C)9, (G)10a, (G)10b, (G)11, (G)12, (C)16(G)8, (C)20, (G)22, (AC)8a, (AC)8b, (AC)9, (CA)6, (TA)8, (TA)9, (TA)10, (TA)13, (AT)10, (AT)15, (AT)17, (TA)18, (GGT)5, (GTA)9, and (AGA)20, presented at least two types of allele. All locus identifications are from Groathouse et al. (8) to maintain integrity. This result validated the comments from Groathouse group on the polymorphic (C)20, (AT)17, (TA)18, (GTA)9, and (AGA)20 and no diversity of (CG)6, albeit (AC)9 was also examined for considerable polymorphism here which was predicted as nonpolymorphic loci by them. However, the last repeat unit of some results was not easily defined for unknown reasons (Table 2).
Allelic diversity of eleven minisatellite loci in M. leprae strains. Throughout the 27 M. leprae strains, four out of 11 selected minisatellite loci showed no diversity while the other seven loci, 6-3a, 6-7, 12-5, 18-8, 21-3, 23-3, and 27-5, exhibited variable characteristics, which also verified Groathouse et al. (8) reports on the polymorphism of loci 6-7, 12-5, 18-8, 21-3, and 27-5 (Table 3).
Variations of allelic diversity at STR loci in M. leprae strains. Forty-four selected STR loci consisted of nine protein coding genes, 19 intergenic regions, and 16 pseudogenes. Polymorphism was revealed in six among nine (67%) protein coding genes, 16 out of 19 (84%) intergenic genes, and 10 of 16 (63%) pseudogenes. Almost all of the STR loci located in intergenic regions or in pseudogenes and were unlikely involved in biological functions. Overall, thirty-two out of 44 STR loci were polymorphic and 12 loci were invariable that might be of limited value as epidemiological markers. The variations were between 2 and 11 alleles (Table 4). Notably, at locus (CA)6, isolate Thai-237 differed from the other 26 strains by one repeat unit (Table 2). Similarly, at locus 23-3, isolate Hoshizuka-1 alone, and at locus 27-5, isolate Airaku-2 alone had one copy difference allele (Table 3). Additionally, at locus 12-5, a five-copy-repeat unit was obtained from M. leprae TN, but all of the strains in this study showed three- and four-copy-repeat units instead of the five-copy-repeat unit (Table 3).
Comparison among the loci fall within protein coding genes with two variations. Locus 6-3a is in the rpoT gene coding RNA polymerase sigma factor in M. leprae contained two variations, three- and four-copy alleles (2, 13). Twelve strains, Zensho-2 to Zensho-12, harbored the three-copy allele whereas 15 strains, Hoshizuka-1 to Kyoto-1, had a four-copy allele in the rpoT gene (Table 3). Geographic distribution of each genotype of M. leprae rpoT revealed the distinguished distribution in several countries in the world (13, 10). Intriguingly, we compared the copy numbers of the other two protein coding genes 12-5 and 18-8 having two alleles with the strains carrying three-copy and four-copy rpoT that were closely associated with geographic distributions and no correlation was exploited.
Discriminatory capacity, stability, and reproducibility of nine potential polymorphic loci detected among serial passage strains by nude mice. Nine polymorphic microsatellite loci, (AC)9, (AC)8b, (AC)8a, (TA)10, (AT)17, (AT)15 (GTA)9, (TA)18, and (AGA)20 (also named TTC) were selected for discriminatory capacity analysis. All 27 strains were divided into two groups, three-copy rpoT and four-copy rpoT, based on the rpoT polymorphism. By adding the conjunction of 9 loci, they were distinguished from each other (Table 5). Then stability and reproducibility testing was carried out among them through serial passage strains by nude mice owing to the nature of susceptibility of replication slippage of microsatellite (28). One generation designated approximately 1 year, these strains have been in passage for 4 to 11 years. Not only the identical profile of allele was shared among the different generations of the strains at each locus, but also the copy number of repeats was in agreement with that in the repeated experiment of the same strains, which ensured these polymorphic microsatellite loci were highly stable and reproducible (Tables 1 and 6). The stability of TTCwas done previously (11)]. It was the rationale for these nine polymorphic microsatellite loci to be a significant source of informative markers for the identification and discrimination of M. leprae strains.
Application of polymorphic microsatellite loci for multicase households. Based on the condition of leprosy patients living in same household and possessing the identical TTC pattern, eleven smears from three household were chosen (Table 7). Five bacterial materials from household I shared identical 13-copy of TTC repeat were also subjected to loci (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15, (AT)17, (TA)18, and (GTA)9 and presented the identical copy number of 12, 7, 10, 10, 13, 15, 14, and 9, respectively. In household II which had 12 copies of the TTC repeat, four smears at the above loci showed equal repeats of 10, 7, 9, 10, 13, 13, 11, and 11. There was an exact match by copy number at each locus between samples 6 and 7 as well as samples 8 and 9 within one individual. In household III, two smears harboring eight copies of the TTC repeat, the copy number of loci (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15 and (TA)18 was 10, 7, 9, 8, 16, and 15. respectively. Strikingly, at locus (AT)17, a difference of 9 and 10 copies and a mismatch of 11 and 12 copies at locus (GTA)9 were found in these two smears, respectively.
Linkage between the growth rate of M. leprae in nude mice footpad and allelic diversity. Shepard et al. (20) reported the hereditary fast-slow growth difference among M. leprae strains in conventional mouse footpads, which was also observed in nude mice footpads in our laboratory. However, in this study, the growth curve of M. leprae in nude mice footpads gave no difference between strains with three-copy rpoT (Zensho-2, Airaku-3, and Thai-53) and those with four-copy rpoT (Izumi and Zensho-4) except Kyoto-2, which revealed the copy number of rpoT was irrelevant to the fastidious growth. In addition, no correlation was found between the VNTR of the other protein coding genes and M. leprae growth.
DISCUSSION
Poor understanding of leprosy transmission has hindered us from eradicating the disease even though M. leprae was identified as the pathogen as early as 1873 by Hansen (19). The unusual biological traits of being especially slow growing as well as the inability to culture M. leprae in vitro have partially accounted for the setbacks of detailed studies on leprosy. However, the determination of the complete M. leprae TN genome sequence (4) is a breakthrough for leprosy research, which has served as a public domain used by the Groathouse study group for the screening of potential VNTR for molecular typing (8). Tandem repeats are usually classified among satellites (spanning megabases of DNA, associated with heterochromatin), minisatellites (repeat units in the range from 6 to 100 bp, spanning hundreds of base pairs) and microsatellites (repeat units in the range from 1 to 5 bp, spanning a few tens of nucleotides) (5, 7). Both microsatellite and minisatellite loci have been selected for this study to substantiate the polymorphic loci as promising candidates used as the molecular typing tools for leprosy epidemiology.
In order to generate comprehensive and reliable data, a battery of 27 laboratory strains was used to develop PCR systems to amplify 44 STR target loci and the PCR products were sequenced. Unexpectedly, the results disclosed that not all the STR loci were polymorphic. Some of the loci were with no polymorphism which may have little or no long-term epidemiological value. In bacteria, loci containing a tandem repeat from the microsatellite class have been called simple sequence contingency loci. Altered number of repeats allows reversible on and off states of expression for the corresponding gene. In such an extreme situation, the microsatellite is not suitable for strain identification, epidemiological, or phylogenetic studies (7). The slight allele shift found in a few loci, (CA)6, 23-3, 27-5, and 12-5, was on one hand supposed to be a result of replication slippage, which can lead to an increase or a decrease in the copy number of the repeat element during cell division and inclined to be attributed to the locus with no polymorphism, and on the other hand, there might be different subpopulations of M. leprae.
As far as we are concerned, the nine combined microsatellite loci with strong discriminatory capacity which have been also tested for its stability and reproducibility in this study were the most likely and reliable sites for genetic diversity for M. leprae. Our previous study showed the 10-copy TTC repeat was the predominant genotype in a leprosy-endemic village (11). Nonetheless, by TTC alone, no conclusion could be drawn that bacteria were from the same population. Meanwhile, the multicase household which was examined the identical genotype for TTC alone does not mean an identical M. leprae isolate at all. As a matter of fact, when eight polymorphic loci rather than TTC, (AC)8a, (AC)8b, (AC)9, (TA)10, (AT)15, (AT)17, (TA)18, and (GTA)9, were added to the genotype, M. leprae could be further subdivided into two populations. Moreover, all 27 strains could also be identified individually by these combined nine polymorphic loci. Hence, this group of locus is anticipated to a wide application for clinical samples as a preliminary molecular typing system in leprosy epidemiology.
It is thought that tandem repeats located within the regulatory region of a gene can constitute an on/off switch of gene expression at the transcriptional level (7). In spite of this, the intergenic or interpseudogenic location of the microsatellite loci makes it unlikely that a difference in repeat copy number would itself confer any biological advantage, but it is possible that it acts as a marker for some biologically distinct subpopulation of bacteria and that it is carried along during selective expansion of this subpopulation (28). On the basis of this, the repeat unit which was differed by one copy number at (AT)17 and (GTA)9 loci when multicase household were subjected to the nine combined polymorphic loci indicated the presence of two subpopulations of M. leprae which have different dominant genotypes and presumably have propagated independently of each other. With regard to the fully matching polymorphism profile in each sample from the same dwelling at each polymorphic microsatellite loci, it was possible that the patients were infected by the same strain of M. leprae that survived in some common infectious sources. Taking these into consideration, there might be certain publicly shared infectious sources other than multibacillary cases played a role in the transmission of leprosy.
The unique function of rpoT was precisely evaluated in tracking the possible worldwide spread of leprosy and its contribution to the study of anthropology and archaeology (10, 13). We analyzed the other two loci with characteristics common to locus 6-3a in rpoT and found no regularity and significance such as rpoT had.
Young et al. (28) compared microsatellite profiles for samples taken from different anatomic sites from the same individual and observed that while skin, blood, and nasal cavity samples consistently generated matching profiles, frequent mismatches were found when bacteria in skin and nerves were compared. Mostly, we obtained identical repeats from different skin lesions on the same individual such as samples 6 (left earlobe), 7 (right earlobe) and 8 (earlobe) and 9 (back). Rarely, different skin lesions from the same patient exhibited mismatch profiles at the TTC locus (data was not shown). This probably suggested different bacterial populations coexisted instead of resulting from replication slippage. Nevertheless, the underlying mechanisms are awaiting to be elucidated.
Molecular typing systems are undergoing rapid technical improvements. Advance in the understanding of the biological basis of microbial biodiversity at subspecies levels will improve the conceptual framework required for proper epidemiologic interpretation of disease transmission. Wider application of these systems will surely contribute to the epidemiology of leprosy transmission and, therefore, allow for more effective control and prevention strategies to reach a world free of leprosy.
ACKNOWLEDGMENTS
This study was supported by a Health Research Grant of Emerging and Re-emerging Infectious Diseases, Ministry of Health, Labor and Welfare, Government of Japan, a grant of the Postdoctoral Fellowship Program, Japan Society for the Promotion of Science, a grant of the Foundation for Hansen's diseases in Asia, and a grant of the U.S.-Japan Cooperative Medical Science Program.
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