Published ahead of print on April 1, 2005, doi:10.1164/rccm.200408-1147OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200408-1147OC
Clinical Relevance of Mycobacterium tuberculosis plcD Gene MutationsDepartment of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan; Arkansas Department of Health; Department of Epidemiology, College of Public Health, and Department of Neurobiology and Developmental Sciences, College of Medicine, University of Arkansas for Medical Sciences; and Central Arkansas Veterans Healthcare Center, Little Rock, Arkansas Correspondence and requests for reprints should be addressed to Zhenhua Yang, M.D., Ph.D., Epidemiology Department, School of Public Health, University of Michigan, 109 S. Observatory Street, Ann Arbor, MI 48109-2029. E-mail: zhenhua{at}umich.edu
To identify Mycobacterium tuberculosis virulence factors, we integrated comparative genomics and epidemiologic data analysis to investigate the relationship between certain genomic insertions and deletions in the phospholipase-C gene D (plcD) with the clinical presentation of tuberculosis (TB). Four hundred ninety-six well-characterized M. tuberculosis clinical isolates were studied. Approximately 30% (147) of the isolates had an interruption of the plcD gene. Patients infected with the plcD mutant were twice as likely to have extrathoracic disease as those infected by a strain without an interruption (adjusted odds ratio, 2.19; 95% confidence interval, 1.27, 3.76). When we limited the analysis to the 275 isolates with distinct DNA fingerprint patterns, we observed the same association (adjusted odds ratio, 2.74; 95% confidence interval, 1.35, 5.56). Furthermore, the magnitude of the association appeared to differ with the type of extrathoracic TB. Our findings suggest that the plcD gene of M. tuberculosis is potentially involved in the pathogenesis of TB, and the clinical presentation of the disease may be influenced by the genetic variability of the plcD region.
Key Words: extrathoracic tuberculosis • pathogenesis • virulence factors Tuberculosis (TB) is a major cause of morbidity and mortality worldwide (1). The lack of an effective vaccine to prevent those who are latently infected with Mycobacterium tuberculosis from developing active disease, the variable results obtained in bacillus Calmette-Guérin vaccine efficacy trials, the emergence of multidrug-resistant TB worldwide, and the increasing coinfection of HIV with M. tuberculosis in many regions of the world (2–4) highlight the need for a highly effective vaccine and new antimicrobial agents. Identification of the M. tuberculosis factors that contribute to the pathogenesis of TB is necessary if the goal to develop a more effective vaccine is to be reached. To date, identification of M. tuberculosis virulence factors has been based primarily on in vitro and animal studies (5). Although these studies have increased our understanding of the function of a number of M. tuberculosis genes and the impact of gene products on the organisms' behavior in various in vitro and in vivo models, correlation of in vitro findings and animal studies with the pathogenesis of human disease remains challenging (5). An alternative strategy to identify M. tuberculosis virulence factors (6–11) combines comparative genomics to identify genome alteration with epidemiologic methods, to assess associations between genetic polymorphisms and clinical characteristics of the disease. Previous comparative genomic studies have identified large sequence deletions in multiple regions of the M. tuberculosis genome (6, 11, 12). However, the epidemiologic and clinical phenotypes of these genomic alterations remain largely unknown. Of the previously reported genomic deletions, the region containing the phospholipase-C gene D (plcD) (6) is of particular interest because of the role of plc in the pathogenesis of disease caused by a number of intracellular bacteria (13–15). Furthermore, the expression of M. tuberculosis plc genes is strongly upregulated during the first 24 hours of macrophage infection, and plc gene mutants are attenuated in the late phase of the infection in a mouse model (16). This article reports an observational study that integrates comparative genomics with epidemiologic methods to investigate the genetic diversity in the plcD gene region among a large set of M. tuberculosis isolates and to assess the association between the observed genetic polymorphisms and the clinical presentation of the patients. Some of the results have been previously reported in the form of a meeting abstract (17).
Study Design and Sample This study was performed in two steps. First, we performed a molecular characterization of the plcD gene region of a sample of M. tuberculosis clinical isolates. Then, we assessed the association of the genetic alterations of the plcD gene with certain patient characteristics, particularly the clinical presentation of the patients. The study sample included 496 isolates of M. tuberculosis obtained from 496 patients with culture-confirmed TB diagnosed in Arkansas between January 1, 1996, and December 31, 2000. During the study period, a total of 973 TB cases were diagnosed in Arkansas. Of these 973 cases, 847 (87.05%) were defined as thoracic TB and 126 (12.95%) as extrathoracic TB using the definitions described later. The proportions of thoracic and extrathoracic TB being culture confirmed were 74.62 (632/847) and 69.05% (87/126), respectively, resulting in a total of 719 culture-confirmed cases. Of these 719 cases, 705 (98%) had a viable isolate at the Mycobacteria Research Laboratory at the Central Arkansas Veterans Healthcare Center. At the laboratory of the University of Michigan, the genomic DNA of 496 retrievable isolates was successfully extracted. All of these isolates were included in this study. Isolates were obtained from extrathoracic sites in 63 of the 68 (92.65%) extrathoracic TB cases studied. Genomic DNA from the isolates was extracted from Lowenstein-Jensen cultures using standard procedures (18). All the isolates were genotyped using a combination of IS6110 fingerprinting and pTBN12 secondary typing (19). Patient data were obtained from the Arkansas Department of Health surveillance records, as described previously (20). This study was approved by the Health Sciences Institutional Review Boards of the University of Michigan and the University of Arkansas for Medical Sciences.
Polymerase Chain Reaction Assays Genomic DNA of the M. tuberculosis laboratory strain H37Rv and the clinical strain CDC1551 served as a negative and a positive control, respectively, in PCR experiments as the plcD gene region is truncated and interrupted in strain H37Rv (6) and is intact in strain CDC1551 (http://www.tigr.org) (22).
Southern Blot Analysis
DNA Sequencing
Definitions
Statistical Analysis
Characteristics of Study Patients Of the 496 patients studied, 428 were defined as having thoracic TB and 68 as having extrathoracic disease, representing, respectively, 78.16 (68/87) and 67.72% (428/632) of the patients with culture-confirmed thoracic and extrathoracic disease in Arkansas during the study period. The anatomic sites of the 68 extrathoracic cases are set out in Table 1. Of the 443 patients who had a chest radiograph report available, 173 (39.05%) were found to have cavitary involvement. Of the 496 study isolates, 221 (44.56%) were contained within 58 fingerprint clusters based on a combination of IS6110 fingerprinting and pTBN12 secondary typing (19). The size of the clusters ranged from 2 to 16 patients. A comparison of the patients' sociodemographic information, including age, sex, race/ethnicity, geographic location and type of residency, homelessness, alcohol consumption, and drug use, showed no significant difference between the study sample and all the 705 patients with culture-confirmed disease who had viable isolates (p > 0.05). The two groups also had comparable clinical characteristics, including HIV status, clinical forms of disease (extrathoracic vs. thoracic), sputum smear positivity, and chest radiography findings (cavitary vs. noncavitary; p > 0.05).
Diversity of plcD Gene On the basis of the results of the plcD-PCR1, the 496 isolates fell into five groups, designated groups I through V. Of the five groups, group I (the wild-type) was the largest, comprising 349 (70.36%) of the 496 isolates. The plcD-PCR1 product size of group I isolates was 1.9 kb, identical to that of the CDC1551 product (Figure 1A). The remaining 147 isolates (29.64%) were designated as mutant-type and categorized into four groups (groups II–V). Isolates in groups II to IV all had a PCR product of different size, ranging from 2.4 to 3.3 kb (Figure 1A). Groups II, III, and IV contained 21 (4.23%), five (1.01%), and one (0.20%) isolate, respectively (Figure 2A). Group V included 120 (24.19%) isolates that, like strain H37Rv, failed to generate a product in plcD-PCR1, despite the fact that the DNA served as a target for the 16S rRNA gene control PCR. Both mutant and wild types were observed among isolates in 20 (34.48%) of the 58 clusters.
Characterization of Groups II, III, and IV Isolates in groups II, III, and IV were examined for the insertion of IS6110 into the plcD gene sequence and compared with the wild-type group and CDC1551 by Southern hybridization using IS6110 as a probe. IS6110 hybridization was observed in all the isolates in these three groups (Figure 1B), whereas group I and CDC1551 showed no IS6110 hybridization. DNA sequence analysis of the plcD-PCR1 products of all the isolates in groups II, III, and IV confirmed that a complete copy of IS6110 was inserted in each isolate. The insertion of IS6110 resulted in a partial deletion of the plcD gene in the isolates in groups III and IV, but an IS6110 insertion without a deletion was found for the isolates sequenced for group II. The site and orientation of the IS6110 insertions varied from isolate to isolate (Figure 2A).
Characterization of Group V Nineteen (15.83%) of the 120 group V isolates were in subgroup Vb. These isolates were not amplified by plcD-PCR2 because of the deletion of the sequence complementary to the forward primer (Figure 2B). However, they were successfully amplified by plcD-PCR3. DNA sequencing of the plcD-PCR3 product of these 19 isolates identified an IS6110 insertion of dual orientations at the right-hand–side end of gene MT1797 followed by the deletion of MT1798 and a partial plcD deletion of 171 to 689 bp long, thereby resulting in the loss of the plcD-PCR1F and plcD-PCR2F priming sites (Figure 2B). The remaining 55 (45.83%) of the group V isolates were placed in subgroup Vc. These isolates failed to be amplified by any of the primers used in the study. However, Southern blotting of the PvuII-restricted genomic DNA using the plcD gene as a probe confirmed that all 55 isolates had an interruption of the plcD gene. Of the 55 isolates, 35 (63.64%) appeared to have a complete deletion of the plcD gene, whereas the remaining 20 were found to have a partial deletion of the plcD gene of approximately 830 to 1,250 bp long.
Clinical Relevance of plcD Gene Region
This study found that approximately 30% of the study isolates had an interruption of the plcD gene by either an insertion of IS6110 inside the plcD gene or an insertion of IS6110 followed by a partial deletion of the plcD gene that most frequently involved a deletion of adjacent genes. The IS6110 insertion and genomic deletion in the plcD gene region of M. tuberculosis was observed in two earlier studies using small selected samples involving 24 and 25 isolates, respectively (12, 25), and in a recent study using a hospital-based sample that included 106 isolates (26). However, the relationship between the genotype of the plcD gene of M. tuberculosis and the clinical phenotype of the infection has not been described previously. Because extensive data regarding the isolates were available for the present study, we were able to assess the association between the plcD gene mutation and the clinical presentations of TB, while confirming the previously reported genetic diversity in this region using a much larger set of isolates. After adjustment for host risk factors (e.g., being female, non-Hispanic black, and HIV positive) for extrathoracic TB identified in a previous study using the same patient dataset (20), we found that infection by a strain having an interrupted plcD gene is associated with the occurrence of extrathoracic TB. One limitation of our study is a lack of information on HIV infection status for about half of the study subjects; thus, the adjustment for the confounding of HIV status in our analysis might be imperfect. However, having unknown HIV status was not found to be associated with extrathoracic TB by multivariable analyses (Table 3). The different degrees of association found between the plcD mutation and the different types of extrathoracic TB may suggest that the pathogenesis of TB in different anatomic sites could be affected differently by genetic changes in M. tuberculosis. Future studies with larger samples of clinical isolates collected from different anatomic sites would generate useful information to enhance our understanding of the mechanisms of host and M. tuberculosis interaction in the pathogenesis of different forms of TB. In the present study, the mutant plcD genotypes were associated with the insertion of IS6110. IS6110 can upregulate downstream genes through an outward-directed promoter in its 3' end (27). Sequence analysis of the mutant isolates showed that all the IS6110 insertions, either within the coding sequence of the plcD gene, or in the flanking region of plcD, resulted in a partial deletion of the coding sequence of the plcD gene. Thus, it is unlikely that the orientation of IS6110 made any difference in plcD gene function of the mutants. However, it is possible that the orientation of the insertion did make a difference in the expression of the adjacent genes, which might contribute to defining the clinical phenotype of the study patients. In addition, the mutant types in our study were attributable to only insertion and deletion events. Thus, it is possible that some wild-type isolates had small genetic alterations, such as point mutations, or small deletions that could reduce gene function. If this did occur, it could cause misclassification of the plcD genotype, which, in turn, might have weakened the strength of the association. Future investigations of single nucleotide changes and small deletions in this region and further functional analysis of different mutants in comparison with the wild-type isolates will add to our understanding of the role of the deleted genes in the virulence of M. tuberculosis. An interesting observation in this study is that a significantly higher proportion of patients having isolates with plcD mutations lived within a city limit. This raises the question of whether or not phospholipase C is involved in the airborne transmissibility of M. tuberculosis. The plcD mutations were not found significantly more frequently in any zip code, city, or county in this study (data not shown). The possibility of other confounders that might differ between city and non–city dwellers was explored by analyzing the geographic distribution of thoracic and extrathoracic TB and their respective proportion included in this study; no significant differences were found in the comparison (p = 0.84). A future study using epidemiologic linkage between/among clustered patients would allow us to study this question more thoroughly. Several host-related risk factors for extrathoracic TB have been reported previously (20, 28–30). However, this report is the first to observe a microbial change acting as an independent risk factor for extrathoracic TB. Like all epidemiologic studies, this study does not prove causal relationship. However, it does provide a rationale for the selection of gene targets for future functional studies aimed at identifying M. tuberculosis virulence factors. The usefulness of combining comparative genomics with epidemiologic data to study the pathogenesis of tuberculosis is exemplified by this report.
The authors thank Annadell H. Fowler, Leonard N Mukasa, Bill Starrett, Deborah Witonski, Peter J. Boldenow, and Patricia C. Juliao for their valuable efforts during the study. They acknowledge Dr. Kashef Ijaz's contribution to the establishment of the Arkansas Department of Health's surveillance database that was used for the study. They also thank Drs. Jack T. Crawford and Laura S. Cowan at the Centers for Disease Control and Prevention, Atlanta, GA, for providing the DNA preparation of CDC1551.
Supported by the National Institutes of Health (grant NIH-R01-AI151975). Conflict of Interest Statement: Z.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Y.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.F.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.H.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; F.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.D.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form August 31, 2004; accepted in final form March 23, 2005
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ABSTRACT
METHODS
2, or Fisher's exact test, as appropriate, we compared the characteristics of the study patients with all the culture-confirmed patients who had viable isolates (n = 705) to address potential selection bias. Then, we tested the differences in demographic and clinical characteristics between the wild and mutant types. The magnitude of the association between the plcD gene region genotype and the disease presentation was estimated using the odds ratio and 95% confidence intervals. To control for potential confounding of previously reported host-related risk factors for extrathoracic TB (
