Search tips
Search criteria 


Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. 2009 November; 77(11): 5071–5079.
Published online 2009 September 8. doi:  10.1128/IAI.00457-09
PMCID: PMC2772522

Cross-Reactive Immunity to Mycobacterium tuberculosis DosR Regulon-Encoded Antigens in Individuals Infected with Environmental, Nontuberculous Mycobacteria[down-pointing small open triangle]


Mycobacterium tuberculosis DosR regulon-encoded antigens are highly immunogenic in M. tuberculosis-infected humans and are associated with latent tuberculosis infection. We have investigated the hypothesis that infection with or exposure to nontuberculous mycobacteria (NTM) can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens since responsiveness has been observed in non-M. tuberculosis-exposed but purified protein derivative-responsive individuals. M. tuberculosis DosR regulon-encoded antigen-specific T-cell responses were studied in peripheral blood mononuclear cells (PBMCs) of NTM-infected/exposed individuals. BLASTP was used to determine the presence of M. tuberculosis DosR regulon-encoded protein orthologs among environmental mycobacteria and nonmycobacteria. Significant gamma interferon production was observed in PBMCs from NTM-infected/exposed individuals in response to M. tuberculosis DosR regulon-encoded antigens. DosR regulon-encoded protein orthologs were prominently present in tuberculous and environmental mycobacteria and surprisingly also in nonmycobacteria. The ubiquitous presence of the highly conserved DosR master regulator protein Rv3133c suggests that this is a general adaptive bacterial response regulator. We report a first series of M. tuberculosis antigens to which cross-reactive immunity is induced by NTM infection/exposure. The high conservation of M. tuberculosis DosR regulon-encoded antigens most likely enables them to induce cross-reactive T-cell responses.

Yearly, tuberculosis (TB) claims 1.7 million lives. Its global incidence exceeds 9 million new cases. TB morbidity and mortality are likely to increase further as a result of TB reactivation in human immunodeficiency virus type 1-Mycobacterium tuberculosis-coinfected individuals, as well as the rising frequencies of multidrug-resistant and extensively drug-resistant M. tuberculosis strains (41). It is estimated that 2 billion people carry a latent TB infection. This vast reservoir forms a major source of new TB cases: 1 out of every 10 M. tuberculosis-infected individuals will develop active TB disease at one point in their lifetime, while the remainder are able to contain the bacilli without any clinical symptoms.

In a series of recent M. tuberculosis antigen discovery studies, aiming at identifying new M. tuberculosis biomarker and vaccine antigens, we found that genes from the M. tuberculosis DosR (Rv3133c) regulon encode antigens that can induce specific T-cell immunity in M. tuberculosis-infected individuals (20, 32). Tubercle bacilli express the DosR regulon under in vitro conditions of hypoxia, low-dose nitric oxide (40), and carbon monoxide, (19, 37), conditions thought to be encountered by persisting intracellular bacilli in the immunocompetent host. Studies have also reported upregulated expression of DosR regulon genes in infected murine macrophages and infected murine lung tissues (19, 35-37). Importantly, immunity to M. tuberculosis DosR regulon-encoded antigens might contribute to the control of persistent M. tuberculosis infection since several DosR regulon-encoded antigens were preferentially recognized by subjects with latent TB infection (20, 32). Finally, we also found that Mycobacterium bovis BCG vaccination does not induce immune responses to M. tuberculosis DosR regulon-encoded antigens, which might partly underlie its inefficacy against late M. tuberculosis infection (16, 21).

During these studies, we unexpectedly observed that some M. tuberculosis DosR regulon-encoded antigens were also recognized by healthy controls (HCs) who had a positive in vitro gamma interferon (IFN-γ) response to purified protein derivative (PPD) or M. tuberculosis lysate. However, none of these tuberculin skin test (TST)-negative HCs had had any known exposure to or infection with M. tuberculosis or had received BCG vaccination (20). We therefore hypothesized that these responses could be due to exposure to environmental, nontuberculous mycobacteria (NTM).

NTM comprise all mycobacterial species that are not included within the M. tuberculosis complex (M. tuberculosis, M. bovis, M. bovis BCG, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canetti) or Mycobacterium leprae (8, 28). NTM are facultative intracellular bacteria with specific niches in the environment and are ubiquitously present and opportunistic. Diagnosis and treatment of lung infections caused by NTM involve careful evaluation of the infection since no reference standard or parameter exists by which the different NTM can be characterized (5). It has long been suspected that exposure to NTM can shape host immunity. For example, neonatal BCG vaccination results in significant protection against pediatric TB but generally fails to protect adequately against pulmonary TB in adults; this latter failure has been attributed in part to the immunomodulatory effects of NTM exposure/infection (2, 7, 10, 26), although the kinetics of the latter as well as the underlying mechanisms involved remain undefined.

Here, we have investigated whether infection with or exposure to NTM can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens. We also studied the presence of M. tuberculosis DosR regulon protein orthologs and homologs among mycobacteria and several nonmycobacterial species.


Study subjects.

One hundred thirty-seven Dutch subjects were included in this study: 80 blood bank donors; 33 healthy, M. tuberculosis-unexposed, BCG-nonvaccinated individuals; 12 NTM-infected subjects; and 12 NTM-exposed subjects. Full characteristics of the latter two groups have been described in detail elsewhere (4). In brief, we included patients for whom Mycobacterium marinum (n = 7) had been cultured from a typical lesion on the hand or forearm. To discriminate between infection and colonization, Mycobacterium kansasii (n = 5) infection was defined by characteristic radiographic abnormalities in combination with a positive culture from a bronchoscopic washing or lung biopsy specimen or at least two positive sputum cultures, in the absence of an alternative diagnosis. Currently, no reference standard or parameter exists by which exposure to environmental mycobacteria can be characterized or quantified. Individuals exposed to NTM were selected by reasoning that exposure would be most pronounced during repeated and intense contact with the natural habitat of environmental bacteria, due to professional or recreational activities. Owners of tropical fish tanks (n = 4), veterinarians (n = 3), and professional flower growers (n = 5) were selected as representative groups. Study subjects were recruited at the Leiden University Medical Center and at the Regional Health Service in Geleen, The Netherlands. The study protocols (P207/99 and P136/97) were approved by the Institutional Review Board of the Leiden University Medical Center.

All study subjects except blood bank donors, answered a questionnaire about BCG vaccination, travel history, and historic TB/NTM contact. None of the study subjects used or had used immunosuppressive treatment or belonged to a risk group for human immunodeficiency virus infection.

Blood was obtained by venipuncture after written informed consent was obtained. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation and stored in liquid nitrogen until use.

M. tuberculosis antigens.

Recombinant proteins (Table (Table1)1) were produced as described previously (15). Briefly, selected M. tuberculosis H37Rv genes were amplified by PCR from genomic H37Rv DNA and cloned by Gateway Technology (Invitrogen, San Diego, CA). Proteins were overexpressed in Escherichia coli strain BL21(DE3) and purified as described previously (15). Recombinant proteins were subjected to quality assurance and quality control assays as described in reference 21, including testing for residual endotoxin levels, absence of nonspecific T-cell stimulation in PPD-nonresponsive donors, and absence of cellular toxicity as assessed by lack of inhibition of mitogen-induced proliferation.

Selected DosR antigens tested in the present study

As reference antigens PPD (RT49; SSI, Denmark) and M. tuberculosis “hypoxic” lysate (31, 43, 44) were used (provided by Peter Andersen, SSI, Denmark).

Synthetic peptides from ESAT-6 and culture filtrate protein 10 (CFP-10) were produced as previously described (17). For both antigens, nine peptides (20 amino acids with a 10-amino-acid overlap) spanning the complete protein sequence were synthesized.

Lymphocyte stimulation assay.

Lymphocyte stimulation assays were performed as previously described (20). Briefly, PBMCs (1.5 × 105/well) were cultured in Iscove's modified Dulbecco's medium (Gibco, Paisley, United Kingdom), supplemented with 10% pooled human serum, 50 U/ml penicillin, and 50 μg/ml streptomycin, in 96-well round-bottom microtiter plates (Nunc, Roskilde, Denmark) at 37°C in 5% CO2, in the absence or presence of stimulant (in triplicate). The following antigen concentrations were used: all M. tuberculosis recombinant proteins at 0.33 μM (average concentration, 8 μg/ml; range, 4.3 to 16.2 μg/ml), M. tuberculosis hypoxic lysate and PPD both at 5 μg/ml, and peptide pools of ESAT-6 and CFP-10 at 1 μg/ml for each peptide. Phytohemagglutinin (2 μg/ml) (Remel, United Kingdom) and medium were used as positive and negative controls, respectively. At day 6, supernatants were harvested and stored at −20°C until use.

Detection of IFN-γ by enzyme-linked immunosorbent assay.

IFN-γ concentrations were measured by enzyme-linked immunosorbent assay (U-CyTech, Utrecht, The Netherlands) as previously described (20). The detection limit of the assay was 20 pg/ml. Values of unstimulated cultures (median concentration, 20 pg/ml; range, 20 to 184 pg/ml) were subtracted from stimulated cultures to obtain the antigenic responses. Responses were regarded positive if the response was ≥100 pg/ml.

Assessment of DosR protein orthologs in environmental (myco)bacteria.

Gene homologs are genes in different species that descended from a common ancestral DNA sequence. Gene homologs are further specified as orthologs when they have retained the same function in the different species. Here, protein orthologs for M. tuberculosis DosR regulon-encoded antigens were identified as reciprocal best matches. Protein sequences that were sourced from the National Center for Biotechnology Information (NCBI) and annotated at Tuberculosis Database (30) were used in this analysis. Using the Basic Local Alignment Search Tool (BLAST) program (1), protein sequences from M. tuberculosis DosR regulon-encoded antigens were compared with other (myco)bacterial species and vice versa. Reciprocal best-matching pairs between M. tuberculosis DosR regulon-encoded antigens and those of others were identified as ortholog pairs. When there was no reciprocal best match, a hit that produced the highest percent similarity score was taken as the best homolog. A cutoff value score of 40% similarity was used.

Statistical analysis.

The Wilcoxon signed-rank test was used to compare the responses per antigen in the study population to those under the unstimulated (medium) condition. The Bonferroni correction was applied as a post hoc adjustment: corrected P value = 1 − (1 − P)n. The Kruskal-Wallis test was used for comparison between groups and followed by Dunn's post hoc test when appropriate. All statistical analyses were performed on the raw data (i.e., not corrected for the medium values). Statistical analyses were performed with SPSS 14.0 and Graph Pad Prism 4.02; P < 0.05 was considered to be statistically significant.


Selection of DosR regulon-encoded antigens.

Eight of the most interesting M. tuberculosis DosR regulon-encoded antigens were selected for further investigation (Table (Table1).1). Selection was based on T-cell recognition profiles in TB patients, TST converters, and uninfected HCs observed in our earlier study (20): Rv1733c, Rv2029c, Rv2032, Rv2627c, and Rv3129 ranked among the top 10 most frequently recognized antigens in all three groups. Antigens Rv2626c and Rv2628 were selected as they ranked among the top 10 antigens in at least two study groups, including the TST converters. Rv2031c (16-kDa heat shock protein, HspX, α-crystallin) (42) was selected since it has been relatively well studied in antimycobacterial immunity. All antigens were tested in equimolar concentrations to allow for direct comparison of immunogenicity between the different proteins, since they varied in size (12.3 to 46.3kDa).

In vitro PPD responses in healthy individuals: a survey among healthy M. tuberculosis-unexposed donors.

Given the ubiquitous nature of NTM, it is likely that humans are exposed to and immunologically primed by NTM (39a). We therefore assessed the in vitro responsiveness to PPD in two different groups of healthy individuals and, for comparison, in NTM-exposed/infected individuals. Of note, PPD is not M. tuberculosis-specific since it contains many antigens that are cross-reactive with other mycobacteria, including BCG and NTM.

The first group of HCs comprised M. tuberculosis-uninfected/unexposed, BCG-unvaccinated individuals (n = 27). Twenty individuals had a negative TST (the remaining 7 were not TST tested). Second, a group of healthy Dutch blood bank donors (n = 66) was included (unknown TST status). This group was less well defined since no specific information was available concerning BCG vaccination and TB exposure/contact history. However, given the low TB incidence in The Netherlands and the lack of a national BCG vaccination policy, none of these parameters would be expected to be of high significance here.

Figure Figure11 shows in vitro IFN-γ responses to PPD in both groups of healthy controls. In both groups, over half of the individuals, 56% and 68%, respectively, responded in vitro to PPD (IFN-γ, >100 pg/ml). Although these responses are most likely the result of exposure to NTM, in the case of older blood bank donors we cannot formally exclude a contribution of remote TB exposure or BCG vaccination. The proportions of in vitro PPD responders in the NTM-infected/exposed group (18/24 of these individuals had been TST tested, overall with a negative TST result) were slightly higher, both 75%. In general, the high levels of in vitro responsiveness to PPD in these selected study groups may be due to the higher sensitivity of in vitro 6-day assays compared to the TST.

FIG. 1.
Results of an in vitro PPD survey in healthy individuals and NTM-infected/exposed individuals. HCs were either a group of well-defined healthy, M. tuberculosis-uninfected, non-BCG-vaccinated individuals (n = 27) or a group of random healthy blood ...

In vitro IFN-γ responses to selected M. tuberculosis DosR regulon-encoded antigens in individuals infected with or exposed to environmental mycobacteria.

We next studied 24 subjects with either proven NTM infection (n = 12) or significant exposure to NTM (n = 12) (Table (Table2).2). Antigens were tested in all 24 study subjects, except for Ag85B, Rv2626c, Rv2032, and CFP-10 (all four antigens, n = 23), and Rv3129 (n = 17), due to insufficient available PBMCs from some donors. For five DosR regulon-encoded antigens, a significant response was found (Rv1733c, P = 0.0026; Rv2029c, P = 0.011; Rv2032, P = 0.0341; Rv2626c, P = 0.0226; and Rv2627c, P = 0.0025; Wilcoxon signed-rank test) (Fig. (Fig.2A).2A). The Bonferroni correction was performed following the Wilcoxon signed-rank test. The Bonferroni correction is regarded as conservative: combined with the small study population, it may result in an underestimation of the significance of the results. Nevertheless, following this correction, responses to two M. tuberculosis DosR regulon-encoded antigens remained significant: Rv1733c (P = 0.033) and Rv2627c (P = 0.032).

FIG. 2.
IFN-γ responses to M. tuberculosis antigens in NTM-primed individuals and in healthy, PPD-negative (PPD) individuals. Responses to M. tuberculosis antigens were measured as follows. M. tuberculosis lysate, Ag85B, ESAT-6, CFP-10, and a ...
Characteristics of NTM-infected or -exposed study subjects included in this study

The highest frequency of responders was detected following stimulation with Rv1733c (42% response), while only 8% responded to Rv2031c (confirming our previous observations (20).

A significant proportion of the donors responded to control antigens Ag85B (43%), ESAT-6 (25%), and CFP-10 (35%). All responses to ESAT-6 and CFP-10 were confined to the individuals who also responded to PPD or M. tuberculosis lysate. As expected, many subjects responded to PPD and M. tuberculosis lysate: 75% and 63%, respectively.

Importantly, the responses and the proportion of responders to M. tuberculosis DosR regulon-encoded antigens were low (nonsignificant) or absent in healthy TST-negative and in vitro PPD-negative individuals (Fig. (Fig.2B)2B) and M. tuberculosis lysate-nonresponsive individuals (20), showing that responses to M. tuberculosis DosR regulon-encoded antigens are associated with mycobacterium responsiveness and exposure.

Taken together, our results show that individuals infected with or exposed to environmental mycobacteria have high and significant IFN-γ responses to individual antigens of the M. tuberculosis DosR regulon-encoded antigens. The majority of these individuals responded to the complex mycobacterial antigens PPD and M. tuberculosis hypoxic lysate in the absence of any detectable M. tuberculosis exposure, compatible with NTM infection/exposure.

IFN-γ responses to M. tuberculosis DosR regulon-encoded antigens in individual groups with specific NTM infection or exposure.

In the previous section, all NTM-infected/exposed individuals were analyzed as a single group. Here, although numbers are relatively small, results are analyzed per subgroup: Figure Figure33 shows IFN-γ responses to PPD, Ag85B, ESAT-6, and CFP-10 and to M. tuberculosis DosR regulon-encoded antigens Rv1733c, Rv2029c, Rv2626c, and Rv2627c in M. marinum patients (n = 7), M. kansasii patients (n = 5), and NTM-exposed individuals (n = 12).

FIG. 3.
IFN-γ responses to M. tuberculosis antigens PPD, Ag85B, ESAT-6, and CFP-10 and M. tuberculosis DosR regulon-encoded antigens Rv1733c, Rv2029c, Rv2626c, and Rv2627c in individuals infected with M. marinum (n = 7) or M. kansasii (n = ...

A number of individuals responded to ESAT-6 and CFP-10, although these responses were low in M. kansasii-infected individuals. Responses to CFP-10 were significantly higher in the M. marinum-infected group (P < 0.05), in line with our previous work (4).

Overall, responses to M. tuberculosis DosR regulon-encoded antigens are found in all different subgroups. Particularly to Rv2029c and Rv2626c, higher responses were found in NTM-exposed rather than NTM-infected individuals.

The presence of M. tuberculosis DosR regulon protein orthologs in environmental (myco)bacteria.

Twenty-seven bacterial genomes were analyzed for the presence of orthologs of the 48 M. tuberculosis H37Rv DosR regulon-encoded antigens. The selection encompassed 4 M. tuberculosis strains (CDC1551, “C,” “F11” and “Haarlem”), 3 tuberculous, non-M. tuberculosis strains (M. bovis, M. bovis BCG, and M. leprae), 8 NTM strains (e.g., M. marinum), and 12 other (environmental) bacteria (e.g., Streptomyces spp.) phylogenetically related to mycobacteria. Results are summarized in Table Table3.3. When an ortholog was lacking, the best homolog was selected. As expected, high degrees of similarities for all M. tuberculosis DosR regulon sequences were found in M. tuberculosis strains: identities were close to or exactly 100%, and few DosR regulon orthologs scored below 99% similarity. In contrast, M. leprae had orthologs/homologs for only six M. tuberculosis DosR regulon-encoded antigens, compatible with its well-known genome downsizing (9). Nevertheless, M. leprae shared a high degree of similarity with other essential, M. tuberculosis non-DosR regulon-encoded proteins, including Rv0440 (Hsp65; 96% identity) and Rv3804 (Ag85A; 90% identity).

Presence of orthologs or homologs of M. tuberculosis H37Rv DosR antigens in tuberculous mycobacteria, NTM, and nonmycobacterial environmental bacteria

M. bovis and M. bovis BCG both contain virtually identical orthologs of all 48 M. tuberculosis DosR regulon-encoded antigens (similarities from 98% to 100%).

Six non-DosR regulon-encoded antigens, TB10.4 (Rv0288), Hsp65 (Rv0440), Ag85B (Rv1886c), TB10.3 (Rv3019c), Ag85A (Rv3804c), and ESAT-6 (Rv3875), were included as reference genes. These genes had orthologs in all seven tuberculous mycobacterial strains, with very high similarity to complete identity. TB10.3 and ESAT-6 formed the exception: for TB10.3, no ortholog was found in M. leprae, and as expected, no ESAT-6 ortholog was present in M. bovis BCG.

Forty-one out of 48 M. tuberculosis DosR regulon-encoded antigens appeared to have orthologs (or homologs) in the NTM species analyzed. The number of DosR regulon orthologs/homologs in the eight NTM species varied from the presence of 27 orthologs/homologs in M. avium to 39 in Mycobacterium vanbaalenii. Interestingly, almost half of the DosR regulon-encoded antigens were present in all eight NTM species. Orthologs of all five reference genes were found in the NTM.

Currently, the genome sequence of M. kansasii is incomplete, and therefore it was not included in Table Table3.3. However, nucleotide BLAST searches with the available sequences from NCBI resulted in the identification of 13 M. tuberculosis DosR antigen homologs/orthologs (similarities of 73% to 84%) in M. kansasii, including Rv2627c (80% similarity), which was tested in this study.

Variable numbers of M. tuberculosis DosR regulon orthologs and homologs were also found in nonmycobacterial species such as Streptomyces coelicolor (present in soil) and Bifidobacterium longum (a gastrointestinal commensal). When assessing the bacterial species with regard to the average number of orthologs per single DosR regulon-encoded antigen, NTM had significantly higher numbers of DosR regulon-encoded protein orthologs (average of 3.8 orthologs per single DosR regulon-encoded antigen among eight examined NTM) than the nonmycobacterial species (average of 2.5 orthologs per single DosR regulon-encoded antigen among 12 examined nonmycobacteria).

In contrast, most reference antigens had few or no orthologs in environmental nonmycobacterial species, with the anticipated exception of the highly conserved gene hsp65 (Rv0440).

An interesting observation concerned the M. tuberculosis DosR sequence (Rv3133c) itself: orthologs of this protein were found in almost all species assessed, with M. leprae forming the exception. Equally interesting is the fact that these Rv3133c orthologs had a strikingly high degree of similarity among all mycobacterial species (from 93 to 100%) and, to a lesser extent, also a considerable degree of similarity in more distantly related nonmycobacterial species (ranging from 50 to 87%).

Taken together, our results demonstrate that M. tuberculosis DosR regulon-encoded proteins have orthologs in both environmental mycobacterial and nonmycobacterial species, with a remarkably high degree of similarity for the dormancy response regulator gene Rv3133c.


This study was designed to investigate the hypothesis that infection with or exposure to NTM can induce cross-reactive immune responses to a recently identified set of M. tuberculosis antigens, namely M. tuberculosis DosR regulon-encoded antigens. Previously, we reported that M. tuberculosis-infected individuals (actively and latently) can respond to M. tuberculosis DosR regulon-encoded antigens (11, 20, 32) and that responsiveness to these late-stage antigens was associated with control of latent M. tuberculosis infection (20, 32). Unexpectedly, we observed that a significant proportion of healthy, M. tuberculosis-uninfected, BCG-unvaccinated individuals also responded to M. tuberculosis DosR regulon-encoded antigens, next to M. tuberculosis lysate (20) and PPD in vitro (21). We therefore hypothesized that these responses might have resulted from exposure to NTM, with concomitant induction of cross-reactive immune responses to M. tuberculosis DosR regulon-encoded antigens.

Of note, very few in vitro PPD-nonresponsive individuals responded to the tested recombinant M. tuberculosis antigens (Fig. (Fig.2B).2B). Possible contamination of the recombinant proteins is difficult to exclude in the absence of a “gold standard” control, but our rigorous quality assurance and quality control procedures and the strong correlation between responses to M. tuberculosis lysate/PPD and tested M. tuberculosis recombinant antigens strengthen the conclusion that responses observed are indeed truly specific. Moreover, the presence of M. tuberculosis DosR regulon-encoded homologs in nonmycobacterial species (Table (Table3)3) suggests that also nonmycobacteria might evoke responses to M. tuberculosis DosR regulon-encoded antigens.

From early to recent publications, it is evident that environmental mycobacteria such as M. kansasii, M. smegmatis, and Mycobacterium avium are capable of adapting to conditions of starvation (3, 23, 38). M. smegmatis not only behaved similarly to slow-growing M. tuberculosis under in vitro oxygen depletion and reactivation conditions (12) but also expressed orthologs of M. tuberculosis DosR antigens Rv2031c (α-crystallin; HspX), Rv3132c, Rv3133c (DosR; devR) and Rv3134c (22, 25). In addition, M. bovis BCG adapted to oxygen starvation in the same fashion as M. tuberculosis by upregulating DosR regulon-encoded antigens Rv2031c, Rv3133c, Rv2623, and Rv2626c (6), although it was recently shown that M. bovis BCG is defective in the induction of the M. tuberculosis dormancy genes Rv1736c and Rv1737c (18).

The observed IFN-γ responses of NTM-infected/exposed individuals to M. tuberculosis antigens, including M. tuberculosis DosR regulon-encoded antigens, support our hypothesis. They also provide indirect evidence that NTM express DosR protein orthologs/homologs in vivo (Fig. (Fig.2A2A and and33).

Recently, a number of mycobacterial genomes have been sequenced, including NTM genomes. We systematically analyzed these genomes for the presence of M. tuberculosis H37Rv DosR regulon-encoded orthologs (or when lacking, the best possible homolog) (Table (Table3).3). Results showed that the majority of mycobacterial genomes indeed encompass orthologs of M. tuberculosis DosR regulon-encoded genes. As expected, the highest similarities (analyzed at the amino acid sequence level in order to assess potential immunological cross-reactivity) were detected among members of the M. tuberculosis complex. Most relevant for our hypothesis was the fact that orthologs of 41 out of 48 different M. tuberculosis DosR regulon-encoded genes were detected in the genomes of NTM. Rather unexpectedly, several environmental nonmycobacterial species were also found to have appreciable numbers of M. tuberculosis DosR regulon-encoded orthologs. Of interest, the DosR response regulator itself, Rv3133c, was found in almost all species examined (with the exception of M. leprae, which is notorious for its downsized genome [9]). Rv3133c encodes a transcription factor that mediates the hypoxic response of M. tuberculosis (14, 27, 33, 34, 39). The widespread presence of Rv3133c orthologs and other members of the dormancy regulon suggests that adaptation to hypoxia is a characteristic shared by both pathogenic and nonpathogenic mycobacteria and even by species outside the genus Mycobacterium.

In the present study, patients with documented M. kansasii and M. marinum infections were included. Both mycobacteria can cause disease in otherwise healthy, immunocompetent individuals. Although M. avium is more ubiquitously present in the environment, patients with M. avium infection were not included since M. avium infections are often associated with preexisting disease or immune deficiency (5). Due to its ubiquitous nature, exposure to M. avium in our study groups, however, cannot be excluded.

Analysis of the responses in the M. marinum- or M. kansasii-infected patients showed that the former patients seemed to respond equally well to Rv1733c and Rv2627c, despite the fact that M. marinum orthologs of these two M. tuberculosis genes vary in the degree of similarity (52.3% and 84.5%, respectively). In contrast, responses to Rv2029c, Rv2626c (Fig. (Fig.3),3), and Rv2032 (data not shown) (29) were practically absent, despite the presence of orthologs of these genes in M. marinum. High responses to M. tuberculosis DosR regulon-encoded antigens Rv1733c, Rv2029c, Rv2626c, and Rv2627c were observed in patients who were intensely exposed to environmental mycobacteria. Our study is not sufficiently powered to explain these particular observations. One limitation is that levels of exposure to NTM in the latter group are unquantified as no valid set of criteria is available. Although the small numbers may have caused some of the observed differences, it also remains possible that different immune recognition profiles may have developed specifically following NTM infection or exposure, due to possible differences in expression of antigens. Additionally, immunomodulatory effects of repeated mycobacterial infection/exposure may have shaped the observed recognition profile. In any case, further studies will be needed to confirm and extend these findings.

In conclusion, we document T-cell immunity (IFN-γ responses) to M. tuberculosis DosR regulon-encoded antigens in individuals infected with or exposed to NTM, in the absence of M. tuberculosis infection, M. tuberculosis exposure, or BCG vaccination. The results lend support to the hypothesis that M. tuberculosis DosR regulon-encoded antigen-directed responses can be the result of exposure to or infection with cross-reacting NTM. The results are corroborated by the presence of orthologs of many different M. tuberculosis DosR antigens in environmental mycobacteria as well as nonmycobacteria. Prior studies have suggested significant effects of prior sensitization by NTM on BCG vaccination and host immunity to M. tuberculosis, but the antigens involved have not been identified (13, 24, 26). Our results warrant more specific studies to analyze the contribution and influence of cross-reactive immunity following NTM infection on host responses to M. tuberculosis, BCG, and potential novel TB vaccines.

Supplementary Material

[Supplemental material]


This work was supported by a grant from the Foundation Microbiology Leiden, the European Commission within the 6th Framework Programme, contract no. LSHP-CT-2003-503367. (The text represents the authors’ views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information.) This work was also supported by the Bill and Melinda Gates Foundation, Grand Challenges in Global Health (GC6#74 and GC12#82).

We thank Corine Prins for efforts in obtaining blood samples for this study, and we thank the group of Jan-Wouter Drijfhout for synthesis of ESAT-6 and CFP-10 peptides.

We have no financial conflicts of interest.


Editor: B. A. McCormick


[down-pointing small open triangle]Published ahead of print on 8 September 2009.

Supplemental material for this article may be found at


1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. [PubMed]
2. Andersen, P., and T. M. Doherty. 2005. The success and failure of BCG—implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 3:656-662. [PubMed]
3. Archuleta, R. J., H. P. Yvonne, and T. P. Primm. 2005. Mycobacterium avium enters a state of metabolic dormancy in response to starvation. Tuberculosis (Edinburgh) 85:147-158. [PubMed]
4. Arend, S. M., K. E. van Meijgaarden, K. de Boer, E. C. de Palou, D. van Soolingen, T. H. Ottenhoff, and J. T. van Dissel. 2002. Tuberculin skin testing and in vitro T cell responses to ESAT-6 and culture filtrate protein 10 after infection with Mycobacterium marinum or M. kansasii. J. Infect. Dis. 186:1797-1807. [PubMed]
5. Arend, S. M., D. van Soolingen, and T. H. Ottenhoff. 2009. Diagnosis and treatment of lung infection with nontuberculous mycobacteria. Curr. Opin. Pulm. Med. 15:201-208. [PubMed]
6. Boon, C., R. Li, R. Qi, and T. Dick. 2001. Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model. J. Bacteriol. 183:2672-2676. [PMC free article] [PubMed]
7. Brandt, L., J. F. Cunha, A. Weinreich Olsen, B. Chilima, P. Hirsch, R. Appelberg, and P. Andersen. 2002. Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis. Infect. Immun. 70:672-678. [PMC free article] [PubMed]
8. Brosch, R., S. V. Gordon, M. Marmiesse, P. Brodin, C. Buchrieser, K. Eiglmeier, T. Garnier, C. Gutierrez, G. Hewinson, K. Kremer, L. M. Parsons, A. S. Pym, S. Samper, D. van Soolingen, and S. T. Cole. 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. USA 99:3684-3689. [PubMed]
9. Cole, S. T., K. Eiglmeier, J. Parkhill, K. D. James, N. R. Thomson, P. R. Wheeler, N. Honore, T. Garnier, C. Churcher, D. Harris, K. Mungall, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. M. Davies, K. Devlin, S. Duthoy, T. Feltwell, A. Fraser, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, C. Lacroix, J. Maclean, S. Moule, L. Murphy, K. Oliver, M. A. Quail, M. A. Rajandream, K. M. Rutherford, S. Rutter, K. Seeger, S. Simon, M. Simmonds, J. Skelton, R. Squares, S. Squares, K. Stevens, K. Taylor, S. Whitehead, J. R. Woodward, and B. G. Barrell. 2001. Massive gene decay in the leprosy bacillus. Nature 409:1007-1011. [PubMed]
10. Demangel, C., T. Garnier, I. Rosenkrands, and S. T. Cole. 2005. Differential effects of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG strain expressing RD1 antigens. Infect. Immun. 73:2190-2196. [PMC free article] [PubMed]
11. Demissie, A., E. M. S. Leyten, M. Abebe, L. Wassie, A. Aseffa, G. Abate, H. Fletcher, P. Owiafe, P. C. Hill, R. Brookes, G. Rook, A. Zumla, S. M. Arend, M. Klein, T. H. Ottenhoff, P. Andersen, T. M. Doherty, and the VACSEL Study Group. 2006. Recognition of stage-specific mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis. Clin. Vaccine Immunol. 13:179-186. [PMC free article] [PubMed]
12. Dick, T., B. H. Lee, and B. Murugasu-Oei. 1998. Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol. Lett. 163:159-164. [PubMed]
13. Edwards, M. L., J. M. Goodrich, D. Muller, A. Pollack, J. E. Ziegler, and D. W. Smith. 1982. Infection with Mycobacterium avium-intracellulare and the protective effects of Bacille Calmette-Guerin. J. Infect. Dis. 145:733-741. [PubMed]
14. Florczyk, M. A., L. A. McCue, A. Purkayastha, E. Currenti, M. J. Wolin, and K. A. McDonough. 2003. A family of acr-coregulated Mycobacterium tuberculosis genes shares a common DNA motif and requires Rv3133c (dosR or devR) for expression. Infect. Immun. 71:5332-5343. [PMC free article] [PubMed]
15. Franken, K. L., H. S. Hiemstra, K. E. van Meijgaarden, Y. Subronto, J. den Hartigh, T. H. Ottenhoff, and J. W. Drijfhout. 2000. Purification of His-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr. Purif. 18:95-99. [PubMed]
16. Geluk, A., M. Y. Lin, K. E. van Meijgaarden, E. M. S. Leyten, K. L. M. C. Franken, T. H. M. Ottenhoff, and M. R. Klein. 2007. T-cell recognition of the HspX protein of Mycobacterium tuberculosis correlates with latent M. tuberculosis infection but not with M. bovis BCG vaccination. Infect. Immun. 75:2914-2921. [PMC free article] [PubMed]
17. Hiemstra, H. S., G. Duinkerken, W. E. Benckhuijsen, R. Amons, R. R. de Vries, B. O. Roep, and J. W. Drijfhout. 1997. The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries. Proc. Natl. Acad. Sci. USA 94:10313-10318. [PubMed]
18. Honaker, R. W., A. Stewart, S. Schittone, A. Izzo, M. R. Klein, and M. I. Voskuil. 2008. Mycobacterium bovis BCG vaccine strains lack narK2 and narX induction and exhibit altered phenotypes during dormancy. Infect. Immun. 76:2587-2593. [PMC free article] [PubMed]
19. Kumar, A., J. S. Deshane, D. K. Crossman, S. Bolisetty, B. S. Yan, I. Kramnik, A. Agarwal, and A. J. Steyn. 2008. Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon. J. Biol. Chem. 283:18032-18039. [PMC free article] [PubMed]
20. Leyten, E. M., M. Y. Lin, K. L. Franken, A. H. Friggen, C. Prins, K. E. van Meijgaarden, M. I. Voskuil, K. Weldingh, P. Andersen, G. K. Schoolnik, S. M. Arend, T. H. Ottenhoff, and M. R. Klein. 2006. Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis. Microbes Infect. 8:2052-2060. [PubMed]
21. Lin, M. Y., A. Geluk, S. G. Smith, A. L. Stewart, A. H. Friggen, K. L. M. C. Franken, M. J. C. Verduyn, K. E. van Meijgaarden, M. I. Voskuil, H. M. Dockrell, K. Huygen, T. H. Ottenhoff, and M. R. Klein. 2007. Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination. Infect. Immun. 75:3523-3530. [PMC free article] [PubMed]
22. Mayuri, G. Bagchi, T. K. Das, and J. S. Tyagi. 2002. Molecular analysis of the dormancy response in Mycobacterium smegmatis: expression analysis of genes encoding the DevR-DevS two-component system, Rv3134c and chaperone alpha-crystallin homologues. FEMS Microbiol. Lett. 211:231-237. [PubMed]
23. Nyka, W. 1974. Studies on the effect of starvation on mycobacteria. Infect. Immun. 9:843-850. [PMC free article] [PubMed]
24. Orme, I. M., and F. M. Collins. 1986. Crossprotection against nontuberculous mycobacterial infections by Mycobacterium tuberculosis memory immune T lymphocytes. J. Exp. Med. 163:203-208. [PMC free article] [PubMed]
25. O'Toole, R., M. J. Smeulders, M. C. Blokpoel, E. J. Kay, K. Lougheed, and H. D. Williams. 2003. A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis. J. Bacteriol. 185:1543-1554. [PMC free article] [PubMed]
26. Palmer, C. E., and M. W. Long. 1966. Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. Am. Rev. Respir. Dis. 94:553-568. [PubMed]
27. Park, H. D., K. M. Guinn, M. I. Harrell, R. Liao, M. I. Voskuil, M. Tompa, G. K. Schoolnik, and D. R. Sherman. 2003. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48:833-843. [PMC free article] [PubMed]
28. Piersimoni, C., and C. Scarparo. 2008. Pulmonary infections associated with non-tuberculous mycobacteria in immunocompetent patients. Lancet Infect. Dis. 8:323-334. [PubMed]
29. Purkayastha, A., L. A. McCue, and K. A. McDonough. 2002. Identification of a Mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr gene within macrophages, in standing versus shaking cultures, and under low oxygen conditions. Infect. Immun. 70:1518-1529. [PMC free article] [PubMed]
30. Reddy, T. B., R. Riley, F. Wymore, P. Montgomery, D. Decaprio, R. Engels, M. Gellesch, J. Hubble, D. Jen, H. Jin, M. Koehrsen, L. Larson, M. Mao, M. Nitzberg, P. Sisk, C. Stolte, B. Weiner, J. White, Z. K. Zachariah, G. Sherlock, J. E. Galagan, C. A. Ball, and G. K. Schoolnik. 2009. TB database: an integrated platform for tuberculosis research. Nucleic Acids Res. 37:D499-D508. [PMC free article] [PubMed]
31. Rosenkrands, I., R. A. Slayden, J. Crawford, C. Aagaard, C. E. Barry III, and P. Andersen. 2002. Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins. J. Bacteriol. 184:3485-3491. [PMC free article] [PubMed]
32. Roupie, V., M. Romano, L. Zhang, H. Korf, M. Y. Lin, K. L. M. C. Franken, T. H. M. Ottenhoff, M. R. Klein, and K. Huygen. 2007. Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice. Infect. Immun. 75:941-949. [PMC free article] [PubMed]
33. Saini, D. K., V. Malhotra, D. Dey, N. Pant, T. K. Das, and J. S. Tyagi. 2004. DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR. Microbiology 150:865-875. [PubMed]
34. Saini, D. K., V. Malhotra, and J. S. Tyagi. 2004. Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis. FEBS Lett. 565:75-80. [PubMed]
35. Schnappinger, D., S. Ehrt, M. I. Voskuil, Y. Liu, J. A. Mangan, I. M. Monahan, G. Dolganov, B. Efron, P. D. Butcher, C. Nathan, and G. K. Schoolnik. 2003. Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J. Exp. Med. 198:693-704. [PMC free article] [PubMed]
36. Shi, L., Y. J. Jung, S. Tyagi, M. L. Gennaro, and R. J. North. 2003. Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc. Natl. Acad. Sci. USA 100:241-246. [PubMed]
37. Shiloh, M. U., P. Manzanillo, and J. S. Cox. 2008. Mycobacterium tuberculosis senses host-derived carbon monoxide during macrophage infection. Cell Host Microbe 3:323-330. [PMC free article] [PubMed]
38. Smeulders, M. J., J. Keer, R. A. Speight, and H. D. Williams. 1999. Adaptation of Mycobacterium smegmatis to stationary phase. J. Bacteriol. 181:270-283. [PMC free article] [PubMed]
39. Sousa, E. H., J. R. Tuckerman, G. Gonzalez, and M. A. Gilles-Gonzalez. 2007. DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis. Protein Sci. 16:1708-1719. [PubMed]
39a. von Reyn, C. F., T. W. Barber, R. D. Arbeit, C. H. Sox, G. T. O'Connor, R. Brindle, C. Gilks, K. Hakkarainen, A. Ranki, C. Bartholomew, J. Edwards, and M. Magnusson. 1993. Evidence of previous infection with M. avium among healthy subjects: an international study of dominant mycobacterial skin tests. J. Infect. Dis. 168:1553-1558. [PubMed]
40. Voskuil, M. I., D. Schnappinger, K. C. Visconti, M. I. Harrell, G. M. Dolganov, D. R. Sherman, and G. K. Schoolnik. 2003. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198:705-713. [PMC free article] [PubMed]
41. WHO. 2006. Fact sheet no. 104. Revised March 2006. World Health Organization, Geneva, Switzerland.
42. Wilkinson, R. J., K. A. Wilkinson, K. A. De Smet, K. Haslov, G. Pasvol, M. Singh, I. Svarcova, and J. Ivanyi. 1998. Human T- and B-cell reactivity to the 16kDa alpha-crystallin protein of Mycobacterium tuberculosis. Scand. J. Immunol. 48:403-409. [PubMed]
43. Yuan, Y., D. D. Crane, and C. E. Barry III. 1996. Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial α-crystallin homolog. J. Bacteriol. 178:4484-4492. [PMC free article] [PubMed]
44. Yuan, Y., D. D. Crane, R. M. Simpson, Y. Q. Zhu, M. J. Hickey, D. R. Sherman, and C. E. Barry III. 1998. The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc. Natl. Acad. Sci. USA 95:9578-9583. [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)