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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 323-331

T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls

1 Department of Animal Sciences, University of Hyderabad - 500 046, Andhra Pradesh, India
2 Government General Chest Hospital, Hyderabad -500 018, Andhra, Pradesh, India

Date of Submission24-Jan-2012
Date of Acceptance04-Apr-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
M Sritharan
Department of Animal Sciences, University of Hyderabad - 500 046, Andhra Pradesh
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Source of Support: The financial support of Institute of Life Sciences University of Hyderabad MoU towards a part of this study is acknowledged., Conflict of Interest: None

DOI: 10.4103/0255-0857.99495

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 ~ Abstract 

Background: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. Objective: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 μg Fe/mL) and low iron (0.02 μg Fe / mL) cultures of M. tuberculosis. Materials and Methods: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. Results: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). Conclusions: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.

Keywords: Culture filtrate proteins, ESAT proteins, IFN-γ, iron, lymphocyte transformation test, M. tuberculosis

How to cite this article:
Duggirala S, Venu K, Subhakar K, Sritharan M. T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls. Indian J Med Microbiol 2012;30:323-31

How to cite this URL:
Duggirala S, Venu K, Subhakar K, Sritharan M. T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls. Indian J Med Microbiol [serial online] 2012 [cited 2019 Aug 17];30:323-31. Available from:

 ~ Introduction Top

Tuberculosis (TB) is a disease of global public concern. It is a major killer among the infectious diseases in India. Globally, there were 5.7 million notifications of new and recurrent cases of TB in 2010. [1] With more than 8 million active cases, 1.1 million deaths (in 2010), latent TB, the problems of HIV and drug-resistance, there is a great need to develop better control measures. The only TB vaccine available today is the live attenuated M. bovis BCG whose efficacy in protection against the disease is questionable. There is thus an immediate requirement for an effective vaccine, in particular, one that can protect individuals residing in an endemic area with a high prevalence of the disease. This can be made possible by a better understanding of host-pathogen interactions and the identification of immuno-protective antigens.

Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are promising vaccine candidates as they are potent inducers of cell-mediated immune response in the mammalian host. This complex mixture of proteins released into the immediate environment by the pathogen was first extensively characterised by Nagai and his group. [2] Today, with modern and sophisticated tools for effective separation, as many as 450 CFPs have been identified in the proteome of M. tuberculosis. [3] Several of these CFPs were shown to be immuno-protective in experimental animals [4],[5] and humans. [6],[7] The secretory proteome of M. tuberculosis consists of numerous actively secreted components; some of these proteins, namely the ESAT-6 and CFP-10 are unique to M. tuberculosis and are not expressed by other mycobacteria.

The T-cell response to the mycobacterial antigens plays an important role in the outcome of an infection. [8] The production of IFN-γ and a shift in favour of Th1 over Th2 immune response are generally considered as correlates of protective immunity. The peripheral blood mononuclear cell (PBMC) responses of tuberculin skin test-reactive healthy contacts of TB patients and those of healed TB (memory immune) subjects are suggested as the model of protective immunity against TB. [8] Accordingly, the antigens recognized by T lymphocytes of this group (i.e., sensitized / infected) but not by those of active TB patients (with disease) should be considered important for vaccine development.

The composition of the CFPs is influenced by factors such as period of growth of the pathogen, [9] temperature, aeration, etc. Here, we studied the influence of iron levels in the medium of growth on the expression profile of the CFPs. Iron, essential for the growth of for M. tuberculosis,[10] is not freely available in vivo as the mammalian host restricts the bio-availability of this essential micronutrient by a process called as 'nutritional immunity'. [11] There is increasing evidence to show that M. tuberculosis faces conditions of iron limitation in vivo. The pathogen responds to iron deprivation in vitro by elaborating the siderophore-mediated iron acquisition machinery. [15],[18] In bacteria, iron levels not only regulate the iron acquisition machinery but also other virulence determinants. [12] In this study, we compared the protein profile of the CPFs from high (CFP-high) and low (CFP-low) iron cultures of M. tuberculosis, fractionated them to obtain CF-Ags from CFP-high (H1-H5) and CFP-low (L1-L5) and compared the in vitro immune response of TB patients and normal endemic controls to these antigens and PPD.

 ~ Materials and Methods Top

Study subjects

A total of 60 newly diagnosed cases of TB patients from Andhra Pradesh, Government General Chest Hospital, Hyderabad, and 20 healthy individuals were included in the study. Approval of the Ethical committee of Government General Chest Hospital and informed consent from the patients were obtained before collection of samples. [Table 1] lists the study group. These subjects were between 30 and 34 years. The Mantoux test was not performed for any of the individuals due to its low specificity in this population and the unwillingness of the patients to remain in the clinic for measuring the induration after 48-72 h. All the TB patients had active TB who denied any prior anti-tubercular treatment. Individuals with history of HIV infection, diabetes, defaulters of treatment, relapse cases and other serious illness were excluded from the study. The control subjects included healthy donors with no clinical evidence of the disease.
Table 1: Study groups and their details

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Growth of M. tuberculosis under high and low iron conditions

M. tuberculosis
(ATCC 27294) was grown in liquid Proskauer and Beck medium under high (8 μg Fe/mL) and low iron (0.02 μg Fe/mL) conditions as described [13] . The siderophores mycobactin and carboxymycobactin were assayed using standard protocols. [13] Carboxymycobactin was expressed as siderophore units/100 mg of cell dry: one siderophore unit was equal to (AC - AS/AC) × 100, where AC represents the absorbance of CAS solution plus medium and AS was the absorbance of the CAS solution plus culture filtrate of the respective sample. Mycobactin was expressed as OD 450 nm units/g of cell dry weight.

Preparation of CFPs from high and low iron cultures

The CFPs from high (CFP-high) and low (CFP-low) iron grown organisms were processed as follows. After the addition of the protease inhibitor cocktail (ready-to-use mixture of 4-(2-amino-ethyl) benzene-sulphonyl fluoride (AEBSF), E-64, bestatin, leupeptin, aprotinin, and sodium EDTA; Sigma Chemical Company, St. Louis, USA) to a final concentration of 1 mM, the bacteria were removed first by centrifugation at 6000 rpm at 4°C for 15 min, followed by filtration of the supernatant through 0.2 μM filter to ensure no viable bacilli were present. CFPs were precipitated by ammonium sulfate (80% final saturation) at 4°C and then desalted by ultrafiltration using Amicon filters (Millipore, India). Protein concentration in the samples was determined by the BCA kit (Sigma Chemical Company, St. Louis, USA) and stored at -80°C till use.

SDS-PAGE and immuno-blotting

The proteins were subjected to 5-20% gradient SDS-PAGE gels using Tris-glycine buffer system [13] or Tris-Tricine buffer system (using 10% gels) for the separation of low-molecular-weight proteins. [14] Western blotting was done by standard protocols with ESAT-6-specific monoclonal antibody HYB 76-8/ MPT64-specific monoclonal antibody (kindly given by Dr. Ida Rosenkrands, Statens Serum Institute, Copenhagen).

Purification of CF-Ags by preparative gel electrophoresis

CF-Ags were prepared by preparative gel electrophoresis of CFP-high/CFP-low. After briefly staining the gel with Coomassie Blue, five fractions (CF-Ags) containing the CFPs between the molecular markers 116-66 kDa, 66-45 kDa, 45-25 kDa, 25-14 kDa, <14 kDa were eluted and precipitated with 5 volumes of ice-cold acetone. A total of 10 CF-Ags, five from CFP-high (H1 to H5) and five from CFP-low (L1 to L5) were thus obtained. Protein concentration in these ten samples were determined and stored at -80°C before use in the immune response studies.

Lymphocyte transformation test

Peripheral venous blood from patients/healthy controls was collected in heparinised tubes and the PBMCs were separated by Ficoll-Hypaque (Sigma Chemical Company, St. Louis, USA) density gradient centrifugation. After determination of the viable cell count by the trypan blue dye exclusion method, aliquots of 2 × 10 5 cells in 0.2 mL of RPMI-1640 medium (Sigma Chemical Company, St. Louis, USA) (supplemented with 100 IU/mL penicillin, 50 μg/mL of streptomycin (Hi-Media, India) and 5% foetal calf serum) was added in a 96-well flat-bottomed sterile tissue culture plate. After standardization for optimal response, the following concentrations of antigens were used in this study: CFP-high and CFP-low (2.5 μg/mL), CF-Ags (2 μg/mL) and PPD (3 μg/mL). Phytohemagglutinin (PHA, 2.5 μg/mL, Sigma Chemical Company, St. Louis, USA) was used as the mitogen. Negative controls included the PBMCs minus antigen/mitogen. Each set was done in triplicates. Cultures were incubated for a period of 72 h at 37°C in an atmosphere of 5% CO 2 . After 72 h, 100 μL of the supernatants was taken and stored at -80°C for assaying IFN-γ. Cell proliferation was measured by the MTT assay. [15] Briefly, 10 μL of MTT {3, (4, 5 dimethyl thiazol-2-yl) 2,5 diphenyl tetrazolium bromide; stock 5 mg/mL} was added to each well and incubated for 4 h at 37°C in an atmosphere of 5% CO 2 . The purple formazan crystals were dissolved by the addition of 100 μL of acidified isopropanol and the OD 570 nm was read in a microplate reader (BioRad, USA).

Assay of IFN-γ

The culture supernatants of PBMC collected above were assayed for IFN-γ by the human IFN-γ ELISA kit (Opt EIA from BD Pharmingen, San Diego) as per the manufacturer's instructions. The detection limit for IFN-γ was 2.35 pg/mL. Data is represented as the mean of the triplicates.

Statistical analysis

All data were analysed with SPSS ver. 15.00 software and Minitab. Non-parametric test Mann-Whitney U test and One-way ANOVA were performed to check the variance among groups. P < 0.05 was considered as significant. Proliferation was expressed as stimulation index (SI), which is the ratio of the test (PBMCs stimulated with antigen) and the control (PBMCs minus the antigen). SI value of 2 was taken as the cut-off for determining the percentage positivity. [15]

 ~ Results Top

Iron-limited (0.02 μg Fe/mL) growth of M. tuberculosis resulted in an increase in the levels of both the siderophores mycobactin and carboxymycobactin [Figure 1]. Electrophoretic separation by SDS-PAGE with the conventional Tris-glycine system did not reveal differences between CFP-high and CFP-low [Figure 2]a. With the Tris-Tricine buffer system, there was good resolution of the low-molecular-weight proteins and two iron-regulated CFPs Irp-1 and Irp-2 were detected upon iron limitation [Figure 2]b. The ESAT-6-specific monoclonal antibody HYB 76.8 reacted strongly with two bands of approximate molecular mass of 8 and 9 kDa in CFP-high lane 1 [Figure 2]c; both these proteins were down-regulated in the corresponding CFP-low [lane 2], [Figure 2]c. The immunoblot developed with the monoclonal antibody against the constitutively expressed MPT64 showed equal protein loading in lanes 1 and 2 [Figure 2]c.
Figure 1: Expression of mycobactin and carboxymycobactin in low iron cultures of M. tuberculosis. Both mycobactin and carboxymycobactin were up-regulated in low (L) iron (0.02 μg Fe/mL) as compared to high (H) iron (8 μg Fe/mL) cultures. The vertical bars represent the standard deviation of the mean from three independent experiments

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Figure 2: Culture fi ltrate proteins of high and low iron M. tuberculosis. Lanes 1 and 2 represent the SDS– PAGE profi le of CFP-high and CFP-low separated with the Tris– glycine (a) and the Tris– Tricine (b) buffer systems. These two panels also show the different CF-Ag pools (H1– H5 and L1– L5). M is the molecular weight marker. The arrows indicate the iron-regulated proteins. (c) is the immunoblot of CFP-high and CFP-low developed with ESAT-6-specifi c monoclonal antibody HYB 76-8 and MPT 64-specifi c monoclonal antibody

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CFP-high and CFP-low were fractionated and the respective CF-Ags (H1-H5 and L1-L5) of defined molecular masses were prepared from each of the samples. These fractions included proteins in the molecular size (in kDa) range of 116-66 (H1, L1), 66-45 (H2, L2), 45-25 (H3, L3), 25-14 (H4, L4) and <14 kDa (H5, L5) respectively. All the ten CF-Ag pools were re-run on SDS-PAGE to confirm the proteins present in each fraction [Figure 2]a and b.

The subjects in the study included pulmonary TB patients (smear positive and smear negative), extra-pulmonary TB patients and normal healthy controls. The PBMCs from these subjects were stimulated with (a) CFP-high and CFP-low, (b) CF-Ags, (c) PPD and (d) mitogen phytohemagglutinin (PHA). PHA served as the positive control and lymphocytes without antigen/mitogen as the negative control. The lymphocyte proliferation was expressed as SI and a case was taken as positive when the SI ≥2.0 and was used to calculate the % recognition of TB cases within the respective group.

The median SI in response to PHA of PBMCs of smear positive and smear negative pulmonary TB, extra-pulmonary TB and normal healthy individuals was 2.9, 2.9, 2.81 and 2.3, respectively. Lymphocytes from normal healthy controls did not respond to PPD, CFP-high and CFP-low [Figure 3] as well as to the different CF-Ags [Figure 4]a. Only 20-35% case recognition was seen with most of the CF-Ags with a maximum of 40% with L1 and H3 fractions. On the other hand, pulmonary TB patients responded to all the mycobacterial antigens, with maximal proliferation of the lymphocytes from both the categories of pulmonary TB patients [Figure 3] and [Figure 4]b. Identical SI values for CFP-high, CFP-low and PPD were observed with smear positive patients, while the smear negative pulmonary TB patients responded better to the CFPs (CFP-high > CFP-low > PPD) [Figure 3]. The proliferating effect of the CF-Ags on the PBMCs of both these groups is shown in [Figure 4]b and % case recognition is summarised in [Table 2]. Significant lymphocyte proliferation was seen with the high-molecular-weight fractions CF-Ags H1 and L1 in both the categories of patients [P < 0.05], [Table 2]; the overall values were however higher with the smear positive cases. Interestingly, the high SI and % recognition observed with CF-Ag L2 (72 and 65 in smear positive and negative patients; P0 = 0.0029, [Table 2]) was not seen with the corresponding CF-Ag H2 (% recognition of 44 and 40, respectively). Similarly, L4 was better than H4 but SI with L5 was lower than H5 (however statistically insignificant); no difference was seen between H3 and L3. The performance of all these CF-Ags was lower than PPD [Table 2]. SI of extra-pulmonary patients was much lower, only marginally higher than the normal healthy controls [Figure 3]. Except PPD (median SI of 2.07; 60% recognition), SI < 2 was seen with all the other antigens in the study; among the CF-Ag pools, the maximum SI of 1.93 was observed with CF-Ag L5 [Figure 4]a.
Figure 3: Effect of CFP-high and CFP-low on the proliferation of PBMCs from TB patients. PBMCs from patients with extra-pulmonary TB (15 cases) and pulmonary TB (25 smear positive and 20 smear
negative cases) and from normal healthy controls (20 subjects) were stimulated with CFP-high, CFP-low and PPD respectively. Standard box-plots with median (25th and 75th percentiles) and whiskers at maximum and minimum values are indicated. Median values (solid bars) are indicated in the box plots

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Figure 4: (a and b) Lymphocyte proliferation response to CF-Ags. (a) SI of normal subjects and extrapulmonary TB cases to defi ned CF-Ags, while (b) SI of pulmonary TB patients (smear positive and smear negative cases). Standard box-plots with median (25th and 75th percentiles) and whiskers at maximum and minimum values are indicated. Median values (solid bars) are indicated in the box plots

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Table 2: Immune recognition of CF-Ags by the PBMCs of pulmonary TB patients

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High levels of IFN-γ were secreted by the PBMCs of normal healthy controls [Figure 5], [PPD> CFP-low> CFP-high] with low levels of the cytokine in all the three categories of TB patients. [Figure 6] represents the level of the cytokine upon stimulation with the CF-Ags. The low-molecular-weight CF-Ag L5 induced the maximal expression of the cytokine (233.1 pg/mL) in the endemic normal subjects that was greater than PPD (177.3 pg/mL) and CF-Ag H5 (140 pg/mL) [Figure 6]b. Significantly low levels of IFN-γ (30-60 pg/mL) were secreted by the lymphocytes from TB patients in response to all the CF-Ags [P < 0.05], [Figure 6]. In the presence of the mitogen (positive control), the PBMCs of TB patients and normal controls released high levels of the cytokine; 231.5, 332.5, 428.7 and 290.75 pg/mL were the median IFN-γ released by the lymphocytes from smear positive, smear negative pulmonary TB cases, extra-pulmonary TB patients and the normal control subjects. The corresponding negative control (minus antigen/mitogen) produced only 24, 26.5, 31 and 24 pg/mL of IFN-γ [Figure 6].
Figure 5: Release of IFN-γ upon stimulation with CFP-high and CFP-low. The box plots represent the IFN-γ levels released by the PBMCs of the three categories of TB patients and endemic normal
healthy controls upon stimulation with CFP-high, CFP-low and PPD. Standard box-plots with median (25th and 75th percentiles) and whiskers at maximum and minimum values are indicated. Median values (solid bars) are indicated in the box plots

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Figure 6: Release of IFN-γ upon stimulation with CF-Ags. The box plots represent the IFN-γ levels released by the PBMCs of the three categories of TB patients and endemic normal healthy controls upon stimulation with the different CF-Ags. (a,b) represent the two groups of pulmonary TB patients and (c,d) represent the normal subjects and the extra-pulmonary TB cases. Standard box-plots with median (25th and 75th percentiles) and whiskers at maximum and minimum values are indicated. Median values (solid bars) are indicated in the box plots. P value, obtained by
comparison of the response with normal subjects was ≤0.02 with all the CF-Ags. With PPD as the antigen, it was 0.001 with the smear positive cases and was statistically insignifi cant with the smear negative (0.0739) and extra-pulmonary TB cases (0.0990)

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 ~ Discussion Top

M. tuberculosis, like other mycobacteria have adapted to iron limitation by expressing the high affinity Fe 3+ -chelating siderophores mycobactin and carboxymycobactin. [13] In this study, the iron-limited growth of M. tuberculosis was evident from the high levels of these siderophores in low iron (0.02 μg Fe/mL) organisms; there was repression of their synthesis when the medium was supplemented with 8 μg Fe/mL. As in other bacterial systems, iron acts as a regulatory signal and switches off the expression of the siderophores under high iron conditions; the intracellular iron binds to the iron regulator IdeR and the IdeR-Fe complex binds to the 'iron box' upstream of the mbt genes resulting in the repression of mycobactin synthesis. It is well understood in several bacterial systems that iron controls not only the expression of the components of the iron acquisition machinery but also virulence determinants/toxins. Earlier, we had reported the effect of iron on the virulence determinant KatG. [16] and here, we studied the influence of iron on the CFPs of M. tuberculosis. The CFPs from high (CFP-high) and low (CFP-low) iron cultures were electrophoretically separated to obtain defined molecular mass fractions whose effect on the immune response of the PBMCs of TB patients was analysed and compared with the traditionally used PPD.

As mentioned earlier, the composition of the CFPs is greatly influenced by the growth conditions and period of growth. Generally, short-term culture filtrates are used to exclude contamination with cytoplasmic proteins released from dead bacteria in late phase cultures. We studied the influence of iron levels on the CFP profile of organisms harvested from shake cultures harvested in the mid-log phase. This was required to ensure that the organisms were under high / low iron status. Two low molecular weight iron-regulated CFPs Irp-1 and Irp-2 were identified in iron-limited cultures upon separation by the Tris-Tricine buffer system. Interestingly, the two low molecular weight proteins of 8 and 9 kDa, belonging to the ESAT family group of proteins were repressed upon iron limitation. Ongoing studies in our lab on the transcriptional profiling of high and low iron organisms showed significant levels of expression of the esx genes; notable was the down-regulation of esx-3, confirmed by real time PCR (unpublished ongoing work); the latter has been reported by other studies. [17]

CFPs are candidate diagnostic antigens widely used in immune response studies. Different methods of antigen preparation were used, including purified proteins, pools of proteins with defined molecular masses, protein eluted from 2D gel spots and transblotted nitrocellulose membrane strips. [18] The effect of these proteins on the immune response of lymphocytes from humans and experimental animals have been studied. [19],[20] In this study, we analysed the immune response of TB patients to CF-Ags prepared from CFP-high and CFP-low samples. The objective was to identify mycobacterial antigens with immuno-protective potential as well as diagnostic antigens for the detection of TB patients in this endemic region. We measured the in vitro proliferation and IFN-γ production by the PBMC, as they are reliable parameters of immuno-competence that reflect the response in vivo.[21] The exposure to environmental mycobacteria was presumed to be similar in all the patients and the control subjects in this endemic region. Values are represented as medians since the data did not approximate normal distributions, which, as pointed out by Demisse and coworkers [21] could be due to the presumption that all the groups contain antigen responsive and antigen un-responsive individuals. The CF-Ags in this study were useful in the identification of both the groups of pulmonary TB patients but performed poorly with the extrapulmonary TB. The high-molecular-weight CF-Ags L1 (66-116 kDa) and L2 (45-66 kDa) evoked a statistically significant lymphocyte proliferation in the pulmonary TB patients, as compared to normal controls. Although the overall performance of H1 was comparable to L1, the proliferation response with the CF-Ags H1 and H2 was not statistically significant. Iron levels did not influence the proteins in the molecular mass between 25-45 kDa (H3 and L3) that presumably contains the strongly immunogenic Ag85 complex. CF-Ag L4 (molecular mass proteins between 14-25 kDa) was significantly better than H4 in the identification of both the groups of pulmonary TB patients. The latter was recognized equally by the low-molecular-weight CF-Ags H5 and L5 and the proliferative response of the PBMCs from the extrapulmonary TB cases to L5 is noteworthy. These observations, coupled to the strong IFN-γ release by L5 in normal healthy controls (discussed below), imply the antigenic potential of this fraction, both for diagnosis and for vaccine. It would be worth separating and purifying the proteins in this fraction and study their individual potential for a better understanding of the host-pathogen interactions.

CF-Ag L5 triggered the maximal expression of IFN-γ in normal controls clearly indicating the presence of immuno-protective nature of this fraction. The lower response by TB patients is suggestive of a lowered immune status that could be due to the suppressive factors secreted by monocytes and lymphocytes resulting in a shift from Th1 to Th2 type of cytokine response. [22] It may be recalled that L5 showed low expression of the 8 and 9 kDa proteins and up-regulation of the iron-regulated protein Irp-2. These proteins would be worth further exploring as there is increasing evidence of immuno-protection associated with high levels of IFN-γ. , Activation of pathways that lead to the induction of the Th-1 immune response conferring protection against the disease has been clearly demonstrated in murine models of experimental TB. [22] IFN-γ is a powerful activator of macrophages, thereby enabling the latter to kill the residing mycobacterium. [23] Demissie et al. [21] showed that the level of IFN-γ was higher in the close contacts of TB patients than the patients. Thus, the level of IFN-γ correlates with protection against disease development though it must be acting in concert with other cytokines and not on its own. [24]

Iron levels are known to influence the immune response of the mammalian host. [25] In TB, the Th1 response has a beneficial effect due to the secretion of the cytokines IL-2, IL-12, IFN-γ and also TNF-α. [23] These cytokines exert strong immunoregulatory effects, and regulate the production of nitric oxide (NO) [25] generated by iNOS. NO is a free, but stable lipophilic radical, which by reacting with electron accepting species forms reactive NO compounds such as ONOO - that are known to cause tissue damage leading to cell death. NO shows antimicrobial activity as it reduces ferric iron to ferrous iron that catalyses the generation of hydroxyl free radicals via the Fenton reaction. [26] NO production is thus an important component of macrophage cytotoxicity. It can also regulate cellular iron metabolism via iron-responsive elements in transferrin receptor and ferritin mRNA. In the Th2 response that counters the Th1 response, an increase in IL-4 expression is associated with increase in the levels of the mRNA transcript of transferrin receptor, resulting in iron assimilation and the suppression of NO. [26] Iron levels therefore greatly influence the balance between the Th1 and Th2 responses and thus control the outcome of the disease. Iron overload seems to tip the scale in the direction of Th2 responses resulting in impaired immunity and increased inflammation.

In conclusion, an outcome of this study is that iron modulates the expression of CFPs, as seen with the ESAT group of proteins and up-regulated a low molecular protein Irp-2, both of which are highly likely to influence the outcome of the immune response of the mammalian host. It is well known that the mammalian host lowers the biologically available iron by a process called 'nutritional immunity' [11] ; withholding iron not only limits the iron for the growth of the invading pathogen but also influences the outcome of the immune response by altering the expression of the secreted proteins.

 ~ Acknowledgments Top

DS is recipient of Senior Research Fellowships thank University Grants Commission of India. The financial support of Institute of Life Sciences - University of Hyderabad MoU towards a part of this study is acknowledged. The authors acknowledge Dr. K. Rajya Lakshmi, Medical Officer, TB control, TU, Hyderabad for her guidance in sample collection and Prof. C. Raghavendra Rao, Department of Mathematics and Computer Information Sciences, University of Hyderabad in statistical analysis of the data. We thank Dr Ida Rosenkrands for providing the antibodies for the immunoblotting studies.

 ~ References Top

1.WHO Library Cataloguing-in-Publication Data. Global tuberculosis control: WHO report 2011.  Back to cited text no. 1
2.Nagai S, Wiker HG, Harboe M, Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun 1991;59:372-82.  Back to cited text no. 2
[PUBMED] Souza GA, Målen H, Søfteland T, Saelensminde G, Prasad S, Jonassen I, et al. High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example. BMC Genomics 2008;9:316.  Back to cited text no. 3
4.Pal PG, Horwitz MA. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun 1992;60:4781-92.  Back to cited text no. 4
5.Roberts AD, Sonnenberg MG, Ordway DJ, Furney SK, Brennan PJ, Belisle JT, et al. Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis. Immunology 1995;85:502-8.  Back to cited text no. 5
6.Schwander SK, Torres M, Carranza CC, Escobedo D, Tary-Lehmann M, Anderson P, et al. Pulmonary mononuclear cell responses to antigens of Mycobacterium tuberculosis in healthy tuberculosis contacts of patients with active tuberculosis and healthy controls from the community. J Immunol 2000;165:1479-85.  Back to cited text no. 6
7.Sable SB, Kumar R, Kalra M, Verma I, Khuller GK, Dobos K, et al. Peripheral blood and pleural fluid mononuclear cell responses to low-molecular-mass secretory polypeptides of Mycobacterium tuberculosis in human models of immunity to tuberculosis. Infect Immun 2005;73:3547-58.  Back to cited text no. 7
8.Barnes PF, Vankayalapati. Th1 and Th2 cytokines in the human immune response to tuberculosis. In: Cole ST, Eisenach KD, McMurray DN, Jacobs WR, editors. Tuberculosis and the tubercle bacillus. ASM Press; 2005. p. 489-96.  Back to cited text no. 8
9.Andersen P, Askgaard D, Ljungqvist L, Bennedsen J, Heron I. Proteins released from Mycobacterium tuberculosis during growth. Infect Immun 1991;59:1905-10.  Back to cited text no. 9
10.Ratledge C. Iron, mycobacteria and tuberculosis. Tuberculosis 2004;84:110-30.  Back to cited text no. 10
11.Kochan I. Role of iron in regulation of nutritional immunity. Bioorg Chem 1976;2:55-7.  Back to cited text no. 11
12.Sritharan M. Iron and bacterial virulence. Int J Med Microbiol 2006;24:163-4.  Back to cited text no. 12
13.Yeruva VC, Duggirala S, Lakshmi V, Kolarich D, Altmann F, Sritharan M. Identification and characterization of a major cell wall-associated iron-regulated envelope protein (Irep-28) in Mycobacterium tuberculosis. Clin Vaccine Immunol 2006;13:1137-42.  Back to cited text no. 13
14.Schagger H, Jagow GV. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368-79.  Back to cited text no. 14
15.Surekha RH, Vijaya Lakshmi V, Sumanlatha G, Murthy KJ. Cell-mediated immune responses in children towards secreted proteins of Mycobacterium bovis BCG. Tuberculosis 2005;85:89-93.  Back to cited text no. 15
16.Sritharan M, Yeruva VC, Sivagami Sundaram CA, Sridevi D. Iron enhances the susceptibility of pathogenic mycobacteria to isoniazid, an anti-tubercular drug. World J Microbiol Biotechnol 2006;22:1357-64.  Back to cited text no. 16
17.Siegrist MS, Unnikrishnan M, McConnell MJ, Borowsky M, Cheng TY, Siddiqi N, et al. Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc Natl Acad Sci U S A 2009;106:18792-7.  Back to cited text no. 17
18.Abou-Zeid C, Filley E, Steele J, Rook GA. A simple new method for using antigens separated by poly-acrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles. J Immunol Methods 1987;98:5-10.  Back to cited text no. 18
19.Brodin P, Rosenkrands I, Andersen P, Cole ST, Brosch R. ESAT-6 proteins: Protective antigens and virulence factors? Trends Microbiol 2004;12:500-8.  Back to cited text no. 19
20.Dietrich J, Doherty TM. Interaction of Mycobacterium tuberculosis with the host: Consequences for vaccine development. APMIS 2009;117:440-57.  Back to cited text no. 20
21.Demissie A, Ravn P, Olobo J, Doherty TM, Eguale T, Geletu M, et al. T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts. Infect Immun 1999;67:5967-71.  Back to cited text no. 21
22.Redford PS, Boonstra A, Read S, Pitt J, Graham C, Stavropoulos E, et al. Enhanced protection to Mycobacterium tuberculosis infection in IL-10-deficient mice is accompanied by early and enhanced Th1 responses in the lung. Eur J Immunol 2010;40:2200-10.  Back to cited text no. 22
23.Cooper AM. T cells in mycobacterial infection and disease. Curr Opin Immunol 2009;21:378-84.  Back to cited text no. 23
24.Sahiratmadja E, Alisjahbana B, De Boer T, Adnan I, Maya A, Danusantoso H, et al. Dynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment. Infect Immun 2007;75:820-9.  Back to cited text no. 24
25.Brock JH. Iron and Infection: Molecular, physiological and clinical aspects. In: Bullen JJ, Griffiths E, editors. Chichester: John Wiley and Sons; 1998.  Back to cited text no. 25
26.Weinberg ED. Modulation of intramacrophage iron metabolism during microbial cell invasion. Microbes Infect 2000;2:85-9.  Back to cited text no. 26


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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2004 - Indian Journal of Medical Microbiology
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