Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2010  |  Volume : 53  |  Issue : 4  |  Page : 718--722

Nonspecificity of 35 kDa protein: A proposed marker for the differential diagnosis of M. avium infection in the Indian population


Kapil Gupta1, Rajiv Mahajan2, Gopal K Khuller3, Indu Verma3,  
1 Department of Biochemistry, AIMSR, Bhatinda - 151 109, India
2 Department of Pharmacology, AIMSR, Bhatinda - 151 109, India
3 Department of Biochemistry, PGIMER, Chandigarh - 160 012, India

Correspondence Address:
Indu Verma
Department of Biochemistry, PGIMER, Chandigarh - 160 012
India

Abstract

Objective: The subunit vaccine strategies and development of various diagnostic reagents for Mycobacterium avium infection relies on the presence of secreted, species-specific mycobacterial antigens. The M. avium 35 kDa protein has been suggested as a candidate for vaccine/diagnostic reagent, specifically for M. avium infection. The present study was conducted to evaluate the diagnostic specificity of the M. avium 35 kDa protein in the Indian population. Materials and Methods: Culture filtrate proteins were isolated by growing the bacilli in modified Youman«SQ»s medium. The 35 kDa protein was purified by high-resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blast search was carried out. Western blotting was performed with either monoclonal antibody CS-38 or serum samples of tuberculosis (TB) patients. The 35 kDa-specific immunoglobulin G antibody titer was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA). Results: Despite the absence of gene for the 35 kDa protein, the sera of TB patients and TB patient«SQ»s contacts nonspecifically recognize it. Of 109 TB patients tested, the sera of 84 patients in ELISA (percentage recognition = 87.5%) and 27 of 29 TB patients tested in western immunoblotting (percentage recognition = 93.10%) recognized the M. avium 35 kDa protein, while with sera of TB patient«SQ»s contacts, the recognition was 50%. Conclusion: Contrary to Western studies, the M. avium 35 kDa protein does not seem to be a good candidate for the specific diagnosis of M. avium infection in the Indian population.



How to cite this article:
Gupta K, Mahajan R, Khuller GK, Verma I. Nonspecificity of 35 kDa protein: A proposed marker for the differential diagnosis of M. avium infection in the Indian population.Indian J Pathol Microbiol 2010;53:718-722


How to cite this URL:
Gupta K, Mahajan R, Khuller GK, Verma I. Nonspecificity of 35 kDa protein: A proposed marker for the differential diagnosis of M. avium infection in the Indian population. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Nov 27 ];53:718-722
Available from: https://www.ijpmonline.org/text.asp?2010/53/4/718/72053


Full Text

 Introduction



Mycobacteria are a group of acid-fast, aerobic, slow-growing bacteria with a widespread nature. [1] Mostly, the mycobacterial diseases are associated with Mycobacterium tuberculosis and M. leprae, the causative agents of tuberculosis (TB) and leprosy, respectively. [2] However, recently, a spurt has been seen in the incidences of infections caused by M. avium also, particularly in human immunodeficiency virus (HIV)-infected patients. [3] Until now, M. avium was rarely known to cause disease in individuals without predisposing lung disease, [4] but various reports state that pulmonary and extrapulmonary Mycobacterium avium complex (MAC) infections are becoming a more prevalent clinical problem in individuals even without predisposing conditions, particularly in the older female population. [3],[5] Furthermore, studies have reported that pulmonary diseases caused by MAC in non-HIV-infected persons are as common as pulmonary tuberculosis in many areas. [6]

MAC, or M. avium, complex consists predominantly of two species, M. avium subspecies avium and M. avium subspecies intracellulare, [5] but in more than 95% of the patients, acquired immunodeficiency syndrome-related disseminated MAC infection is caused by M. avium subspecies avium. [7] Owing to its increasing medical importance and differential chemotherapy from M. tuberculosis, research has been directed toward its diagnosis, vaccination as well as chemotherapy. The various diagnostic methods used thus far are purified protein derivative (PPD) based skin testing, biochemical differentiation, high performance liquid chromatography (HPLC) based studies and various nucleic acid hybridization and polymerization assays. [8],[9],[10],[11],[12],[13] However, these conventional identification methods of M. avium infection are complex, not very specific and require growth of mycobacterial cultures from patient's specimen, which is time consuming or requires radioisotopic facilities, restriction enzymes and extensive instrumentation.

Because proteins secreted by mycobacteria have been thought as the primary target during the initial phase of the infection, development of sensitive and specific immunodiagnositic procedures based on specific secretory proteins for the rapid detection of M. avium infections would greatly facilitate clinical management of active disease. The various antigens reported to be M. avium specific are 14 kDa and 35 kDa. [14],[15] In case of 14 kDa, the homologous gene is present in M. tuberculosis, while the 35 kDa gene is absent in both M. tuberculosis and M. bovis Bacille Calmette Guerin (BCG), indicating that this 35 kDa protein is not recognized by TB patients and can be used for the differential diagnosis of M. avium, as reported by Western studies. [15]

Accordingly, the present study was planned to evaluate the specificity of the 35 kDa protein in the differential diagnosis of M. avium infection from that of M. tuberculosis by taking the mouse model as well as human TB patients in the Indian population.

 Materials and Methods



Growth of M. avium subspecies avium, M. tuberculosis H 37 Rv, M. smegmatis, M. bovis BCG and isolation of culture filtrate proteins (CFPs)

CFPs of M. avium subspecies avium (MTCC 1723 IMTECH, NCTC 8551 London), M. tuberculosis H 37 Rv (NCTC London), M. smegmatis (ATCC 607) and M. bovis BCG were isolated by growing the bacilli in modified Youman's liquid synthetic medium as a stationary pellicle culture. [16] Bacilli were harvested after 4-5 weeks, supernatants were filter-sterilized (0.22 μm pore size membrane filter), desalted and concentrated 100 times by ultrafiltration on an amicon YM-3 membrane (Millipore, Bedford, MA, USA). These mycobacterial CFPs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10-12% resolving gel followed by silver staining. [17],[18]

Antiserum

Antisera against various mycobacterial species were obtained by infection of mice with the above-mentioned bacilli. Briefly, mice were infected with 1 x 10 6 bacilli through the intravenous route and bled on the 2 nd , 4 th , 6 th , 8 th and 10 th weeks postinfection. Serum was separated and stored at -20 0 C till further use.

Purification of the 35 kDa Protein

The protein was purified by high-resolution preparative SDS-PAGE (Hoefer SE 600, Amersham Pharmacia, Biotec Inc., San Francisco, CA, USA) (16 cm x 4 cm x 1.5 mm) at 250 V using Tris-glycine (25 mM, 192 mm) as the electroelution buffer. The protein concentration was estimated by the micro BCA (Sigma, St Louis, MO, USA) method and the purified protein was analyzed by SDS-PAGE followed by silver staining. The identity of the protein was confirmed by reactivity with the known monoclonal antibody (MoAb) and stored at -20 0 C till further use.

N-terminal Sequencing of the Purified Protein

The purified protein was subjected to N-terminal sequencing (ladder sequencing, concentration dependent) in Ultraflex MALDI TOF/TOF (Bruker Daltonics Inc., Billerica, MA 01821, USA). The sequence so obtained was carried for blast search to detect the homology with other mycobacterial species.

Study Population

One hundred and nine confirmed TB patients (both sex, mean age 40 ± 5 years) before the commencement of antibiotic chemotherapy were included in the study after taking due informed written consent. Blood was drawn from patients under aseptic conditions and sera was separated and stored at -20 0 C till further use. The serum samples (n = 20) from healthy individuals were also taken and these served as controls for monitoring the antibody responses against M. avium-specific secretory proteins. The individuals included in this category were non-TB, non-HIV without any evident contact of TB patients or their biological samples and without any sign of immunocompromization. Serum samples of healthy household contacts (n = 6) of confirmed sputum-positive TB patients as well as medical, paramedical and laboratory subjects directly in contact with TB patients or their biological samples/cultures for more than 6 months with no clinical symptoms or previous history suggestive of TB and normal chest X-ray were evaluated for antibodies present against M. avium-specific secretory proteins.

Immunoreactivity with MoAb and Serum Samples of TB Patients

M. avium CFPs (70 μg/well) or purified 35 kDa (4-5 μg/well) were resolved on 12% denaturing SDS-PAGE and transferred to a 0.45 μm nitrocellulose membrane (Millipore). Western blotting was carried out with either MoAb CS-38 (a specific MoAb for the M. avium 35 kDa protein) or serum samples of TB patients at 1:100 dilutions.

Detection of the 35 kDa-specific Serum Total Immunoglobulin (Ig) G Antibody Levels

IgG antibody specific for the 35 kDa protein of M. avium was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA) at 1:100 dilutions. The mean absorbance of healthy individuals + 3 SD was taken as the cut-off value to detect the TB patients recognizing the 35 kDa protein.

 Results



The 4-week stationary culture of M. avium subspecies avium, M. tuberculosis H 37 R V , M. smegmatis and M. bovis BCG isolated the CFPs with a mean yield of 15 ± 0 2 mg/L of the secretary proteins.

M. avium CFPs, M. smegmatis CFPs, M. bovis BCG CFPs and M. tuberculosis CFPs when applied to 12% denaturing SDS-PAGE and immunoblotted with M. avium antisera raised in mice, the antiserum was found to react mainly with M. avium CFPs including 35 kDa, while in other mycobacterial CFs, the 35 kDa protein was not present. In the reverse experiment, when M. avium CFPs were made to react with antiserum raised against different mycobacterial species, i.e. M. avium, M. tuberculosis H 37 R V and M. bovis BCG, the 35 kDa was recognized by all other mycobacterial antisera, giving a clear distinct band. When M. avium CFP was immunoblotted with TB patient and TB patient's contact sera, to evaluate the diagnostic potential of 35 kDa, 27 of 29 TB patients [Figure 1] and three of six contacts [Figure 2] recognized the protein, giving a percent recognition of 93.10 and 50%, respectively, indicating the presence of antibodies against the 35 kDa protein in TB patients as well as in contacts.{Figure 1}{Figure 2}

Further incubation of MoAb CS-38 with the purified 35 kDa protein gave a distinct band thus confirming its identity [Figure 3].{Figure 3}

The MALDI TOF/TOF showed the protein to be the MMP-1 of M. avium, with the N-terminal sequence of MTSAQNESQA. On performing a BLAST search with various mycobacterial species, it showed 100% homology with the 35 kDa protein of M. avium paratuberculosis, M. leprae, M. smegmatis and M. avium 104 (the most prevalent clinical isolate of M. avium known to cause opportunistic infections in humans), but no homology with the proteome of M. tuberculosis and M. bovis BCG in the entire Blast search result [Table 1].{Table 1}

The seroreactivity of this purified protein was also checked with the serum samples of TB patients by western immunoblotting. Of the 14 TB patients evaluated, 12 showed positive immunoreactivity, giving a percent recognition of 85.7% [Figure 4].{Figure 4}

Of the 109 TB patients, IgG antibody levels against the 35 kDa protein were present above the cut-off in 93 patients (at 492 OD), giving a percent recognition of 83.32% [Figure 5]. The difference in the OD represented by recognition of the 35 kDa by M. avium antisera and M. tuberculosis antisera was not significant.{Figure 5}

 Discussion



Although the western immunoblotting of myobacterial CFPs with M. avium antisera raised in mice showed that the gene for the 35 kDa protein is only present in the M. avium genome, the nonspecific recognition of this protein by TB patients and TB patient's contacts in the Indian population nullifies its earlier suggested utility as a marker for the serodiagnostic test for the differential diagnosis of M. avium infection.

Early diagnosis of M. avium in clinical samples is becoming increasingly important because of the greater frequency of MAC infection, impeding availability of new drugs and differential chemotherapy as compared to M. tuberculosis. [19],[20],[21],[22] The development of sensitive and specific immunodiagnositic procedures for the rapid detection of M. avium disease would greatly facilitate the clinical management of active disease. The diagnosis of M. avium disease on the basis of distinct and specific antigens would be sensitive and specific as only M. avium-infected patient's sera will recognize the species-specific proteins. The 35 kDa protein was taken as the differential serodiagnostic marker because earlier it has been suggested to be specific for M. avium infection. The genomic data also demonstrate that the gene for this protein is only present in M. avium and M. leprae, while it is absent from M. tuberculosis and M. bovis BCG. [23] The western immunoblotting of mycobacterial CFPs with M. avium antisera raised in mice also confirmed the earlier reports that the gene and the protein are only present in the M. avium genome. [15] However, the recognition of M. avium 35 kDa by different mycobacterial antisera indicated that the protein may be present only in M. avium but may still have some cross-reactive epitopes that are recognized by M. tuberculosis H 37 Rv or M. bovis BCG antisera.

The specificity of M. avium-specific proteins can be validated only if they do not share the B-cell epitopes with the M. tuberculosis secretory proteome, i.e. it must not be recognized by the sera of confirmed TB patients or TB patient's contacts. Hence, the selected M. avium 35 kDa protein in the CF were probed with the sera of sputum-positive, culture-positive TB patients and TB patient's contacts and the nonspecific recognition of the 35 kDa protein by both categories raised doubts for its reported feasibility for the differential serodiagnosis of M. avium infection [24] and indicated the presence of cross-reactive epitopes within this protein.

Considering the existence of multiple proteins of overlapping molecular mass in the CF of M. avium as the possibility for cross-reactive epitomes, the 35 kDa protein was purified and its reactivity with MoAb CS-38 confirmed its identity. Further characterization by MALDI TOF/TOF and BLAST search demonstrated it to be the MMP-1 protein of molecular weight 35 kDa only present in the MAC species and M. leprae, as used in earlier studies. [2],[24] However, the positive recognition of purified protein by the sera of TB patients by western immunoblotting (n = 14, [Figure 4]) and by ELISA (n = 109, [Figure 5] again indicated it to be a nonspecific protein for M. avium infection. This nonspecific recognition demonstrated in TB patients is supported by a study carried out in the early nineties demonstrating the recognition of 35 kDa by all TB patients. [6] It is noteworthy to mention that many deleted genes encoding large molecules are reported to contain limited stretches (with possible B- and T-cell epitopes) that are homologous to genes outside the deleted regions. [25] Thus, it may be possible that the 35 kDa protein possess cross-reactive epitopes in M. tuberculosis and M. bovis BCG proteome, resulting in the nonspecific recognition.

Further, the nonspecific recognition of the 35 kDa by the sera of TB patients indicates its nonutility in the differential diagnosis of M. avium infection from that of M. tuberculosis in the Indian population. Hence, the present study emphasizes the need for some new antigenic target other than 35 kDa that can be used for the development of a serological diagnostic assay for M. avium infection.

 Acknowledgment



We thank Dr. Jaikishan and Dr. Neeru of TB and Chest Diseases Hospital, Patiala, for providing clinical assistance for the study.

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