Mycobacterium leprae hsp18 promoter-EGFP transcriptional fusion construct: Environmental stress and strain-specific expression.

The Hsp18 protein is a major T-cell antigen of Mycobacterium leprae belonging to the family of small heat-shock proteins. The protein is specifically regulated at post-translational level during the intracellular growth of M. leprae within macrophages due to auto-phosphorylation, indicating its importance in the survival of the bacterium. The promoter and regulatory sequences that control hsp18 expression are located within a 256-bp sequence upstream of the translation start site. However, there are no studies describing either characterization of the hsp18 promoter or its genetic regulation. Therefore, we constructed an hsp18-EGFP transcriptional fusion in an E. coli-Mycobacterium shuttle vector. A 168-bp sequence comprising the hsp18 promoter was cloned upstream of the EGFP gene and transformed in M. smegmatis, and the integration of the construct was confirmed by Southern hybridization. hsp18 promoter activity was measured by analyzing EGFP expression in M. smegmatis and Escherichia coli grown under different environmental stress conditions normally encountered by M. leprae in vivo. We found that the 168-bp upstream sequence of hsp18 could function as a promoter, and the regulation of hsp18 expression was host-, environmental stress-, and temperature-dependent. Appreciable EGFP expression was detected in M. smegmatis grown under normal conditions, and theexpression was significantly increased by environmental stress. However, EGFP expression was observed in E. coli only under stress conditions. Comparative sequence analysis revealed the putative sigma factor C (SigC)-binding site within the 168-bp promoter sequence of hsp18, which might be involved in the regulation of hsp18 expression during stress conditions in M. leprae. Thus, our data demonstrated the transcriptional regulation of hsp18 expression in response to different environmental stress conditions, possibly through SigC in Mycobacterium. Further, this shuttle vector could be used for the functional characterization of M. leprae genes in heterologous systems.

A S52P mutation in the 'α-crystallin domain' of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function.

Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 (HSP18P(52)) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 (HSP18S(52)) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S(52) is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P(52) had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S(52). Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts.

Protective role of Mycobacterium leprae small heat-shock protein in heterologous hosts, Escherichia coli and Mycobacterium smegmatis, grown under stress.

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.

alpha1-acid glycoprotein as a putative biomarker for monitoring the development of the type II reactional stage of leprosy.

Leprosy, a spectral disease manifested on the basis of host immune responses, is complicated by its reactional stages, namely type I reversal reaction (RR) and type II erythema nodosum leprosum (ENL). These reactional stages are characterized by uncontrolled and aberrant immune responses. Biomarkers for reactional stages would aid in early diagnosis, efficient treatment, prevention of neurological complications and prediction of predisposition to reactional stages. In this study, comparative analysis of the serum proteome of leprosy patients by two-dimensional electrophoresis (2DE) followed by mass spectrometry showed differential expression of acute-phase protein alpha (1)-acid glycoprotein (AGP; also known as orosomucoid). AGP levels in untreated ENL cases were significantly higher than in lepromatous leprosy (LL; a non-reactional disease stage) (P=0.0126), RR (P=0.0176) and healthy controls (P=0.0030). These data were confirmed using ELISA. The levels of AGP decreased to normal levels after treatment with multidrug therapy and thalidomide (P =0.0167). In a follow-up study, AGP levels, which were high in the untreated ENL stage, decreased significantly at 5 days ( P=0.0084) and 21 days (P=0.0027) post-treatment. A stage-dependent increase in AGP in an LL patient who progressed into the ENL stage was also shown. Glycosylation analysis by 2DE showed differential expression of acidic glycoforms of AGP in untreated ENL cases. Changes in AGP concentration and differential expression of isoforms correlated with the inflammatory condition in ENL and also with the treatment regimen. Thus, initial validation of AGP as an ENL-specific biomarker and treatment indicator was shown in this study.

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases.

Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.

Functional characterization of a small heat shock protein from Mycobacterium leprae.

BACKGROUND: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty. RESULTS: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins. CONCLUSION: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae.

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52(nd) position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52(nd) position is observed in beta4 strand and Proline in 52(nd) position is observed in loop. The number of residues contributing alpha helix at N-terminal region varies in both models. In 18S more number of residues is present in alpha helix when compared to 18P. The loop regions between beta3 and beta4 strands of both models vary in number of residues present in it. Number of residues contributing beta4 strand in both models vary. beta6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.

Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin.

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.

Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae.

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of slashed circleC31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNA(Gln) amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also.

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.