Kuppamuthu Dharmalingam

• Madurai Kamaraj University

Corneal mycotic ulceration is predominantly due to Aspergillus and Fusarium solani infection in tropical countries. In this study, we examined the proteome profile of tear samples from A. flavus keratitis patients at various stages of infection. The proteome was profiled using 2D PAGE and the protein levels were quantified using 2D DIGE. Alpha-1-an...

Abstract Aspergillus flavus is one of the predominant causative organisms of mycotic keratitis in tropical parts of the world. Extracellular proteins are the earliest proteins that come in contact with the host and have a role in the infection process. Exoproteins of A. flavus isolated from infected cornea, sputum and a saprophyte were pooled and i...

Supplementary material

Interleukin 17A (IL-17) production by peripheral blood neutrophils was examined in patients with fungal keratitis and in uninfected individuals in southern India, which has high levels of airborne Aspergillus and Fusarium conidia. Il17a gene expression and intracellular IL-17 were detected in all groups, although levels were significantly elevated...

M ycobacterium leprae HSP 18 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 HSP 18 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 al...

The aim of this study is to examine the in vivo role of a small heat shock protein (sHsp18) from M. leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environments that are normally encountered by M. leprae during its infection of the human host. Using an E. coli system where shsp18 expr...

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients cl...

Tear sample details used in this study. (DOCX)

Classification of differentially regulated proteins according to biological process, cellular & molecular. (DOCX)

Differentially Expressed Proteins in Tears of keratitis patients. (DOCX)

Identified Tear proteins and their functions. (DOCX)

BACKGROUND: In the field of biomarker discovery, when the human host sample is not available for experimentation, for example, during the pre-symptomatic stages of diseases and where the onset of infection can not be predicted accurately, animal models are the only option available. Proteome analysis of infectious disease models will be of importan...

Filamentous fungi of the genera Aspergillus and Fusarium are major causes of corneal ulcers in the United States and in the developing world and result in significant visual impairment and blindness. RNA was extracted from 110 patients with corneal ulcers in southern India within 1 week of infection with either Fusarium solani or Aspergillus flavus...

The proteomic profile of tear fluid is of fundamental interest in eye research. In this study we optimized the tear sample preparation method for two-dimensional (2D) analysis and determined the protein profile of tear fluid from healthy males and females. To find the most efficient method for tear sample preparation, four widely applied precipitat...

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 diagnosi...

Vitreoscilla hemoglobin (VHb) is the best studied prokaryotic hemoglobin. Its expression in recombinant host is known to enhance the growth and oxygen utilization properties under oxygen limited conditions. The expression of VHb is shown to be tightly regulated under the control of an oxygen responsive promoter, fnr. Expression of hemoglobin was co...

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 an...

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 carrie...

Purification and identification of sHsp18 of M. leprae in E. coli. (A) SDS-PAGE showing the expression of sHsp18 in E. coli. The protein profile of the E. coli cells over expressing the sHsp18 protein were checked on a 12% SDS-PAGE before and after IPTG induction. Lanes represent, Molecular weight marker (lane 1), Total cell lysate, uninduced and i...

Localization of sHsp18 in E. coli. To determine the localization of the sHsp18 protein on expression in E. coli, the cells were fractionated and the protein profile of each fraction was checked on a 12% SDS-PAGE for the presence of the sHsp18 protein. Lanes represent 1-Purified sHsp18 protein (Control), 2-Periplasmic fraction, 3-Cytoplasmic fractio...

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 m...

Mycotic keratitis is a major cause of corneal blindness in India. A proper understanding of the pathogenesis may help in refining the existing treatment. The purpose of this study is to examine the total tear protein profile of fungal keratitis patients, which may have a bearing on pathogenesis and disease progression. Tear samples were collected f...

Since Mycobacterium leprae is uncultivable in artificial cell free medium, experimental animals alone could be used for a greater understanding of host-pathogen relationship. Mouse footpad model, with the local and limited growth of M. leprae, have been successfully exploited to study the bacteriology, chemotherapy, immunology and pathogenesis of l...

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 mediate...

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 sequenc...

Lipoarabinomannans (LAMs) are major lipoglycans of the mycobacterial envelope and constitute immunodominant epitopes of mycobacteria. In this paper, we show that mannose-capped (ManLAM) and non-mannose-capped (PILAM) mycobacterial lipoglycans insert into T helper cell rafts without apparent binding to known receptors. T helper cells modified by the...

Extracellular proteins of Streptomyces peucetius that bind to a red pigment were identified during the course of isolation of mutants defective in daunorubicin production. Two pigment-protein complexes were partially purified and this complex inhibited the growth of Bacillus subtilis. Routine solvent extraction could not separate the pigment from p...

Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restrict...

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using...

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...

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively...

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (...

Streptomyces peucetius, producer of the antitumor anthracycline antibiotic daunorubicin, was mutagenized, and mutants defective in daunorubicin biosynthesis were screened. One mutant (SPVI), which failed to produce daunorubicin, was found to overproduce an extracellular chitinase. Time course analyses of chitinase production and of the extracellula...

Using the multilocus minisatellite probe 33.6 in combination with the HinfI restriction enzyme, the extent of genetic variation detected by DNA fingerprinting was estimated in 102 unrelated individuals of the Dravidian Hindu population of Tamil Nadu, South India. In this first study of its kind on an anthropologically defined Indian population, DNA...

Using the multilocus minisatellite probe 33.6 in combination with the HinfI restriction enzyme, the extent of genetic variation detected by DNA fingerprinting was estimated in 102 unrelated individuals of the Dravidian Hindu population of Tamil Nadu, South India. In this first study of its kind on an anthropologically defined Indian population, DNA...

Allele frequency distribution of the VNTR locus D2S44 was studied in Tamil Nadu (South India) population. Randomly chosen individuals (Tamils of the plains, Madras City; N = 142) were tested for HaeIII-generated polymorphism detectable by probe YNH24, and the allele sizes and frequencies were determined. Heterozygosity (93.6%) observed in the Tamil...

Allele frequencies were determined in unrelated individuals of Tamil speaking population from the Madras City (Tamil Nadu, South India) area for the polymorphic DNA locus D10S28 using the probe TBQ7. Membranes hybridized with the probe YNH24 were subjected to deprobing and were subsequently hybridized with random priming - labeled, purified inserts...

The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA+/RglA+ phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA+/RglA- phenotype and directed production of a 23-kDa polypeptid...

The chromosome of Streptomyces coelicolor A3(2) carries unstable DNA sequences hybridising with DNA sequences from an unstable chromosomal region of the related species S. lividans. These S. coelicolor sequences are nearly identical to those of S. lividans TK23 in organisation but differ from those of S. lividans 66 TK64 which harbours a tandem dup...

Chloramphenicol resistance is an unstable character in Streptomyces fradiae, since spontaneous chloramphenicol-sensitive (Cmls) mutants arose at very high frequencies. One such Cmls mutant, DM14, showed DNA amplification as well. Extracellular protease activity was tenfold higher in DM14 when compared with its wild-type parent. Protease activity de...

The Mcr systems (previously known as Rgl systems) ofEscherichia coli recognize and cleave specific sequences carrying methylated or hydroxymethylated cytosines. We have cloned the mcrA gene and determined its nucleotide sequence. An 831 base pair sequence encodes the McrA protein. Analysis of the sequence data reveals that there arc additional ORFs...

Streptomyces lividans arginine auxotrophs which show amplification of a 5.7-kb DNA sequence, arose at a very high frequency, varying from 10% to 25% of Cmls spores. The amplifiable DNA sequence was shown to be stable over many generations. However, treatment of Cmls arg mutants with subinhibitory concentrations of antibiotics such as spectinomycin,...

The restriction of non glucosylated phage T4 DNA is reduced significantly in host bacterial strains carrying recBCsbcA mutations even in the presence of a functional rgl gene. In recBCsbcA hosts a high frequency of phage mutations are observed both in the glucosyl transferase genes and in the DNA sequences recognized by the rgl restriction enzymes....

The rglA and rglB genes code for two different proteins which cleave the hydroxymethylated cytosine residues of T-even phages. We isolated Tn10 and Tn5 insertion mutants of the above genes and of the genes in and around the rglA and rglB loci. These insertions were used to construct a detailed genetic map. Our results show that the rglA gene maps a...

We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein. Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in th...

Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degrad...

In this and the following paper the control of gene expression from different T4 DNA templates is examined. Absence of glucosylation did not grossly alter T4 early gene expression. Late gene expression, on the other hand, was altered extensively. The absence of late gene expression in DNA negative or maturation defective mutants was overcome to an...

Under restricting conditions expression of early T4 genes is determined in part by the location of the particular gene relative to the cleavage site. Some genes are inactivated directly by cleavage of the DNA. Other genes are inactivated by secondary exonucleolytic degradation of cleaved DNA. A third class of genes continues to be expressed from th...

The primary action of restriction endonuclease, cleaving infecting DNA, has been demonstrated in vivo. This primary cleavage is followed rapidly by hydrolysis of the cleaved DNA at its newly exposed termini. Infecting viruses can inactivate cytoplasmic and membrane restriction endonucleases to prevent cleavage of unmodified DNA replicas.

HOST-controlled restriction modification systems usually prevent the survival of unmodified genomes in bacterial cells1-4. Phage DNA can be modified by methylation (lambda, and f1)5-7 or by glucosylation (T-even phages3). Modification of the lambda genome is mediated by a host methylase8 whereas modification of the T4 genome is by phage coded alpha...