Parthasarathi Ajitkumar

Expression profiling of sodium butyrate (NaB)-treated cells: identification of regulation of genes related to cytokine signaling and cancer metastasis by NaB

Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (NaB), a short chain fatty acid, is a HDAC inhibitor and is produced in the colonic lumen as a consequence of microbial degradation of dietary fibers. In order to dissect out the mechanism of NaB-induced growth inhibition of cancer cells, we carried out expression profiling of a human lung carcinoma cell line (H460) treated with NaB using a cDNA microarray. Of the total 1728 genes analysed, there were 32 genes with a mean expression value of 2.0-fold and higher and 66 genes with a mean expression value 3.0-fold and lower in NaB-treated cells. For a few selected genes, we demonstrate that their expression pattern by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis is matching with the results obtained by microarray analysis. Closer view at the expression profile of NaB-treated cells revealed the downregulation of a total of 16 genes associated with cytokine signaling, in particular, interferon γ (IFNγ) pathway. In good correlation, NaB-pretreated cells failed to induce interferon regulatory factor 1, an INFγ target gene, efficiently upon IFNγ addition. These results suggest that NaB inhibits proinflammatory cytokine signaling pathway, thus providing proof of mechanism for its anti-inflammatory activity. We also found that NaB induced three genes, which are known metastatic suppressors, and downregulated 11 genes, which have been shown to promote metastasis. Upregulation of metastatic suppressor Kangai 1 (KAI1) by NaB in a time-dependent manner was confirmed by RT–PCR analysis. The differential regulation of metastasis-associated genes by NaB provides explanation for the anti-invasive properties of NaB. Therefore, our study presents new evidence for pathways regulated by NaB, thus providing evidence for the mechanism behind anti-inflammatory and antimetastatic activities of NaB.

The Multifunctional PE_PGRS11 Protein from Mycobacterium tuberculosis Plays a Role in Regulating Resistance to Oxidative Stress

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H2O2-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella Typhimurium and Salmonella Typhi

OBJECTIVES Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. METHOD We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RT-PCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. RESULTS Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. CONCLUSIONS The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon γ (IFNγ), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumins ability to hinder the bactericidal action of ciprofloxacin and IFNγ might significantly augment Salmonella pathogenesis.

Bacterial cell division protein FtsZ is a specific substrate for the AAA family protease FtsH

The role of AAA (ATPases Associated to a variety of cellular Activities) family protease FtsH in bacterial cell division is not known, although mutations in ftsH were found to inhibit cell growth and division (1, 6, 13). Overexpression of heterologous FtsH in Escherichia coli results in the formation of multinucleate ®lamentous cells due to the abolition of cell septation (8). Further, independent studies on FtsH (15) and FtsZ (2), which is the key regulator of bacterial cell division, have shown that FtsH protease and FtsZ protein are localized to the mid-cell site during septation. FtsZ protein is the prokaryotic homologue of tubulin (5, 10, 12), possessing GTP-dependent polymerization activity (4, 11). igni®cantly, the AAA family ATPase member katanin disassembles tubulin polymers in an ATP-dependent manner (7). Based on these observations, we reasoned that an interaction similar to that between katanin and tubulin might hold true for FtsH and FtsZ in prokaryotes as well. To verify this hypothesis, we examined whether the FtsH protease of Escherichia coli

Transcriptional Analysis of the Principal Cell Division Gene, ftsZ, of Mycobacterium tuberculosis

(FtsH_{Ec})

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis

could degrade FtsZ of E. coli

Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv

(FtsH_{Ec})

Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis.

in vitro.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

Multiple promoters drive the expression of the principal cell division gene, ftsZ, in bacterial systems. Primer extension analysis of total RNA from Mycobacterium tuberculosis and a Mycobacterium smegmatis transformant containing 1.117 kb of the upstream region of M. tuberculosis ftsZ and promoter fusion studies identified six ftsZ transcripts and their promoters in the ftsQ open reading frame and ftsQ-ftsZ intergenic region. The presence of multiple promoters reflects the requirement to maintain a high basal level of, or to differentially regulate, FtsZ expression during different growth conditions of the pathogen in vivo.

Unveiling Unusual Features of Formation of Septal Partition and Constriction in Mycobacteria—an Ultrastructural Study

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.