Kammara Rajagopal

Latency-Associated Protein Acr1 Impairs Dendritic Cell Maturation and Functionality: A Possible Mechanism of Immune Evasion by Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

Single-step overlap-primer-walk polymerase chain reaction for multiple mutagenesis without overlap extension

We present a simple, single-step, single-tube, and rapid method for introducing a series of mutations into cloned DNA. Polymerase chain reaction (PCR)-based mutagenesis methods have become very prevalent due to their simplicity and efficiency for introducing mutations. Our method, overlap-primer-walk PCR, has several advantages over other published methods. It uses two common oligodeoxyribonucleotides and a series of overlapping primers specific for various mutations. Once common flanking primers are selected, two to three mutations require only one additional primer. Therefore, this method is very useful for introduction of multiple mutations in various sites of the target DNA. We illustrate the usefulness of the method by introducing several mutations into the human TNF-alpha encoding gene.

Efficient ligation and cloning of DNA fragments with 2-bp overhangs.

Various methods of ligation are currently available and routinely used by molecular biologists, such as blunt end ligation, cohesive end (two and four overhangs), and ligation of Taq polymerase-derived products. However, there is no efficient method for the cloning of DNA fragments with 2-bp overhangs. We present a simple method for the efficient ligation of DNA fragments with 2-bp overhanging ends, ranging in size from 0.7 to 2.5 kbp. Our method involves the initial heating and flash freezing of the vector-insert DNA mix, and a subsequent unique ligation reaction. This method provides a new molecular biology tool for researchers.

Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli

The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD(600)) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system - no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.

Draft Genome Sequence of Enterococcus raffinosus Strain CFTRI 2200,Isolated from Infant Fecal Material

ABSTRACT The complete genome sequence of Enterococcus raffinosus strain CFTRI 2200, isolated from the fecal material of a 7-month-old infant, is reported. The complete genome consists of 4.237 Mbp with a G+C content of 39.47% and 4,242 protein-coding genes, 54 tRNAs, and 46 rRNAs.