Aug
12
Mon
2013
Delegate Talk: Protoplast fusion and transformation: A tool for activation of latent gene clusters @ Sathyam Hall
Aug 12 @ 3:15 pm – 3:35 pm
Delegate Talk: Protoplast fusion and transformation: A tool for activation of latent gene clusters @ Sathyam Hall | Vallikavu | Kerala | India

Abhijeet Kate, Arpana G Panicker, Diana Writer, Giridharan P, Keshav K V Ramamoorthy, Saji George, Shailendra K Sonawane


Protoplast fusion and transformation: A tool for activation of latent gene clusters

In the quest to discover new bioactive leads for unmet medical needs, actinomycetes present a treasure trove of undiscovered molecules. The ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to sparse studies of cryptic gene clusters. These gene clusters can be tapped to explore scaffolds hidden in them. The up-regulation of the dormant genes is one of the most important areas of interest in the bioactive compounds discovery from microbial resources. Genome shuffling is a powerful tool for the activation of such gene clusters. Lei Yu, et al.1, reported enhancement of the lactic acid production in Lactobacillus rhamnosus through genome shuffling brought about by protoplast fusion. D. A. Hopwood et al.2 suggested that an interspecific recombination between strains producing different secondary metabolites, generate producers of ‘hybrid’ antibiotics. They also mentioned that an intraspecific fusion of actinomycetes protoplast bring about random and high frequency recombination. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol (PEG). In our study we had undertaken random genome shuffling by protoplast fusion of two, rather poorly expressed actinomycetes strains A (Figure 1) & B (Figure 2), mediated by PEG; and also by naked DNA transformation of Strain A protoplast with the DNA of Strain B. We generated eight protoplast fusants and seven transformants from parents considering their morphological difference from the two parent strains. These 15 recombinants were checked for their same colony morphologies for five generations to ensure phenotypic stability. Antibiotic resistance pattern was established by using antibiotic octodisc to generate a marker profile of the recombinants and the parent strains. Eight fusants (AP-18, AP-25, AP-2, AP-11, AP-14, AP-19, AP-11 and AP-27) and four transformants (TAP-30, TAP-31, TAP-32 and TAP-33) (Table 1) have shown a different antibiotic sensitivity pattern as compared to the parent strains. We envisage that these recombinants harbor shuffled gene clusters. To support array of conditions to express such shuffled/cryptic genes the recombinants were fermented in 11 different nutrient stress variants. The extracts generated were subjected to metabolite profiling by HPLC-ELSD, bioactivity screening for cytotoxicity and anti-infective capabilities. Two fusants AP-11 (Figure 3) and AP-25; one transformant TAP-32 (in growth media MBA-5 and MBA-7) displayed antifungal activity unlike parent strains (Table 2) Fusant AP-11 (Table 5) exhibited significant cell growth inhibition of five different cancer cell lines. The parents Strain A and Strain B did not exhibit any cell growth inhibition of these cell lines (Table 5). The metabolite profiling of fusant AP-11 and transformant TAP-32 was done by HPLC-ELSD. AP-11 showed the presence of five additional peaks (Figure 5 & Figure 6); TAP-32 extract from medium MBA-5 (Figure 7 & Figure 8) showed the presence of four additional peaks and TAP-32 extract from MBA-7 (Figure 9 & Figure 10) showed 14 additional peaks as compared to parent strains in similar medium and media controls. The study indicated that protoplast fusion and transformation have not only caused morphological changes but also shuffled genes responsible for synthesis of bioactive molecules. Further characterization of these new peaks is warranted.

Aug
13
Tue
2013
Invited Talk: Pertubation of DNA topology in mycobacteria @ Acharya Hall
Aug 13 @ 11:50 am – 12:12 pm

NagarajaV. Nagaraja Ph.D.
Professor, Indian Institute of Science, Bengaluru, India


Perturbation of DNA topology in mycobacteria

To maintain the topological homeostasis of the genome in the cell, DNA topoisomerases catalyse DNA cleavage, strand passage and rejoining of the ends. Thus, although they are essential house- keeping enzymes, they are the most vulnerable targets; arrest of the reaction after the first trans-esterification step leads to breaks in DNA and cell death.  Some of the successful antibacterial or anticancer drugs target the step ie arrest the reaction or stabilize the topo -DNA covalent complex. I will describe our efforts in this direction – to target DNA gyrase and also topoisomerase1 from mycobacteria. The latter, although essential, has no inhibitors described so far. The new inhibitors being characterized are also used to probe topoisomerase control of gene expression.

In the biological warfare between the organisms, a diverse set of molecules encoded by invading genomes target the above mentioned most vulnerable step of topoisomerase  reaction, leading to the accumulation of double strand breaks. Bacteria, on their part appear to have developed defense strategies to protect the cells from genomic double strand breaks. I will describe a mechanism involving three distinct gyrase interacting proteins which inhibit the enzyme in vitro. However, in vivo all these topology modulators protect DNA gyrase from poisoning effect by sequestering the enzyme away from DNA.

Next, we have targeted a topology modulator protein, a nucleoid associated protein(NAP) from Mycobacterium tuberculosis to develop small molecule inhibitors by structure based design. Over expression of HU leads to alteration in the nucleoid architecture. The crystal structure of the N-terminal half of HU reveals a cleft that accommodates duplex DNA. Based on the structural feature, we have designed inhibitors which bind to the protein and affect its interaction with DNA, de-compact the nucleoid and inhibit cell growth. Chemical probing with the inhibitors reveal the importance of HU regulon in M.tuberculosis.

Invited Talk: Regulation of the MHC complex and HLA solubilisation by the Flavivirus, Japanese Encephalitis Virus @ Acharya Hall
Aug 13 @ 12:13 pm – 12:40 pm

ManjunathR. Manjunath, Ph.D.
Associate Professor, Dept of Biochemistry, Indian Institute of Science, Bengaluru, India


REGULATION OF THE MHC COMPLEX AND HLA SOLUBILISATION BY THE FLAVIVIRUS, JAPANESE ENCEPHALITIS VIRUS

Viral encephalitis caused by Japanese encephalitis virus (JEV) and West Nile Virus (WNV) is a mosquito-borne disease that is prevalent in different parts of India and other parts of South East Asia. JEV is a positive single stranded RNA virus that belongs to the Flavivirus genus of the family Flaviviridae. The genome of JEV is about 11 kb long and codes for a polyprotein which is cleaved by both host and viral encoded proteases to form 3 structural and 7 non-structural proteins. It is a neurotropic virus which infects the central nervous system (CNS) and causes death predominantly in newborn children and young adults. JEV follows a zoonotic life-cycle involving mosquitoes and vertebrate, chiefly pigs and ardeid birds, as amplifying hosts. Humans are infected when bitten by an infected mosquito and are dead end hosts. Its structural, pathological, immunological and epidemiological aspects have been well studied. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral neutrophil leucocytosis in the brain and leads to elevated levels of type I interferon, macrophage-derived chemotactic factor, RANTES,TNF-α and IL-8 in the serum and cerebrospinal fluid.

Major Histocompatibility Complex (MHC) molecules play a very important role in adaptive immune responses. Along with various classical MHC class I molecules, other non-classical MHC class I molecules play an important role in modulating innate immune responses. Our lab has shown the activation of cytotoxic T-cells (CTLs) during JEV infection and CTLs recognize non-self peptides presented on MHC molecules and provide protection by eliminating infected cells. However, along with proinflammatory cytokines such as TNFα, they may also cause immunopathology within the JEV infected brain. Both JEV and WNV, another related flavivirus have been shown to increase MHC class I expression. Infection of human foreskin fibroblast cells (HFF) by WNV results in upregulation of HLA expression. Data from our lab has also shown that JEV infection upregulates classical as well as nonclassical (class Ib) MHC antigen expression on the surface of primary mouse brain astrocytes and mouse embryonic fibroblasts.

There are no reports that have discussed the expression of these molecules on other cells like endothelial and astrocyte that play an important role in viral invasion in humans. We have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecules, HLA-E as well as HLA-F in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial cell line (ECV304), human glioblastoma cell line (U87MG) and human foreskin fibroblast cells (HFF). Nonclassical MHC molecules such as mouse Qa-1b and its human homologue, HLA-E have been shown to be the ligand for the inhibitory NK receptor, NKG2A/CD94 and may bridge innate and adaptive immune responses. We show that JEV infection of HBMEC and ECV 304 cells upregulates the expression of HLA-A, and –B antigens as well as HLA-E and HLA-F. Increased expression of total HLA-E upon JEV infection was also observed in other human cell lines as well like, human amniotic epithelial cells, AV-3, FL and WISH cells. Further, we show for the first time that soluble HLA-E (sHLA-E) was released from infected ECV and HBMECs. In contrast, HFF cells showed only upregulation of cell-surface HLA-E expression while U87MG, a human glioblastoma cell line neither showed any cell-surface induction nor its solubilization. This shedding of sHLA-E was found to be dependent on matrix metalloproteinase (MMP) and an important MMP, MMP-9 was upregulated during JEV infection. Treatment with IFNγ resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Also, sHLA-E was shed upon treatment with IFNβ and both IFNβ and TNFα, when present together caused an additive increase in the shedding of sHLA-E. HLA-E is an inhibitory ligand for CD94/NKG2A receptor of Natural Killer cells. Thus, MMP mediated solubilization of HLA-E from infected endothelial cells may have important implications in JEV pathogenesis including its ability to compromise the blood brain barrier.

Manjunath (2)

Aug
14
Wed
2013
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall
Aug 14 @ 11:35 am – 11:45 am
Delegate Talk: Intrinsic modulation of cytokine response by mycobacteria @ Acharya Hall | Vallikavu | Kerala | India

Sukhithasri V, Nisha N, Vivek V and Raja Biswas


The host innate immune system acts as the first line of defense against invading pathogens. During an infection, the host innate immune cells recognize unique conserved molecules on the pathogen known as Pathogen Associated Molecular Patterns (PAMPs). This recognition of PAMPs helps the host mount an innate immune response leading to the production of cytokines (Akira et al. 2006). Peptidoglycan, one of the most conserved and essential component of the bacterial cell wall is one such PAMP. Peptidoglycan is known to have potent proinflammatory properties (Gust et al. 2007). Host recognize peptidoglycan using Nucleotide oligomerization domain proteins (NODs). This recognition of peptidoglycan activates the NODs and triggers downstream signaling leading to the nuclear translocation of NF-κB and production of cytokines (McDonald et al. 2005). Pathogenic bacteria modify their peptidoglycan as a strategy to evade innate immune recognition, which helps it to establish infection in the host. These peptidoglycan modifications include O-acetylation and N-glycolylation of muramic acid and N-deacetylation of N-acetylglucosamine (Davis et al. 2011). Modification of mycobacterial peptidoglycan by N-glycolylation prevents the catalytic activity of lysozyme (Raymond et al. 2005). Additionally, mycobacterial peptidoglycan is modified by amidation for unknown reasons.

Here, we have investigated the role of amidated peptidoglycan in Mycobacterium sp in modulating the innate immune response. We isolated amidated peptidoglycan from Mycobacterium sp and non-amidated peptidoglycan from Escherichia coli. We made a comparative analysis of the cytokine response produced on stimulation of innate immune cells by peptidoglycan from E. Coli and Mycobacterium sp. Macrophages and whole blood were treated with peptidoglycan and the cytokines secreted into spent medium and plasma respectively were analyzed using ELISA. Our results show that peptidoglycan from Mycobacterium sp is less effective in stimulating innate immune cells to produce cytokines. This intrinsic modulation of the cytokine response suggests that mycobacteria modify their peptidoglycan by amidation to evade innate immune response.