This information is current as Survival in Macrophages Autophagy Expression to Enhance Bacterial TLR2-Dependent Cathelicidin and LprE Suppresses Mycobacterium tuberculosis

Among the synthesized 4′′-alkyl EGCG derivatives, 4′′-C14 EGCG inhibited EGF stimulated phosphorylation of EGFR and its downstream signaling pathways, ERK and Akt. 4′′-C14 EGCG showed significantly improved stability than EGCG and induced apoptosis.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal β-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11β, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Introduction Acute Lymphoblastic Leukemia (ALL) accounts for 20% of all hematological malignancies. L-Asparaginase has been a mainstay of ALL for the last 6 decades and is also included in the WHO list of essential medicines for ALL. Escherichia coli L-asparaginase (EcA) was the first asparaginase to be approved for clinical use. However being isolated from bacteria, EcA has many side-effects which in turn affects the tolerability and efficacy of the drug. EcA administration may cause strong immunogenic and hypersensitive reactions in the patients, necessitating withdrawal of the drug. Sensitive individuals react to repeated EcA administration with formation of anti-drug antibodies (ADAs) that bind to and inactivate the enzyme leading to inadequate plasma levels of EcA. Another serious drawback of EcA is the glutaminase activity which leads to neurotoxicity. Other side effects include hepatotoxicity, thromboembolism and pancreatitis. Although a number of attempts have been made to alleviate these problems by rational protein engineering, the optimization of therapy with EcA for ALL patients still remains a challenge. In an attempt to deal with these problems, we created several EcA mutants. On the basis of their activity, stability and antigenicity we short-listed four EcA mutants (Mutant A, B, C and D) having favourable properties for further development. Methods We identified and mutated several B-cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Enzyme activity was measured at 37 oC (optimum temperature for EcA). Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA variants was verified in ALL sensitive REH cell lines by performing MTT assay after 24 h incubation. Further the antigenicity of the mutants was assessed by performing indirect ELISA where the binding of the mutants to the commercially available l-asparaginase antibody was analysed. Further, in vivo immunogenicity was evaluated by immunizing Balc C mice with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild-type EcA and mutants to pre-existing anti-asparaginase antibodies in serum isolated from primary and relapsed ALL patients receiving asparaginase therapy was studied by indirect ELISA. Pharmacokinetics of the mutants was evaluated in female Balb C mice by plotting the asparaginase activity-time curve till 24 h following administration of a single i.v. dose of 50 IU/kg and compared with the wildtype. Finally the safety of the EcA mutants was determined by performing single-dose acute toxicity study at 3 dose levels in Balb C mice. Results At 37 oC, we did not find any significant difference in asparaginase activity of any EcA variant with the wild-type. All four variants showed markedly reduced glutaminase activity as compared to wild-type EcA (P<0.05). In MTT assay Mutant D showed 34.02%, Mutant B (32.4%), Mutant C (31.4%), Mutant A (24.22%), and wild type EcA (24.37%) reduction in REH cell viability in comparison to untreated cells. Binding to commercially available anti-asparaginase antibody was 49.09%, 32.63%, 27.43% less for Mutant D, Mutant B and Mutant C respectively compared to wild type EcA. Mice immunized with Mutant D showed 5-fold lower titres of IgG and 4-fold lower titres of IgM in comparison to wild type. Similarly, when compared to wild type, mice immunized with Mutant C showed 2.5-fold lower titres of IgG and 3.5-fold lower titres of IgM. At the same time Mutants B, C and D showed 2-3 fold less binding to pre-existing anti-asparaginase antibodies in samples collected from primary ALL patients undergoing asparaginase therapy. Similarly mutants B, C and D showed approximately 2-fold less binding to pre-existing anti-asparaginase antibodies in samples collected from relapsed ALL patients. Pharmacokinetic profiling showed that half life of Mutant A (267.28 ± 9.74), Mutant B (213.29 ± 6.53) and Mutant D (273.83 ± 35.45) was significantly longer than the wild type (102.17 ± 7.7). In acute toxicity study, we did not observe any significant toxicity of the mutants over the wildtype EcA. The findings are summarized in the figure. Conclusion Considering the immunogenicity, antigenicity and pharmacokinetics, mutant D emerged as a potent drug candidate for further development in the treatment of ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.