Artesunate/Chlorproguanil/Dapsone

Chitosan is a promising biopolymer with wide range of applications. It is the deacetylated product of chitin. Commercially, it is produced from chitin via a harsh thermochemical process that has several shortcomings and heterogenous deacetylation product. Chitin can be transformed into chitosan through enzymatic deacetylation using chitin deacetylase (CDA), enabling the production of chitosan with a specific degree of deacetylation. CDA is primarily extracted from fungi followed by bacteria and insects. The extraction of CDA from fungus is more complex, possess several health risks for human including skin lesions. Therefore, screening of potent bacterial CDA is the need of the hour. In this study, for the first time we have isolated a bacterial strain Aneurinibacillus aneurinilyticus from the rinsed water of marine crab shell, and it was found to be a potent CDA producer. The extracellular CDA from A. aneurinilyticus has been partially purified and the specific activity of the enzyme was found to be 569.73 U/ mg protein. SDS-PAGE profiling of the purified sample depicts two isomers of CDA with molecular weights of 27 kD and 45 kD. The pH and temperature optima of the purified CDA were found to be 7.4 and 37 °C, respectively. The partially purified enzyme has Km and Vmax values of 98.455 µM and 909.09 µmole/min, for non-chitinous substrate such as p-nitroacetanilide. For chitinous substrates like glycol chitin, N-acetyl glucosamine hexamer and colloidal chitin, the enzyme exhibited K_m of 96.96, 111.75 and 127.86 µM, respectively, V_max for these substrates were 23.31, 10.12 and 10.772 µmole/min, respectively. Metal ions like Mn and Mg considerably boost the production and activity of CDA, whereas Cd and Co strongly inhibit its activity. Insights from this study further substantiate that this enzyme follows Michaelis-Menten equation and has potential for industrial applications.

Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection globally, and a vaccine is urgently needed to stop transmission and disease. Chlamydial Protease Activity Factor (CPAF) is an immunoprevalent and immunodominant antigen for CD4 T cells and B cells, which makes it a strong vaccine candidate. Due to the tolerogenic nature of the female genital tract (FGT) and its lack of secondary lymphoid tissue, effective induction of protective cell-mediated immunity will likely require potent and safe mucosal adjuvants. To address this need, we produced CPAF in a cell-free protein synthesis platform and adjuvanted it with the TLR9-agonist CpG1826, a synthetic cyclic-di-AMP (CDA) STING (stimulator of interferon genes) agonist ADU-S100, and/or the squalene oil-in-water nanoemulsion, AddaS03. We determined that intranasal immunization with CPAF plus ADU-S100 was well tolerated in female mice, induced CD4 T cells characterized by TNFα production alone or in combination with IL-17 A or IFNγ, significantly reduced bacterial shedding, and shortened the duration of infection in mice intravaginally challenged with Chlamydia muridarum. These data demonstrate the potential for CDA as a mucosal adjuvant for vaccines against Chlamydia genital tract infection.