Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.

Discovery of two ß-1,2-mannoside phosphorylases showing different chain-length specificities from Thermoanaerobacter sp. X-514.

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-ß-oligomannan using ß-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-ß-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-ß-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-ß-oligomannans containing a DP ≥3 and ß-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-ß-oligomannan. Here, we propose 1,2-ß-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-ß-oligomannan phosphorylase as the short name for Teth514_1788 and ß-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and ß-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Improvement of regio-specific production of myricetin-3-O-α-L-rhamnoside in engineered Escherichia coli.

Myricetin is an important flavonol whose medically important properties include activities as an antioxidant, anticarcinogen, and antimutagen. The solubility, stability, and other biological properties of the compounds can be enhanced by conjugating aglycon with sugar moieties. The type of sugar moiety also plays a significant role in the biological and physical properties of the natural product glycosides. Reconstructed Escherichia coli containing thymidine diphosphate-α-L-rhamnose sugar gene cassette and Arabidopsis-derived glycosyltransferase were used for rhamnosylation of myricetin. Myricetin (100 µM) was exogenously supplemented to induced cultures of engineered E. coli. The formation of target product-myricetin-3-O-α-L-rhamnoside-was confirmed by chromatographic and NMR analyses. The yield of product was improved by using various mutants and methylated cyclodextrin as a molecular carrier for myricetin in combination with E. coli M3G3. The maximal yield of product is 55.6 µM (3.31-fold higher than the control E. coli MG3) and shows 55.6 % bioconversion of substrate under optimized conditions.