Metabolic Network for the Biosynthesis of Intra- and Extracellular alpha-Glucans Required for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis synthesizes intra- and extracellular alpha-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the alpha-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of alpha-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in alpha-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that alpha-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block alpha-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some alpha-glucan is exported to form the alpha-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate alpha-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADPglucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying alpha-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of alpha-glucan could be constructed, showing a direct link between the GlgE pathway, alpha-glucan biosynthesis and virulence in a mouse infection model

with nitrogen gas (N-2)

Prolonged cold storage of raw milks favors the growth of psychrotrophs, which produce heat-resistant exoenzymes of considerable spoilage potential; the bacterial proteases and lipases affect raw milk quality; among them phospholipases (PLs) may target the milk fat globule. More importantly, bacterial PLs are key virulence factors for numerous species.Two studies examined the use of nitrogen (N-2) gas and examined its effect on psychrotrophs, proteases and lipase producers when the milk was stored in closed vessels; however, the effect on PLs producers is unknown. Here we show that by considering an open system the PLs producers were sooner or later excluded in raw milk (whereas the PLs producers in the non-treated controls culminated at 10(8) CFU/ml), by effective gas treatments that bring oxygen (O-2) levels in milk tower than 0.1 ppm. No increase of the PLs producers among the anaerobes was noticed during the course of the experiments. In the experiments performed at 6.0 degrees C, the delay after which the PLs producers were no longer detectable seemed independent of the initial Level of PLs producers in raw milk (lower than 10(3) CFU/ml).We anticipate that flushing pure N-2 gas in raw milk tanks, considered as open systems, along the cold chain of raw milk storage and transportation, may be an additional technique to control psychrotrophs, and may also constitute an interesting perspective for Limiting their spoilage and pathogenic potential in food materials in general. (C) 2008 Elsevier GmbH. All rights reserved

Potential of nitrogen gas (N2) to control psychrotrophs and mesophiles in raw milk

Pure N2 gas was introduced in the headspace of test bottles containing raw milk that were then stored at either 6.0, 7.0, or 12.0 °C. Treatment with N2 significantly reduced the growth of total bacteria, and the growth of bacterial subgroups such as psychrotrophs, enterobacteria, protease- and lipase-producing bacteria, and Listeria spp, and completely excluded Bacillus cereus growth on MYP plates. The inhibitory effect was maximal at 6.0 °C, and bacterial growth could be halted at this temperature for 11 days. At 12.0 °C, N2 was able to inhibit growth during the first 48 h. No alarming or undesirable effects, such as the excessive growth of anaerobes or lactobacilli, were observed during the course of the study. The same treatments also halted the growth of one bacterial isolate in pure culture that expressed multiresistance to antibiotics. The continuous flushing of raw milk with pure N2 gas in a so-called open system that allows gas exchanges with the environment positively impacted the microbiological quality of the raw milk at a temperature range of 6.0-12.0 °C. This procedure should therefore be considered as a possible complementary method to refrigeration in controlling bacterial growth and the spoilage potential of both psychrotrophs and mesophiles in raw milk. © 2009 Elsevier GmbH. All rights reserved