Autophagy restricts Mycobacterium tuberculosis during acute infection in mice.

Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.

Bacillus anthracis Virulence Regulator AtxA Binds Specifically to the pagA Promoter Region.

Anthrax toxin activator (AtxA) is the master virulence gene regulator of Bacillus anthracis It regulates genes on the chromosome as well as the pXO1 and pXO2 plasmids. It is not clear how AtxA regulates these genes, and direct binding of AtxA to its targets has not been shown. It has been previously suggested that AtxA and other proteins in the Mga/AtxA global transcriptional regulators family bind to the curvature of their DNA targets, although this has never been experimentally proven. Using electrophoretic mobility shift assays, we demonstrate that AtxA binds directly to the promoter region of pagA upstream of the RNA polymerase binding site. We also demonstrate that in vitro, CO2 appears to have no role in AtxA binding. However, phosphomimetic and phosphoablative substitutions in the phosphotransferase system (PTS) regulation domains (PRDs) do appear to influence AtxA binding and pagA regulation. In silico, in vitro, and in vivo analyses demonstrate that one of two hypothesized stem-loops located upstream of the RNA polymerase binding site in the pagA promoter region is important for AtxA binding in vitro and pagA regulation in vivo Our study clarifies the mechanism by which AtxA interacts with one of its targets.IMPORTANCE Anthrax toxin activator (AtxA) regulates the major virulence genes in Bacillus anthracis The bacterium produces the anthrax toxins, and understanding the mechanism of toxin production may facilitate the development of therapeutics for B. anthracis infection. Since the discovery of AtxA 25 years ago, the mechanism by which it regulates its targets has largely remained a mystery. Here, we provide evidence that AtxA binds to the promoter region of the pagA gene encoding the main central protective antigen (PA) component of the anthrax toxin. These data suggest that AtxA binding plays a direct role in gene regulation. Our work also assists in clarifying the role of CO2 in AtxA's gene regulation and provides more evidence for the role of AtxA phosphorylation in virulence gene regulation.

Genome engineering in Bacillus anthracis using tyrosine site-specific recombinases.

Tyrosine site-specific recombinases (T-SSR) are polynucleotidyltransferases that catalyze cutting and joining reactions between short specific DNA sequences. We developed three systems for performing genetic modifications in Bacillus anthracis that use T-SSR and their cognate target sequences, namely Escherichia coli bacteriophage P1 Cre-loxP, Saccharomyces cerevisiae Flp-FRT, and a newly discovered IntXO-PSL system from B. anthracis plasmid pXO1. All three tyrosine recombinase systems were used for creation of a B. anthracis sporulation-deficient, plasmid-free strain deleted for ten proteases which had been identified by proteomic analysis as being present in the B. anthracis secretome. This strain was used successfully for production of various recombinant proteins, including several that are candidates for inclusion in improved anthrax vaccines. These genetic tools developed for DNA manipulation in B. anthracis were also used for construction of strains having chromosomal insertions of 1, 2, or 3 adjacent atxA genes. AtxA is a B. anthracis global transcriptional regulator required for the response of B. anthracis virulence factor genes to bicarbonate. We found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen, when strains were grown in a carbon dioxide atmosphere. These results demonstrate that the three T-SSR systems described here provide effective tools for B. anthracis genome editing. These T-SSR systems may also be applicable to other prokaryotes and to eukaryotes.