Enolase of Mycobacterium tuberculosis is a surface exposed plasminogen binding protein.

BACKGROUND: Enolase, a glycolytic enzyme, has long been studied as an anchorless protein present on the surface of many pathogenic bacteria that aids in tissue remodeling and invasion by binding to host plasminogen. METHODS: Anti-Mtb enolase antibodies in human sera were detected using ELISA. Immunoelectron microscopy, immunofluorescence microscopy and flow cytometry were used to show surface localization of Mtb enolase. SPR was used to determine the affinity of enolase-plasminogen interaction. Plasmin formation upon plasminogen binding to enolase and Mtb surface was measured by ELISA. Mice challenge and histopathological studies were undertaken to determine the protective efficacy of enolase immunization. RESULTS: Enolase of Mtb is present on its surface and binds human plasminogen with high affinity. There was an average of 2-fold increase in antibody mediated recognition of Mtb enolase in human sera from TB patients with an active disease over control individuals. Substitution of C-terminal lysine to alanine in rEno decreased its binding affinity with human plasminogen by >2-folds. Enolase bound plasminogen showed urokinase mediated conversion into plasmin. Binding of plasminogen to the surface of Mtb and its conversion into fibrinolytic plasmin was significantly reduced in the presence of anti-rEno antibodies. Immunization with rEno also led to a significant decrease in lung CFU counts of mice upon infection with Mtb H37Rv. CONCLUSIONS: Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge. GENERAL SIGNIFICANCE: Plasminogen binding has been recognized for Mtb, however, proteins involved have not been characterized. We show here that Mtb enolase is a moonlighting plasminogen binding protein.

Surface localized and extracellular Glyceraldehyde-3-phosphate dehydrogenase of Bacillus anthracis is a plasminogen binding protein.

The role of anchorless proteins on the surface of most pathogenic microorganisms has long been studied in context to their interactions with multiple host proteins, facilitating the dissemination of pathogen within the host tissues. In order to gain more insights into anthrax pathogenesis, we hereby report the presence of a prominent moonlighting enzyme, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface and in the extracellular medium of Bacillus anthracis. Out of the three heterologously expressed recombinant isoforms, rGapA (334 amino acids in native form; GapA) showed a significant NAD+ mediated GAPDH activity, whereas rGapB (342 amino acids in native form; GapB) showed a slight activity with NADP+. The rGapN (479 amino acids in native form; GapN) was enzymatically inactive with either NAD+ or NADP+. GapA was ascertained to be present in the extracellular medium and on the surface of B. anthracis. On the other hand, GapN was absent from both the surface and extracellular medium, whereas GapB was scarcely present on the surface of B. anthracis. Human plasminogen predominantly interacted with the rGapA isoform at physiological concentrations and the interaction was found to be lysine dependent. Immunization with rGapA resulted in a significant protection upon challenge with Bacillus anthracis in the murine model.

AKT mediated glycolytic shift regulates autophagy in classically activated macrophages.

Autophagy is considered as an innate defense mechanism primarily due to its role in the targeting of intracellular pathogens for lysosomal degradation. Here we report inhibition of autophagy as an adaptive response in classically activated macrophages that helps achieve high cellular ROS production and cell death-another hallmark of innate mechanisms. We show prolonged classical activation of Raw 264.7 macrophages by treating them with IFN-γ and LPS inhibited autophagy. The inhibition of autophagy was dependent on nitric oxide (NO) production which activated the AKT-mTOR signaling, the known negative regulators of autophagy. Autophagy inhibition in these cells was accompanied with a shift to aerobic glycolysis along with a decline in the mitochondrial membrane potential (MOMP). The decline in MOMP coupled with autophagy inhibition led to increased mitochondrial content and considerably elevated cellular ROS, eventually causing cell death. Next, using specific siRNA mediated knockdowns we show AKT was responsible for the glycolytic shift and autophagy inhibition in activated macrophages. Surprisingly, AKT knockdown in activated macrophages also rescued them from cell death. Finally we show that AKT mediated autophagy inhibition in the activated macrophages correlated with the depletion of glucose from the extracellular medium, and glucose supplementation not only rescued autophagy levels and reversed other phenotypes of activated macrophages, but also inhibited cell death. Thus we report here a novel link between AKT mediated glycolytic metabolism and autophagy in the activated macrophages, and provide a possible mechanism for sustained macrophage activation in vivo.

Mycobacterium tuberculosis Inhibits RAB7 Recruitment to Selectively Modulate Autophagy Flux in Macrophages.

Here we report a novel regulatory mechanism for autophagy-mediated degradation of Mycobacterium tuberculosis (Mtb) and specific strategy exploited by the virulent Mtb to evade it. We show while both avirulent (H37Ra) and virulent (H37Rv) mycobacteria could readily localize to autophagosomes, their maturation into autolysosomes (flux) was significantly inhibited by the latter strain. The inhibition of autophagy flux by the virulent strain was highly selective, as it did not perturb the basal autophagy flux in the macrophages. Selective inhibition of flux of Mtb-containing autophagosomes required virulence regulators PhoP and ESAT-6. We show that the maturation of Mtb-containing autophagosomes into autolysosomes required recruitment of the late endosome marker RAB7, forming the intermediate compartment amphisomes. Virulent Mtb selectively evaded their targeting to the amphisomes. Thus we report a crosstalk between autophagy and phagosome maturation pathway and highlight the adaptability of Mtb, manifested by selective regulation of autophagy flux.