Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection

Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence

OxLDL accumulates in pulmonary alveolar macrophages of <i>M. tuberculosis</i>-infected guinea pigs.

<p>A and B represent OxLDL immunostaining in BAL cells collected from <i>M. tuberculosis</i> infected guinea pigs at day 30 and day 60 after infection respectively (1000× magnification). Predominantly macrophages (arrows) and occasionally granulocytes (arrowhead) show intracellular staining.</p

<i>M. tuberculosis</i> infection of guinea pigs results in progressive lung lesions that are less severe in BCG vaccinated animals.

<p>The lung lesion burden increases with time in guinea pigs infected by aerosol with the H37Rv strain of <i>M. tuberculosis</i>. BCG vaccination prior to challenge decreases the rate and severity of lung granulomas as determined by lesion scores. The bars represent median values plus range (when present) for each group (n = 5). The asterisks denote statistically significant increase compared to day 5 after infection (* = p<0.05, *** = p<0.001 and **** = p<0.0001).</p

Sequence analysis of cloned extracellular mycobacterial RNA fragments present in <i>M. tuberculosis</i> CF.

1<p>The specific regions of the RNA sequences represented by each clone are depicted in Figure S1.</p>2<p>The number of clones represents the total number of sequences observed for each RNA species.</p

<i>M. tuberculosis</i> H37Rv RNA altered human monocyte's ability to control <i>M. tuberculosis</i> infection.

<p>CFUs were determined for human monocytes infected with <i>M. tuberculosis</i> H37Rv and incubated for four days in the presence of 1 µg/ml of <i>M. tuberculosis</i> H37Rv CF, purified RNA (gpRNA), or purified RNA digested with RNaseV1 (gpRNA+RNaseV1). The presence of CF or gpRNA resulted in a significant increase in CFUs as compared to the untreated infected monocytes (Control). Data represent the mean ± SEM of 3 replicates of the same experiment (*p<0.03).</p

Serum OxLDL levels increase in guinea pigs infected with <i>M. tuberculosis</i> infection.

<p>In guinea pigs sham-vaccinated with saline, serum OxLDL levels increased with the progression of disease as determined by a competitive ELISA. BCG vaccination of guinea pigs prior to virulent challenge abrogated the increase in serum OxLDL levels. Data is expressed as the mean values for each treatment group (n = 5). The asterisks denote statistically significant increase compared to the naive animals (* = p<0.05 and *** = p<0.001).</p