The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD(+). Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD(+) glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a new NAD(+) glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms

Evaluating the evidence for models of life course socioeconomic factors and cardiovascular outcomes: a systematic review

BACKGROUND: A relatively consistent body of research supports an inverse graded relationship between socioeconomic status (SES) and cardiovascular disease (CVD). More recently, researchers have proposed various life course SES hypotheses, which posit that the combination, accumulation, and/or interactions of different environments and experiences throughout life can affect adult risk of CVD. Different life course designs have been utilized to examine the impact of SES throughout the life course. This systematic review describes the four most common life course hypotheses, categorizes the studies that have examined the associations between life course SES and CVD according to their life course design, discusses the strengths and weaknesses of the different designs, and summarizes the studies' findings. METHODS: This research reviewed 49 observational studies in the biomedical literature that included socioeconomic measures at a time other than adulthood as independent variables, and assessed subclinical CHD, incident CVD morbidity and/or mortality, and/or the prevalence of traditional CVD risk factors as their outcomes. Studies were categorized into four groups based upon life course design and analytic approach. The study authors' conclusions and statistical tests were considered in summarizing study results. RESULTS: Study results suggest that low SES throughout the life course modestly impacts CVD risk factors and CVD risk. Specifically, studies reviewed provided moderate support for the role of low early-life SES and elevated levels of CVD risk factors and CVD morbidity and mortality, little support for a unique influence of social mobility on CVD, and consistent support for the detrimental impact of the accumulation of negative SES experiences/conditions across the life course on CVD risk. CONCLUSIONS: While the basic life course SES study designs have various methodologic and conceptual limitations, they provide an important approach from which to examine the influence of social factors on CVD development. Some limitations may be addressed through the analysis of study cohorts followed from childhood, the evaluation of CVD risk factors in early and middle adulthood, and the use of multiple SES measures and multiple life course analysis approaches in each life course study

Discovery of a Siderophore Export System Essential for Virulence of <em>Mycobacterium tuberculosis</em>

<div><p>Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an <em>Mtb</em> mutant lacking the <em>mmpS4</em> and <em>mmpS5</em> genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of <em>mmpS4</em> and <em>mmpS5</em> did not change uptake of carboxymycobactin by <em>Mtb</em>. Thin layer chromatography showed that the Δ<em>mmpS4/S5</em> mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in <em>Mtb</em> and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the <em>mmpS4/S5</em> deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type <em>Mtb</em>. This is the strongest attenuation observed so far for <em>Mtb</em> mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of <em>Mtb</em>.</p> </div

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis

Effect of <i>mmpS4</i> and <i>mmpS5</i> on the survival of mice infected with <i>M. tuberculosis</i>.

<p>Survival of mice infected with wt <i>Mtb</i> H37Rv (ML617), Δ<i>mmpS4/S5</i> (ML618), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS5</i> (ML619), Δ<i>mmpS4/S5</i> singly complemented with <i>mmpS4</i> (ML620), or Δ<i>mmpS4/S5</i> fully complemented with <i>mmpS4</i> and <i>mmpS5</i> (ML624). Thirteen mice were infected with each strain. Mice were euthanized at day 169.</p

MmpS4 and MmpS5 are not involved in iron sensing or uptake of siderophores.

<p><b>A.</b> GFP fluorescence was measured in wt <i>Mtb</i> mc²6230, Δ<i>mmpS4/S5</i>, and Δ<i>mbtD</i>::<i>loxP</i> strains containing a <i>gfp</i>-based iron-regulated reporter construct. Strains were grown in 7H9 media and fluorescence was measured two days after the addition of carboxymycobactin (cMBT) (black bars) or blank control (grey bars). Experiments were performed in triplicate and are shown with standard deviations. <b>B.</b> Uptake of ⁵⁵Fe loaded cMBT by <i>Mtb</i> Δ<i>mmpS4/S5</i> (black circles) and Δ<i>mmpS4/S5</i> Δ<i>mbtD::hyg</i> (white triangles). Assays were performed at 37°C using a final concentration of 0.25 µM cMBT, 0.45 µCi ⁵⁵Fe in triplicate. Standard deviations are shown.</p

MmpS4 and MmpS5 are required for siderophore secretion in <i>M. tuberculosis</i>.

<p>TLC of cell-associated and secreted siderophores extracted from cultures of wt <i>Mtb</i> H37Rv parent strain ML617, Δ<i>mmpS4</i> single deletion mutant ML472, Δ<i>mmpS5</i> single deletion mutant ML405, <i>mmpS4/S5</i> double deletion mutant ML618, Δ<i>mmpS4/S5</i> double deletion mutant fully complemented with <i>mmpS4</i> and <i>mmpS5</i> ML624, and the siderophore biosynthetic mutant Δ<i>mbtD</i>::<i>hyg</i> ML1424. Cultures were labelled with 7-[¹⁴C]-salicylic acid, which was run on the TLC as a control alongside ⁵⁵Fe-loaded cMBT and mycobactin (MBT). Lanes containing cell-associated extracts were loaded with 5,000 cpm, while media extracts were loaded with 7,500 cpm.</p

Structure of the C-terminal soluble domain of MmpS4 (residues 52–140).

<p><b>A.</b> NMR structure of MmpS4_52–140 showing the backbone superposition of the final 20 conformers. The coordinates for the structures have been deposited in the Protein Data Bank (PDB accession code 2LW3). <b>B.</b> Cartoon depiction of a representative structure.</p

MmpS4, MmpS5 and MbtG are membrane-associated proteins.

<p>Proteins of subcellular fractions of wt <i>Mtb</i> (ML878) were extracted with 3% SDS and analyzed by SDS-polyacrylamide (10%) gel electrophoresis and Western blot using protein specific antibodies. <b>A.</b> Subcellular localization of MmpS4 and MmpS5. OmpATb, IdeR and Ag85 were used as controls for membrane, water-soluble cytoplasmic or periplasmic proteins and secreted proteins, respectively. MmpS4 and MmpS5 were detected using rabbit polyclonal antibodies. <b>B.</b> Subcellular localization of MbtG. MctB and IdeR were used as controls for membrane and water-soluble cytoplasmic or periplasmic proteins, respectively. MbtG was expressed with a C-terminal fusion of the Human influenza hemagglutinin (HA) tag which was detected using an HA-specific antibody.</p