Role of metal-dependent regulation of ESX-3 secretion in intracellular survival of Mycobacterium tuberculosis

More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB. In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis

Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity

Single cell preparations of Mycobacterium tuberculosis damage the mycobacterial envelope and disrupt macrophage interactions

For decades, investigators have studied the interaction o

Fetal Myocardial Performance Index in the Third Trimester of Pregnancy: Feasibility and Reproducibility of Conventional Spectral Doppler versus Spectral Tissue Doppler Technique

Objective: This study aims to compare completion rates and reproducibility of myocardial performance index (MPI) using conventional spectral Doppler versus tissue Doppler in an unselected high-risk third trimester population. Study Design: This was a prospective cross-sectional study of high-risk pregnancies at ≥28 + 0 weeks’ gestation. Conventional spectral and tissue Doppler MPI of the left ventricle (LV) and right ventricle (RV) was attempted on all patients. Results: Seventy-nine pregnancies were evaluated. LV tissue Doppler MPI was completed more frequently than LV conventional spectral Doppler MPI (63/79, 79.7% vs. 45/79, 55.7%), p-value Conclusion: Tissue Doppler had statistically higher completion rates than conventional spectral Doppler, including the obese subgroup, with evidence of strong reproducibility in the third trimester

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes

Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems

Inhibition of fatty acid oxidation promotes macrophage control of Mycobacterium tuberculosis

Macrophage activation involves metabolic reprogramming to support antimicrobial cellular functions. How these metabolic shifts influence the outcome of infection by intracellular pathogens remains incompletely understood