Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis.

Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo. By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host

Internal structure and pigment granules in colored alpaca fibers

Alpaca fibers have some distinct properties such as softness and warmth, which have not been fully understood in combination with the fiber internal structures. In the present investigation, the internal structures of alpaca fibers have been closely examined under the scanning electron microscope (SEM), especially in the longitudinal direction. The results showed that numerous pigment granules reside loosely inside pockets in brown and dark-brown alpaca fibers. These pigment granules were mainly distributed inside the cortical cells, the medullation regions as well as underneath the cuticles. Their size in the brown alpaca fibers was smaller and more uniformly round than in the dark-brown fibers. These granules in colored alpaca fibers loosen the bundle of cortical cells, providing many crannies in the fibers which may contribute to the superior flexibility, warmth and softness of the fibers. Moreover, there are no heavy metal elements found in the granules. The mordant hydrogen peroxide bleaching employed could eliminate the pigment granules and create many nano-volumes for further dyeing of fibers into more attractive colors

A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages.

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR-Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism.

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required leakage of lysosomal cathepsins and was independent of mitophagy, mitoproteases and proteasome degradation. In an in vivo mouse model of endomembrane damage, live lung macrophages that internalised crystals displayed impaired mitochondrial function. Single-cell RNA-sequencing revealed that lysosomal damage skewed metabolic and immune responses in alveolar macrophages subsets with increased lysosomal content. Functionally, drug modulation of macrophage metabolism impacted host responses to Mycobacterium tuberculosis infection in an endomembrane damage dependent way. This work uncovers an inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage

Comparative analysis of viral genome detection via real-time RT-PCR. Mean C_q values from duplicate runs.

<p>RABV: <i>Rabies virus</i>; ;EBLV: <i>European Bat Lyssavirus</i>;neg.: negative control; −: negative result; #: cross-reactivitiy with other <i>Lyssavirus</i> species; ?: doubtful result;</p>*<p>doubtful result was retested; i.h.: in-house assay; dilution series (0), (I), (II), (III); 10⁰, 10⁻¹, 10⁻², 10⁻³; mod.: assay modified; r: RABV-specific detection; e1: EBLV-1 specific; e1+2: EBLV-1+−2 specific; r13, r14, r13/14: R13, R14, duplex R13/14 assay by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058372#pone.0058372-Hoffmann1" target="_blank">[17]</a>; <i>fn</i>: false negative results; <b>exp</b>: expected negative results from previous publication; ts: two-step systems; no duplicates for assays D2, D3 and M; 2) Orlowska et al., 2008 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058372#pone.0058372-Orlowska1" target="_blank">[22]</a>.</p

Mean quantification cycle (C_q) values of viral genome detection via EBLV-1 and EBLV-2 specific real-time RT-PCR.

<p>RABV: <i>Rabies virus</i>;;EBLV: <i>European Bat Lyssavirus</i>;neg.: negative control; –: not applicable; #: cross–reactivitiy with other <i>Lyssavirus</i> species; ?: inconclusive based on curve shape; dilution series (0), (I), (II), (III); 10⁰, 10⁻¹, 10⁻², 10⁻³;</p>*<p>Hoffmann and Müller personal communication; ts: two-step systems; no duplicates for assays M1 and M2; all laboratories except J1 used separate species-specific real-time PCRs to detect EBLV-1 or -2.</p

Complex Epidemiology of a Zoonotic Disease in a Culturally Diverse Region: Phylogeography of Rabies Virus in the Middle East

<div><p>The Middle East is a culturally and politically diverse region at the gateway between Europe, Africa and Asia. Spatial dynamics of the fatal zoonotic disease rabies among countries of the Middle East and surrounding regions is poorly understood. An improved understanding of virus distribution is necessary to direct control methods. Previous studies have suggested regular trans-boundary movement, but have been unable to infer direction. Here we address these issues, by investigating the evolution of 183 rabies virus isolates collected from over 20 countries between 1972 and 2014. We have undertaken a discrete phylogeographic analysis on a subset of 139 samples to infer where and when movements of rabies have occurred. We provide evidence for four genetically distinct clades with separate origins currently circulating in the Middle East and surrounding countries. Introductions of these viruses have been followed by regular and multidirectional trans-boundary movements in some parts of the region, but relative isolation in others. There is evidence for minimal regular incursion of rabies from Central and Eastern Asia. These data support current initiatives for regional collaboration that are essential for rabies elimination.</p></div