High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages

Abstract

Viewplates 96-well glass bottom (PerkinElmer, Rodgau, Germany; 6005430) or Olefin-bottomed 96-well plate (PerkinElmer, 6055302). 12 mm or 22 mm aperture glass bottom dishes (WillCo-dishÒ, Amsterdam, the Netherlands; Cat#GWST-3512-3522).Autoclaved glass beads 2.5-3.5 mm (VWR Chemicals, Lutterworth, UK; Cat#332124G). iPSDM complete medium consisting of X-VIVO15 (Lonza, Cat#BEBP02-061Q) containing 1% (v/v) Glutamax (Gibco, Cat#35050061). NH 4 Cl (Sigma-Aldrich, Cat#A9434) quenching solution at 50 mM in 19 DPBS pH 7.2. DAPI (Invitrogen, Cat#D1306) staining solution in 19 DPBS (1 : 10 000).LysoTracker TM Red DND-99 (Invitrogen, Cat#L7528) staining solution in iPSDM complete medium (1 : 5000).Opera Phenix high-content imaging system (PerkinElmer) equipped with a 639 1.15NA water immersion objective. Harmony 4.9 software (PerkinElmer). MethodsA. Preparation of embryonic bodies and monocyte-producing factories N.B. The following steps require the use of a class II biosafety cabinet, the strict respect of conventional cell culture aseptic techniques and standard operating procedures to minimise contamination.iPSC culture 1 Maintain iPSC in Vitronectin XF coated plates with Essential 8 TM Medium.

High-content high-resolution microscopy and deep learning assisted analysis reveals host and bacterial heterogeneity duringShigellainfection

“…Here, to dissect the complexity of S. flexneri infection in epithelial cells, we use high-content high-resolution microscopy coupled to automated image analysis. High-content microscopy enables the automated acquisition of thousands of microscopy images and therefore is useful to capture the heterogeneity of individual host and bacterial cells during the infection process for quantitative analysis (Aylan et al, 2023;Brodin & Christophe, 2011;Deboosere et al, 2021;Dramé et al, 2023;Fisch et al, 2019a;Lensen et al, 2023;Pylkkö et al, 2021). We analyse multiple parameters, such as morphological host cell features, the de novo synthesis of DNA and 5 proteins in both host and bacterial cells, as well as activation of the bacterial T3SS.…”

Preliminary crystallographic study of the molybdenum cofactor biosynthesis protein MocA complexed with CTP from Mycobacterium sp. strain JC1 DSM 3803

“…Therefore, developing new drugs to combat latent TB infection is essential in preventing the spread of TB. M. tuberculosis controls and subverts the fusion of phagosomes and lysosomes, to thereby remain latent within macrophages, and is resistant to carbon monoxide (CO) (Aylan et al, 2023). As CO is produced by macrophages as part of their antimicrobial response, such resistance allows M. tuberculosis to survive within macrophages (Zacharia and Shiloh, 2012).…”

FEBS fellowships: supporting excellent science for over four decades

“…In the first, FEBS fellow Z. Faidon Brotzakis provides clear and detailed instructions on how to set up and analyse a Metadynamics electron microscopy metaInference (MEMMI) protocol, which combines cryo‐electron microscopy electron density maps with metadynamic‐enhanced‐sampling molecular dynamics to investigate the conformational heterogeneity of protein structures [ 5 ]. The second Research Protocol, by FEBS fellow Pierre Santucci and colleagues, describes how to use fluorescence microscopy‐based approaches to study the responses of Mycobacterium tuberculosis to acidic microenvironments within human macrophages, which will aid investigation into the pathogenesis of tuberculosis [ 6 ]. In the third article, FEBS fellow Geula Hanin together with Anne C. Ferguson‐Smith, describe the use of flow cytometry to enable the isolation and subsequent analysis of mammary adipocytes [ 7 ].…”