Biology subjects

Augenstreich, J.

Publications and source records attributed to Augenstreich, J..

7 recordsLinked to original sources

bioRxiv2026

Characterization of programmed cell death pathways activated in Mycobacterium tuberculosis-infected human macrophages

Mycobacterium tuberculosis (Mtb) primarily infects human lung macrophages, which serve as its major replication niche. Mtb can manipulate host macrophage cell death pathways to its advantage by inhibiting apoptosis and inducing necrotic cell death. However, the specific necrotic cell death pathway activated in human macrophages after Mtb infection remains unclear. Here, we used the THP-1 cell line and primary human monocyte-derived macrophage (hMDM) to analyze multiple programmed cell death pathways during days 1-3 after Mtb infection. Confocal microscopic analysis demonstrates that Mtb-infected THP-1 cells or hMDMs rarely exhibited apoptosis. Immunoblotting shows that Mtb induces significant CASP3 and GSDME activation in THP-1 cells, but not in hMDMs. We show that Mtb, in THP-1 cells but not hMDM, induces a significant increase in GSDMD cleavage, a hallmark of pyroptosis. MLKL phosphorylation was not observed in THP-1 cells or hMDMs during Mtb infections, indicating an absence of necroptosis. No changes in ferroptosis markers such as GPX4 expression or lipid peroxidation levels were detected. Time-lapse live-cell imaging revealed no lysosomal membrane permeabilization prior to plasma membrane rupture (PMR). However, we observed DNA release from Mtb-infected THP-1 cells and hMDMs after PMR. The DNA released from THP-1 cells exhibits low levels of myeloperoxidase and histone H3 citrullination. High-resolution confocal imaging shows that Mtb is associated with the released DNA. We demonstrate that pyroptosis induction in THP-1 cells is dispensable for the DNA release and cell death induction. In conclusion, our results reveal that Mtb-triggered cell death in hMDMs bypasses canonical cell death pathways like apoptosis, pyroptosis, necroptosis, and ferroptosis. Instead, cell death in both THP-1 cells and hMDMs correlates with DNA release, potentially through a pathway similar to NETosis in neutrophils.

cell biology↗

bioRxiv2024

da_Tracker: Automated workflow for high throughput single cell and single phagosome tracking in infected cells.

Time-lapse microscopy has emerged as a crucial tool in cell biology, facilitating a deeper understanding of dynamic cellular processes. While existing tracking tools have proven effective in detecting and monitoring objects over time, the quantification of signals within these tracked objects often faces implementation constraints. In the context of infectious diseases, the quantification of signals at localized compartments within the cell and around intracellular pathogens can provide even deeper insight into the interactions between the pathogen and host cell organelles. Existing quantitative analysis at a single-phagosome level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs with limited bias, high-throughput cell segmentation and quantitative tracking of both single cell and single bacterium/phagosome within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep-learning-based segmentation from Cellpose with tracking algorithms from Trackmate. Our workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-phagosome level (i.e. assessment of phagosome maturation over time). Its capabilities in both single-cell and single-phagosome quantification, its flexibility and open-source nature should assist studies that aim to decipher for example the pathogenicity of bacteria and the mechanism of virulence factors that could pave the way for the development of innovative therapeutic approaches.

cell biology↗

bioRxiv2024

Salmonella Typhimurium infection inhibits macrophage IFNbeta signaling in a TLR4-dependent manner

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila, Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFN{beta}, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFN{beta} has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFN{beta} signaling but not IFN{gamma} signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFN{beta}-mediated signaling and STm-infected, TLR4-deficient BMDMs do not exhibit inhibited IFN{beta} signaling. In summary, we show that macrophages exposed to STm have reduced IFN{beta} signaling via crosstalk with TLR4 signaling, and that IFN{beta} signaling does not affect cell autonomous host defense against STm.

microbiology↗

bioRxiv2023

BBQ methods: Streamlined workflows for Bacterial Burden Quantification in infected cells by confocal microscopy

Accurate quantification of bacterial burden within macrophages, termed Bacterial Burden Quantification (BBQ), is crucial for understanding host-pathogen interactions. Various methods have been employed, each with strengths and weaknesses. This article addresses limitations in existing techniques and introduces two novel automated methods for BBQ within macrophages based on confocal microscopy data analysis. The first method refines total fluorescence quantification by incorporating filtering steps to exclude uninfected cells, while the second method calculates total bacterial volume per cell to mitigate potential biases in fluorescence-based readouts. These workflows utilize PyImageJ and Cellpose software, providing reliable, unbiased, and rapid quantification of bacterial load. The proposed workflows were validated using Salmonella enterica serovar Typhimurium and Mycobacterium tuberculosis models, demonstrating their effectiveness in accurately assessing bacterial burden. These automated workflows offer valuable tools for studying bacterial interactions within host cells and provide insights for various research applications.

cell biology↗

bioRxiv2022

Spatio-temporal analysis of LC3 association to Mycobacterium tuberculosis phagosomes in human macrophages

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on mycobacterial phagosomes remain partially understood. In this study, we employed time-lapse confocal microscopy to investigate in real time the recruitment of LC3B (LC3), a key autophagy marker, to Mtb-containing vacuoles (MCVs) at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. The results reveal that approximately 70% of MCVs exhibited LC3 recruitment but was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. In addition, interferon-gamma (IFN-{gamma}) pre-treatment did not affect LC3 recruitment frequency or autophagosome maturation, but increased the susceptibility of the macrophage to Mtb-induced cell death. Instead, LC3 recruitment and lysotracker staining were mutually exclusive events alternating on some MCVs multiple times showing a new reversible aspect of this autophagy response. It also suggested a role of autophagy in membrane repair of the MCV. Consistently, LC3 recruitment was strongly associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. However, knockdown of ATG7 did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment and autophagy to the MCVs over time during Mtb infection. Our data suggests that there is no major role of autophagy in cell autonomous defense against Mtb nor membrane repair of the MCV in human macrophages. However, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for future research focused on directly investigating the damage and repair processes of phagosomal membranes.

cell biology↗

bioRxiv2020

Phthiocerol dimycocerosates from Mycobacterium tuberculosis increase the membrane activity of bacterial effectors and host receptors

Mycobacterium tuberculosis (Mtb) synthesizes a variety of atypical lipids that are exposed at the cell surface and help the bacterium infect macrophages and escape elimination by the cells immune responses. In the present study, we investigate the mechanism of action of one family of hydrophobic lipids, the phthiocerol dimycocerosates (DIM/PDIM), major lipid virulence factors. DIM are transferred from the envelope of Mtb to host membranes during infection. Using the polarity-sensitive fluorophore C-Laurdan, we visualized that DIM increase the membrane polarity of a supported lipid bilayer put in contact with mycobacteria, even beyond the site of contact. We observed that DIM activate the complement receptor 3, a predominant receptor for phagocytosis of Mtb by macrophages. DIM also increased the activity of membrane-permeabilizing effectors of Mtb, among which the virulence factor EsxA. This is consistent with previous observations that DIM help Mtb disrupt host cell membranes. Taken together, our data show that transferred DIM spread within the target membrane, remodel lipid organization and increase the activity of host cell receptors and bacterial effectors, diverting in a nonspecific manner host cell functions. We therefore bring new insight into the molecular mechanisms by which DIM increase Mtbs capability to escape the cells immune responses.

microbiology↗

bioRxiv2019

The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages

Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modelling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM molecules are transferred from the Mtb envelope to macrophage membranes during infection. Multi-scale molecular modeling and 31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregate in the stalks formed between two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological function of macrophages.

biophysics↗