Biology subjects

Aepfelbacher, M.

Publications and source records attributed to Aepfelbacher, M..

11 recordsLinked to original sources

bioRxiv2026

MINFLUX-nanoscopy of hNAIP/NLRC4 inflammasome activation in single human macrophages

The biochemical and structural basis of NAIP/NLRC4 inflammasome activation is well understood. Far less well known are the spatiotemporal processes within cells that play a role in the activation of the NAIP/NLRC4 inflammasome. We used super-resolution imaging technology, including MINFLUX nanoscopy, to investigate the human hNAIP/hNLRC4 inflammasome in primary human macrophages following the phagocytosis of a specific Yersinia enterocolitica strain. hNAIP, which senses components of the bacterial type III secretion system (T3SS) and activates NLRC4, was recruited to bacteria-containing phagosomes after they were disrupted by the T3SS. Disruption of a single phagosome within a macrophage was sufficient to cause the accumulation of NLRC4 and NLRP3 (a sensor of another inflammasome), as well as the adapter protein ASC, in a dense condensate known as a speck. This subsequently led to plasma membrane permeation and pyroptosis. It appeared that, with increasing infection time, clusters containing NLRC4 and NLRP3/NLRC4 gradually acquired ASC for speck formation. MINFLUX-nanoscopy was able to visualise hNAIP at disrupted phagosomes with nanometre resolution, and also revealed the number and organisation of NLRC4 inflammasome discs within a speck, as well as their hNAIP content, for the first time. Surprisingly, the number of NLRC4 inflammasome discs varied considerably, ranging from 35 to 249 per speck. Also, only a small fraction of these seemed to contain hNAIP. Our results significantly improve our understanding of the process by which the hNAIP/NLRC4 inflammasome is activated in macrophages that have ingested bacteria containing a T3SS.

cell biology↗

bioRxiv2026

Genes controlling polysaccharide synthesis, protein folding and basic metabolism ensure intra-host cell adaptation and replication of Yersinia enterocolitica

Pathogenesis of yersiniosis comprises cell-invasive events in which the bacteria subvert the endocytic-autophagolysosomal pathway of infected host cells to create an intracellular niche for bacterial replication. To determine potential virulence factors that enable intracellular proliferation of Yersinia enterocolitica, we performed a high-throughput transposon-directed insertion site sequencing approach (TraDIS) with infected epithelial HeLa cells. In this screen, we identified seven particular genes that have either metabolic activities in disulfide bond formation (dsbA), glutamine synthesis (glnA), and energy generation (acnB), or dictate synthesis of enterobacterial common antigen ECA (webB/C/F) and LPS (wzx). Individual knockout of the identified genes impaired intracellular replication of Yersinia. Furthermore, these mutants differentially affected host cell autophagy and the acidification of Yersinia-containing vacuoles (YCVs). The results indicate that glnA, acnB, dsbA, wecB, and wecC support autophagy-promoted, intracellular replication of Yersinia. Folding of yet to be defined proteins dependent on dsbA seems to be most critically required for mediating these events. A wzx-controlled LPS synthesis pathway appears implicated in preventing YCV acidification. These results suggest that autophagy induction and adjustment of Yersinia to the intravacuolar environment result from a multifactorial adaptation process that involves formation of a protective carbohydrate envelope and basic metabolic enzymes needed for the refinement of productive intracellular replication.

microbiology↗

bioRxiv2025

Immune activation of primary human macrophages is suppressed by the coordinated action of Yersinia effectors

In order to suppress the host immune response, numerous bacterial pathogens utilise a type 3 secretion system (T3SS) that injects effector proteins into host target cells. We investigated the T3SS effectors of Yersinia enterocolitica (Yops) for their individual and combined effects on gene expression, inflammasome formation and calcium signaling in primary human macrophages. YopP efficiently suppressed the up- and down-regulation of thousands of macrophage genes induced by the bacterias inflammatory stimuli. This was accompanied by parallel changes in histone 3-serine 10 phosphorylation, suggesting a higher-level regulatory mechanism. Surprisingly, YopM and YopQ counteracted selected YopP effects on gene expression, e.g., of cytokine pathways. A combination of YopP and YopQ, but not other combinations of Yops or any single Yop, reduced inflammasome formation. YopH alone blocked calcium transients in infected macrophages. We propose that the T3SS effectors of Yersinia antagonistically, synergistically or individually subdue major immune pathways of human macrophages to jointly suppress macrophage activation.

cell biology↗

bioRxiv2023

A Yersinia effector activates JAK-STAT signaling in human macrophages

The multifunctional Yersinia effector YopM inhibits effector triggered immunity and increases production of the anti-inflammatory cytokine Interleukin-10 (IL-10) to suppress the host immune response. Previously it was shown that YopM induces IL-10 gene expression by elevating phosphorylation of the serine-threonine kinase RSK1 in the nucleus of human macrophages. Using transcriptomics, we now show that YopM affects expression of genes encoding components of the JAK-STAT signaling pathway. Further analysis revealed that YopM mediates nuclear translocation of the transcription factor Stat3 in Y. enterocolitica infected macrophages and that knockdown of Stat3 inhibited YopM-induced IL-10 gene expression. YopM-induced Stat3 translocation did not depend on autocrine IL-10, activation of RSK1 or tyrosine phosphorylation of Stat3. Thus, besides activation of RSK1, stimulation of nuclear translocation of Stat3 is another mechanism by which YopM increases IL-10 gene expression in macrophages.

cell biology↗

bioRxiv2022

Live imaging of Yersinia translocon formation and immune recognition in host cells

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

microbiology↗

bioRxiv2021

Visualization of bacterial type 3 secretion system components down the molecular level by MINFLUX nanoscopy

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

microbiology↗

bioRxiv2021

Epigenetic reprogramming of human macrophages by an intestinal pathogen

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61 % of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response. Author SummaryHuman pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages.

microbiology↗

bioRxiv2021

C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

microbiology↗

bioRxiv2020

SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

microbiology↗

bioRxiv2020

Cargo-specific recruitment in clathrin and dynamin-independent endocytosis

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis, and cancer invasion and is often hijacked by viral infections 1. Unlike clathrin-mediated endocytosis, which exploits cargo-specific adaptors for selective protein internalization, the clathrin and dynamin-independent endocytic pathway (CLIC-GEEC, CG-pathway) has until now been considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids 2,3. Although the core molecular players of CG endocytosis have been recently defined, no cargo-specific adaptors are known and evidence of selective protein uptake into the pathway is lacking 3. Here, we identify the first cargo-specific adaptor for CG-endocytosis and demonstrate its clinical relevance in breast cancer progression. By combining unbiased molecular characterization and super-resolution imaging, we identified the actin-binding protein swiprosin-1 (EFHD2) as a cargo-specific adaptor regulating integrin internalization via the CG-pathway. Swiprosin-1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery, IRSp53 and actin. Swiprosin-1 is critical for integrin endocytosis, but not for other CG-cargo and supports integrin-dependent cancer cell migration and invasion, with clinically relevant implications for breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG-pathway and opens the possibility to discover more adaptors regulating it.

cell biology↗

bioRxiv2018

Visualization and regulation of translocons in Yersinia type III protein secretion machines during host cell infection

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery - also named injectisome - assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their assembly have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the putatively operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology we provide novel information about the spatiotemporal organisation of T3SS translocons and their regulation by host cell factors.\n\nAuthor SummaryMany human, animal and plant pathogenic bacteria employ a molecular machine termed injectisome to inject their toxins into host cells. Because injectisomes are crucial for these bacterias infectious potential they have been considered as targets for antiinfective drugs. Injectisomes are highly similar between the different bacterial pathogens and most of their overall structure is well established at the molecular level. However, only little information is available for a central part of the injectisome named the translocon. This pore-like assembly integrates into host cell membranes and thereby serves as an entry gate for the bacterial toxins. We used state of the art fluorescence microscopy to watch translocons of the diarrheagenic pathogen Yersinia enterocolitica during infection of human host cells. Thereby we could for the first time - with fluorescence microscopy - visualize translocons connected to other parts of the injectisome. Furthermore, because translocons mark functional injectisomes we could obtain evidence that injectisomes only become active when the bacteria are almost completely enclosed by host cells. These findings provide a novel view on the organisation and regulation of bacterial translocons and may thus open up new strategies to block the function of infectious bacteria.

microbiology↗