Biology subjects

Enninga, J.

Publications and source records attributed to Enninga, J..

16 recordsLinked to original sources

bioRxiv2026

A cilia-dependent inflammatory programme links bacterial detection to kidney disease

The primary cilium is a microtubule-based sensory organelle projecting from the plasma membrane of most mammalian cells. Genetic defects in ciliary components cause chronic kidney disease (CKD) characterized by heightened production of inflammatory and fibrogenic mediators by tubular epithelial cells. Yet, whether this reflects a physiological role of the primary cilium remains unknown. Here, we show that primary cilia on kidney tubular cells bind uropathogenic Escherichia coli and, in response to bacterial components, initiate a fibro-inflammatory program reminiscent of CKD. Integrating single-cell transcriptomics with conditional mouse models, we observed that epithelial cilia orchestrate a similar fibro-inflammatory response in the absence of infection during CKD. This convergence reveals a shared cilia-dependent signalling axis governing both host-pathogen responses and CKD progression. Mechanistically, cilia ablation selectively impairs tubular responses to ADP-heptose, a pathogen-associated molecular pattern that activates NF-{kappa}B via the cytosolic innate immune receptor ALPK1. In human kidney organoids, ADP-heptose induces robust fibro-inflammation, and genetic or pharmacological inhibition of ALPK1 attenuates this response in a rodent CKD model. Together, these findings identify primary cilia as central orchestrators of a fibro-inflammatory program linking pathogen detection to kidney disease progression.

physiology↗

bioRxiv2025

Nitrate-responsive Mycobacterial Intracytoplasmic Membranes dampen Inflammation during Mycobacterium tuberculosis Infection

Subcellular compartmentalization of metabolic processes is a main feature of prokaryotic and eukaryotic architecture. Environmental bacteria generate intracytoplasmic membranes (ICMs) as a crucial strategy to adapt their metabolism to environmental changes. While pathogenic intracellular bacteria also perceive various stressful stimuli during host interactions, the subsequent re-organization of their internal architecture has not been explored. Using cryo-electron tomography, we show that Mycobacterium tuberculosis (Mtb), a major human pathogen, is able to form ICMs outside and inside host cells in a strain-dependent manner. We characterize these Mycobacterial intracytoplasmic Membranes (MIMs) as nitrate-induced structures involved in regulation of metabolism. Furthermore, we uncover that MIM formation during macrophage infection correlates with the ability of Mtb to dampen cellular inflammatory responses. Our findings reveal a previously uncharacterized cytoplasmic structure in Mtb and link it to a functional mechanism that enables the bacterium to adapt to its intracellular niche.

microbiology↗

bioRxiv2025

Bridging the resolution gap in cryo-CLEM by introducing cryo-SXT: cryo-CLXEM

Cryo-imaging in cellular biology provides the means to visualize the cellular interieur at close-to-native conditions. A cornerstone in the field has been cryo-correlative light and electron microscopy (cryo-CLEM), with cryo-visible light fluorescent microscopy (cryo-VLFM) providing the specificity by tagging macromolecules or structures and cryo-electron tomography (cryo-ET) for the structural details at molecular level. The large resolution gap between these techniques, however, is limiting this correlative workflow as cryo-ET targets are often smaller than the resolution limit of cryo-VLFM. Here we introduce cryo-soft X-ray tomography (cryo-SXT) as an intermediate step that can compensate for the partial view and limited resolution of cryo-VLFM by providing invaluable cellular context information in 3D to the cryo-ET dataset within an integrated workflow.

cell biology↗

bioRxiv2025

The VapBC Toxin Antitoxin System Enhances Shigella flexneri Fitness Through Coordination of Metabolic Stress Adaptation and Virulence

Shigella flexneri is a facultative intracellular pathogen that causes bacillary dysentery by invading and replicating within intestinal epithelial cells. Successful intracellular survival requires the bacterium to balance metabolic adaptation with the sustained expression of virulence programs by the type III secretion system (T3SS). Toxin- antitoxin (TA) systems, including the highly conserved type II VapBC module, are classically associated with plasmid maintenance via post-segregational killing, but their broader roles during infection remain poorly understood. Here, we investigate the function of the VapBC system during S. flexneri infection. We show that the vapBC operon is activated in response to intracellular stress and that VapC-dependent cleavage of initiator tRNAfMet occurs specifically during infection. The stringent response, primarily mediated by SpoT, appears to influence operon responsiveness by maintaining vapB expression levels, while iron limitation emerges as a strong activator of vapC transcription and activity. Deletion of vapBC impairs T3SS activation and bacterial dissemination, despite normal invasion, and induces a strong host interferon response, including the upregulation of guanylate-binding proteins (GBPs), known to restrict bacterial spread. Transcriptomic profiling of the{Delta} vapBC mutant reveals downregulation of core metabolic genes and upregulation of envelope stress and other TA modules, indicating a loss of intracellular homeostasis. These findings uncover a novel role for VapBC in promoting Shigella fitness by coordinating stress adaptation, virulence expression, and immune evasion, thereby sustaining the bacteriums intracellular lifestyle.

microbiology↗

bioRxiv2025

SifA-mediated Remodeling of the Salmonella-Containing Vacuole Prevents Bacterial Dormancy by Promoting Nutrient Accessibility

Salmonella exploits a range of intracellular niches within host cells, adapting to different microenvironments that support its survival and replication. This niche diversity is mediated by bacterial effectors injected via the two type 3 secretion systems (T3SS). Salmonella resides either within membrane-bound Salmonella containing vacuoles (SCV) or they grow rapidly within the cytosol upon SCV rupture. Recently, we identified a third intracellular subpopulation, dormant Salmonella within modified vacuoles in epithelial cells, which can survive for up to 7 days in vitro. To explore how bacterial effectors influence the balance of these three subpopulations, we constructed a panel of mutants lacking genes encoding key effectors and examined their intracellular behaviors at 6 and 24 hours post-infection (hpi). Deletion of the T3SS-2 effector SifA significantly increased the dormant subpopulation at later infection time points, identifying SifA as the first known effector regulating bacterial dormancy in epithelial cells. SifA-induced Salmonella-induced filaments (SIFs) characterize the mature Salmonella-containing vacuole (SCV), and we observed that SIFs were needed for intravacuolar growth of Salmonella. We could show that SIF formation was critical for nutrient acquisition within the SCV; inhibition of SIF formation resulted in a higher proportion of dormant bacteria, akin to the effect of reduced glucose availability during infection. Both bacterial and host glycolysis pathways were required to prevent dormancy, as proper nutrient scavenging through SIF-mediated modification of the SCV is essential for maintaining a replicative, non-dormant state. These results underscore the importance of nutrient access, facilitated by reprogramming host endomembrane trafficking, for Salmonella to avoid dormancy and sustain active replication within its intracellular niche. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/631959v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d4e9cdorg.highwire.dtl.DTLVardef@419d70org.highwire.dtl.DTLVardef@1c75fb0org.highwire.dtl.DTLVardef@5b5d59_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Intranasal delivery of a broadly neutralizing single domain antibody targeting ACE2 protects against SARS-CoV-2 infection

SARS-CoV-2 accumulates mutations over time leading to the emergence of variants, which become largely resistant to existing vaccines and spike protein-targeted antiviral treatment. Therefore, there is a need for other therapies with broad efficiency. Here, we targeted the angiotensin-converting enzyme 2 (ACE2), the major entry receptor for SARS-CoV-2. We purified three single domain heavy chain antibodies (VHHs) after immunization of an alpaca with the ectodomain of ACE2. These VHHs bound ACE2 with nanomolar affinity and specifically detected membrane-anchored ACE2. Two of them (B07 and B09) neutralized by a competitive mechanism multiple SARS-CoV-2 isolates, including Omicron variants (XBB.1.16.1; EG.5.1.3; BA.2.86.1), without impacting the proteolytic activity of the enzyme. Fusion of B07 with conventional Fc domain markedly improved its binding and neutralizing efficacy. This dimeric Fc-conjugated B07 (B07-Fc) recognized specific residues of the N-terminal helix 1 of ACE2. When administrated prophylactically and intranasally, B07-Fc induced a strong dose-dependent protection of mice expressing human ACE2 (K18-hACE2) from SARS-CoV-2 Omicron. Hamsters were weakly protected due to low binding of B07-Fc to hamster ACE2. These single domain antibodies targeting hACE2 represent potential broad-spectrum therapeutic candidates against any emerging viruses using ACE2 as a receptor. These inhalable neutralizing single domain antibodies also represent a non-invasive approach against respiratory viral infection.

microbiology↗

bioRxiv2024

TYPE THREE SECRETION SYSTEM-INDUCED MECHANOPORATION OF VACUOLAR MEMBRANES

SO_SCPLOWUMMARYC_SCPLOWEndomembrane breaching is a crucial strategy employed by intracellular pathogens enclosed within vacuoles to access the nutrient-rich cytosol for intracellular replication. While bacteria use various mechanisms to compromise host membranes, the specific processes and factors involved are often unknown. Shigella flexneri, a major human pathogen, accesses the cytosol relying on the Type Three Secretion System (T3SS) and secreted effectors. Using in-cell correlative light and electron microscopy, we tracked the sequential steps of Shigella host cell entry. Moreover, we captured the T3SS, which projects a needle from the bacterial surface, in the process of puncturing holes in the vacuolar membrane. This initial puncture ensures disruption of the vacuolar membrane. Together this introduces the concept of mechanoporation via a bacterial secretion system as a crucial process for bacterial pathogen-induced membrane damage. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/613840v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1669028org.highwire.dtl.DTLVardef@8259cborg.highwire.dtl.DTLVardef@1d522caorg.highwire.dtl.DTLVardef@1df0f2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Ca2+ Regulation of Myosin II and Myosin VI during Rupture of the Shigella-Containing Vacuole

Shigella, the causative agent of bacillary dysentery, invades epithelial cells to colonize the intestinal mucosa. Following invasion, Shigella is enclosed in a vacuole that needs to rupture for bacterial intra-cytosolic replication. We show here that rupture of the Shigella vacuole requires Ca2+ influx leading to long lasting local Ca2+ increases that regulate actin dynamics affecting the Shigella vacuole integrity. These Ca2+ increases promote vacuolar rupture by activating myosin II associated with actin filaments in membrane ruffles distant from the vacuole, while tethering myosin VI at the actin coat-surrounded vacuole. Ca2+ depletion and myosin II inhibition impair formation of the actin coat and vacuole rupture. Inhibition of myosin VI also delays rupture of vacuoles but lead to their tumbling. These findings highlight a role for Ca2+ in coordinating actin-based forces and constraints during early rupture steps of bacterial vacuole, that pull on vacuolar membranes and tether them to the actin cortex via myosin II and VI, respectively, a process relevant to intracellular pathogen and endomembrane trafficking.

microbiology↗

bioRxiv2023

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection- associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Finally, hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successive BCV disassembly. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

microbiology↗

bioRxiv2022

Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies.

NF-{kappa}B driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-{kappa}B tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 isolate and demonstrate, through comparative mass spectrometry of the p65 interactome, TIGR4 challenge triggers a novel interaction of COMMD2 with p65 and p62. Mechanistically, we show this complex mediates export of p65 for degradation and COMMD2 is necessary for altering host cellular immunity. With these results, we reveal for the first time a new bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 and p65 degradation while presenting a paradigm for diverging NF-{kappa}B responses to pneumococcus.

microbiology↗

bioRxiv2020

Salmonella endorses a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. Within the infected cells S. Typhimurium colonizes multiple intracellular niches, and it is able to either actively divide at various rates, or remain dormant to persist. A comprehensive tool to monitor these distinct S. Typhimurium lifestyles has not been available so far. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry for quantification at the single-bacterium level. Using SINA, we identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This newly identified subpopulation remained dormant within a vesicular compartment distinct from either conventional Salmonella-containing vacuoles (SCV) or the previously reported niche of dormant S. Typhimurium inside macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later infection time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 expression but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche as it provides an alternative strategy for S. Typhimurium pathogenicity and persistence. Author SummarySalmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella-containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocytes that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We found that the molecular pathway suppressing S. Typhimurium dormancy in enterocytes is the one that has been shown to promote dormancy in macrophages. This suggests a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators depending on the challenged host cell type. Altogether, our work demonstrates the potential of fluorescence reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that is distinct from those observed in macrophages and fibroblasts.

microbiology↗

bioRxiv2020

Salmonella subverts autophagy balancing bacterial fate and cellular inflammation

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of host cell types and adopting different intracellular lifestyles for survival. Host endocytic trafficking and autophagy have been implied to regulate the S. Typhimurium subcellular localization and survival. To reveal alternative host regulators on S. Typhimurium lifestyle, we combined a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA) with haploid forward genetic screening. This identified transcription factor c-MYC as a negative regulator of S. Typhimurium cytosolic lifestyle via stabilizing the Salmonella-containing vacuole (SCV). We further confirmed that c-MYC downstream regulated LC3 acts to maintain SCV stability and limits S. Typhimurium cytosolic lifestyle. We demonstrated that LC3 is recruited to the SCV prior to the endomembrane damage marker Galectin 3, and it regulates SCV stability independent of the autophagosome adaptor NDP52. The LC3 processing enzymes ATG3 and ATG4 reciprocally act on SCV stability, where the loss of LC3-PE conjugation in the absence of ATG3 limits SCV damages. We further identified the dosage-dependent function of the S. Typhimurium effector SopF in mediating SCV stability by actively avoiding LC3 recruitment to the proximity of the SCV to reduce its catastrophic rupture and host cell death. Altogether, we offer insights on the significance of cellular transcription profile in the determination of S. Typhimurium pathophysiology as well as the underlying host-evasion strategy of S. Typhimurium.

microbiology↗

bioRxiv2020

A new form of actin assembly around the Shigella-containing vacuole regulates its intracellular niche

The enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterial containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host Cdc42, Toca-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV. This represents a novel microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon protects Shigellas niche from canonical maturation or host recognition.

cell biology↗

bioRxiv2019

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyosteliumdiscoideum

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1 and T3SSSPI-2, respectively). Recently, we reported that S. Typhimurium requires T3SSSPI-1 and T3SSSPI-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the {Delta}sopB and {Delta}sifA mutant strains of S. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae. ImportanceThe molecular mechanisms involved in Salmonella survival to predation by phagocytic amoebae, such as D. discoideum, remains poorly understood. Although we established that S. Typhimurium requires two specialized type-three secretion systems to survive in D. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containing S. Typhimurium in D. discoideum, and purified the D. discoideum SCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival of S. Typhimurium in D. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used by Salmonella to survive in the environment exploiting phagocytic amoebae as a reservoir.

microbiology↗

bioRxiv2019

Pneumococcal carriage requires KDM6B, a histone demethylase, for its unique inflammatory signature

Streptococcus pneumoniae is a natural colonizer of the human upper respiratory tract and an opportunistic pathogen. After colonization, bacteria either remain in the human upper respiratory tract, or may progress to cause pneumococcal disease. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-{kappa}B signature response distinct from an invasive disease causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 (RelA) and requires a histone demethylase, KDM6B. At the molecular level, we show that interaction of the 6B strain with epithelial cells leads to chromatin remodeling within the IL-11 promoter in a KDM6B dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 di-methyl. Chromatin remodeling of the IL-11 locus facilitates p65 access to three NF-{kappa}B sites, which are otherwise inaccessible when stimulated by IL-1{beta} or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B, with GSK-J4 inhibitor, and through exogenous addition of IL-11 that the host responses to 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies hereby reveal how a chromatin modifier governs cellular responses during infection.

microbiology↗

bioRxiv2017

Identifying Parameters Of Host Cell Vulnerability During Salmonella Infection By Quantitative Image Analysis And Modeling

Salmonella target and enter epithelial cells at permissive entry sites: some cells are more likely to be infected than others. However the parameters that lead to host cell heterogeneity are not known. Here, we quantitatively characterized host cell \"vulnerability\" towards Salmonella infection based on imaged parameters. We performed successive infections of the same host cell population followed by automated high-throuput microscopy and observed that infected cells have higher probability of being re-infected. Establishing a predictive model we identified two combined origins of host cell vulnerability: the pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stage of the infection, and the host cell-inherent vulnerability. We linked the host cell inherent vulnerability with its morphological attributes such as the local cell crowding, and with host cell cholesterol content. This showed that the probability of Salmonella infection success can be forecast from morphological or molecular host cell parameters.

microbiology↗