Biology subjects

Hensel, M.

Publications and source records attributed to Hensel, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Human-restricted Salmonella exploit preassembled flagella for intracellular motility and vacuolar escape

Human-restricted typhoidal Salmonella enterica serovars cause systemic infections known as enteric fever, whereas most non-typhoidal serovars cause self-limiting gastroenteritis. Both groups rely on flagella-mediated motility during intestinal colonization and invasion, yet their intracellular flagellar programs appear to diverge after host cell entry. Non-typhoidal Salmonella are thought to rapidly silence flagellar expression intracellularly, whereas human-restricted S. Paratyphi A maintains flagellar gene expression and uses motility to evade xenophagy. However, the fate and function of flagella assembled before invasion have remained unclear. Here, we used fluorescence microscopy to follow preassembled flagella during epithelial cell invasion across typhoidal and non-typhoidal Salmonella. We show that intracellular bacteria remain flagellated during invasion across epithelial cell lines, serovars, and invasion conditions, indicating that Salmonella do not universally shed or lose preassembled flagella upon host cell entry. We demonstrate that internalized flagella are disassembled within Salmonella-containing vacuoles, whereas cytosolic flagella are targeted by autophagy. We further show that S. Paratyphi A uses flagella assembled before invasion to power intracellular motility and promote escape from the Salmonella-containing vacuole, and that intracellular motility is a phenotype shared by typhoidal Salmonella. Together, these findings reveal that human-restricted Salmonella can exploit preassembled flagella inside host cells as part of an intracellular pathogenic strategy. We propose that intracellular motility represents an adaptation to the human epithelial environment that may promote vacuolar escape and host restriction. ImportanceFlagella are best known for driving bacterial motility before host cell invasion, but their fate and function after intracellular entry remain poorly understood. This study shows that Salmonella do not universally lose their flagella during epithelial cells invasion. Instead, both typhoidal and non-typhoidal Salmonella serovars enter host cells with intact preassembled flagella, but only human-restricted typhoidal serovars use them for intracellular motility. This distinction separates flagellar presence from flagellar function and identifies intracellular motility as a serovar-specific behavior associated with human-restricted systemic disease. The findings suggest that typhoidal Salmonella have adapted to the human epithelial intracellular environment by maintaining a motile state that may promote escape from the Salmonella-containing vacuole and favor cytosolic lifestyle. More broadly, this work reframes flagella not only as extracellular colonization factors but also as intracellular determinants of pathogen behavior, host adaptation, and disease outcome.

microbiology↗

bioRxiv2026

Improved HaloTag for analyses of translocation of type III secretion system effector proteins

Effector translocation during host-pathogen interactions is a prerequisite for the entry of Salmonella into non-phagocytic cells and establishment of a replication permissive intracellular niche. Deciphering the dynamics and kinetics of translocation and subcellular localization demands live-cell imaging and tagging approaches that do not introduce detection delays or perturb the translocation process via the type III secretion system (T3SS). Effector fusions with self-labelling enzymes (SLE), such as HaloTag, allow localization and tracking at high temporal and spatial resolution. However, interference with T3SS-dependent translocation has hampered analyses of the process of translocation and early subcellular distribution and dynamics. Herein, we report that amino acid substitutions of the HaloTag can reduce the thermodynamic stability, resulting in less steric hindrance during translocation of effector-HaloTag fusions by the T3SS in mammalian cells. The top variant, HT-SP5, showed reduced retention in Salmonella, enabling more sensitive and earlier detection of translocated effector proteins of the SPI1 and SPI2 T3SS of Salmonella and of the T3SS effector Map of enteropathogenic Escherichia coli (EPEC). We applied the improved HaloTag HT-SP5 to single molecule tracking, and to follow effector protein dynamics in living host cells early after translocation by invading and intracellular bacteria. Taken together, the improved HaloTag variant HT-SP5 represents a robust and versatile SLE tag for dynamic real-time analyses of delivery and fate of T3SS-translocated effector proteins in living cells host. Application of HT-SP5 will facilitate research on effectors throughout the entire infection process at native effector levels to understand host-pathogen interactions.

microbiology↗

bioRxiv2026

Novel pESI-encoded autotransporter adhesin PeaP of epidemic Salmonella strains mediates adhesion, atypical biofilm formation, and poultry colonization

Salmonella enterica serovar Infantis (SIN) has rapidly become the dominant serovar in poultry worldwide, a success largely linked to the acquisition of the 285 kb megaplasmid pESI. While pESI-encoded antibiotic-resistance and iron-uptake systems are well characterized, pESI-mediated adhesion mechanisms remain poorly understood. Here we identify a novel pESI-encoded monomeric autotransporter adhesin, designated PeaP (pESI-encoded autotransporter protein), and demonstrate its pivotal role in atypical biofilm formation, interference with motility, and colonization of the chicken host. Biofilm assays revealed that pESI-harboring strain SIN 119944 forms robust biofilms at 37 {degrees}C and 42 {degrees}C, temperatures at which CsgD-dependent biofilm formation is negligible. Deletion of csgD did not impair this phenotype, whereas deletion of peaP abolished high-temperature biofilm development and restored motility to wild-type levels. Proteomic profiling of sessile versus planktonic cells highlighted PeaP as the most abundant pESI-derived protein in the biofilm fraction. AlphaFold-based modelling and negative-stain transmission electron microscopy showed that PeaP comprises a C-terminal {beta}-barrel and a 1,500 aa passenger domain with three tandem repeats, projecting filamentous appendages [~]37-nm from the outer membrane. Antibody blockade of PeaP reduced surface adhesion >6-fold, confirming its adhesive function. In an infection model of 2 day-old chicken, the peaP mutant displayed significantly lower colonization, indicating PeaP-mediated adhesion in vivo. Collectively, pESI-positive SIN deploys PeaP for CsgD-independent, temperature-tolerant biofilm formation and enhanced gastrointestinal colonization, providing a mechanistic basis for the epidemic spread of this multidrug-resistant pathogen in poultry.

microbiology↗

bioRxiv2026

Staphylococcus aureus triggers isolate-specific host transcriptional responses alongside TNF-R1 regulated cell death

BackgroundStaphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. MethodsFour S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. ResultsAll S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolatespecific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronicinfection isolate EDCC 5464. Cell death outcomes were likewise isolatedependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNFR1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNFR1 as a key regulator of U937 infection outcome. SaOS2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. ConclusionsThese results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summaryThe bacterium Staphylococcus aureus (S. aureus) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptors role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/723175v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1b4214org.highwire.dtl.DTLVardef@18f4ee6org.highwire.dtl.DTLVardef@1851742org.highwire.dtl.DTLVardef@ba0359_HPS_FORMAT_FIGEXP M_FIG Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937wt, SaOS-2wt, and A549wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937wt cells. S. aureus-induced cell death and caspase activation is abolished in (B) U937{Delta}TNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential ({Delta}{Psi}m) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells ({uparrow} [~]20-40%, {uparrow} {uparrow} 40-60%, {uparrow} {uparrow} {uparrow} >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com. C_FIG

microbiology↗

bioRxiv2026

Crystal structure of E. coli Nissle 1917 flagellin reveals novel features that modulate bacterial motility but not TLR5 recognition

The probiotic E. coli Nissle 1917 (EcN) strain is known to promote intestinal homeostasis via flagellin, the protomer of its motility apparatus, the flagellum. The flagellin of EcN shows atypical features, namely a hypervariable region (HVR), whose structure and significance have remained elusive. We therefore determined the crystal structure of the E. coli Nissle 1917 flagellin FliC at a resolution of 1.2 [A] which revealed an unusual domain architecture: the canonical D1 domain was found connected by an extended linker to an extensive HVR whose D2, D3 and D4 domains form an outer domain (OD) which surrounds the filament core comprised of conserved domains D0-D1. Using both recombinant proteins and gene-edited EcN strains expressing mutant flagellins, the functional requirement for these unique features was subsequently studied for effects on immune recognition on intestinal epithelial and immune cells, as well as on flagellar protein expression, assembly and bacterial motility. While human and mouse TLR5 immune recognition of flagellar proteins or intact bacteria was only moderately affected by removal of linker or D4, especially linker removal reduced protein stability and bacterial motility in both soft agar and liquid media swimming assays. Interestingly, depending on the environment, D4 or HVR removal had different effects on motility and surface structure. Finally, a site-directed mutagenesis approach highlighted that loss of TLR5 recognition strictly entails loss of motility but not vice versa. Our data indicate that specific HVRs/OD might be relevant for motility of E. coli Nissle 1917 in specialized environments, but not for immune recognition. Moreover, we find mutational tolerance is greater for immune recognition than for motility, providing new insights into bacterial adaptation to the host environment.

microbiology↗

bioRxiv2025

Localisation of Apicomplexa motor Myosin A with axial nanometric precision using graphene energy transfer

Migration in Apicomplexan parasites, a phylum that includes Plasmodium spp. responsible for malaria and the zoonotic Toxoplasma, is explained by the linear migration motor model. This model predicts that Myosin A is confined beneath the plasma membrane and above the inner membrane complex (IMC), a membranous barrier separating the cell membrane and the cytoplasm. Cumulative data, using mutant cell lines and biochemical data support this model. Paradoxically, a proof of the precise localization of Myosin A is still lacking due to limitations in resolving the motor with nanometric precision within the space comprising the membrane and the IMC. Here, we implement graphene energy transfer (GET), a novel axial nanometric ruler with a resolution of [~]1 nm, to determine the relative axial position of Myosin A and IMC1 (a reference protein defining IMC position). Using GET, we estimate the IMC dimensions with precision matching electron microscopy (EM) data and the added advantage of identifying specific bauplan proteins. We complement these measurements with 2D STED microscopy in MIRA confiners, uExMIC, and cryo-immunolabeling. Our data present the first direct localization of Myosin A populations with nanometric resolution in the third dimension, which is compatible with the linear motor model. One sentence summaryUsing the novel GET method, this study presents the first direct localization of Myosin A populations in Toxoplasma gondii with 1-4 nm axial precision, supporting the linear model.

biophysics↗

bioRxiv2024

Phosphatase-independent suppression of mucosal inflammation and disease progression by Salmonella SopB

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) translocates effector molecules via its Salmonella pathogenicity island (SPI)1 encoded type 3 secretion system (T3SS) to induce internalization by intestinal epithelial cells and manipulate cellular responses. Among these effector molecules, the Salmonella outer protein B (SopB) was shown to possess phosphatidyl-inositol phosphatase activity and induce bacterial internalisation, promote cell survival, influence endosomal trafficking and alter host cell signalling. Using a neonatal S. Typhimurium infection model, we here show that SopB in vivo suppresses early epithelial chemokine expression, delays mucosal immune cell recruitment, reduces barrier impairment by enterocyte necroptosis, and prevents disease progression and premature death. Unexpectedly, this immunosuppressive effect was independent of the phosphatidyl-inositol phosphatase and phosphotransferase activity of SopB but required an intact N-terminal domain. Thus, SopB exerts a potent phosphatase-independent immunosuppressive effect to delay local tissue inflammation and disease progression likely to promote host transmission.

microbiology↗

bioRxiv2024

Intracellular lipopolysaccharide regulates ER remodeling upon bacterial infection

Selective autophagy of the endoplasmic reticulum (ER), termed ERphagy or reticulophagy, plays a key role in organelle remodeling and cellular homeostasis. However, whether and how ERphagy is regulated during Gram-negative bacteria infection to influence host responses remains unclear. Here, we show that Salmonella enterica serovar Typhimurium releases lipopolysaccharide (LPS) that colocalizes with RETREG1/FAM134B, a reticulon-like ER-resident receptor for ERphagy. Cytosolic delivery of LPS, either during infection or via transfection, markedly increases RETREG1- and LC3B-decorated ER fragments. Mechanistically, affinity-isolation assays demonstrate that LPS directly binds RETREG1 through interactions between lipid A and positively charged residues within its amphipathic helices and C-terminal region. This interaction promotes RETREG1 oligomerization and drives ER membrane fragmentation, a process further amplified by the O-antigen moiety of LPS. The resulting ER fragments accumulate around LC3-positive Salmonella-containing vacuoles, facilitating bacterial clearance. Importantly, both intracellular and extracellular Salmonella exploit outer membrane vesicles (OMVs) to deliver LPS into the host cytosol, triggering RETREG1 activation and ER remodeling. Collectively, our findings reveal a previously unrecognized host response by which LPS of Gram-negative bacteria are sensed by the host ERphagy machinery to promote xenophagy and enhance antibacterial defense.

cell biology↗

bioRxiv2023

Rapid in-EPON CLEM for everyone: Combining fast and efficient labeling of self-labeling enzyme tags with EM resistant Janelia Fluor dyes

Correlative light and electron microscopy (CLEM) allows to link light microscopy (LM) of living cells to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between the LM and EM modalities due to chemical and physical distortions. Post-embedding CLEM enables precise registration of fluorescent structures directly on thin resin sections. However, in-resin CLEM techniques require fluorescent markers withstanding EM sample preparation. Most fluorescent proteins lose their fluorescence during EM sample preparation. Synthetic dyes present an alternative as their photostability and brightness exceed those of fluorescent proteins. Together with self-labeling enzymes (SLE) as protein tags, these fluorophores can be used to precisely label cellular structures of interest. By applying SLE labelling for post-embedding CLEM, we compared Janelia Fluor dyes and TMR to identify most suitable fluorophores. Epithelial cells expressing HaloTag fusion proteins were stained with various ligand-conjugated dyes, and fluorescence preservation was quantified after conventional room temperature sample preparation with embedding in EPON. The results obtained show that only the red dyes TMR, JF549, JFX549 and JFX554 retain their fluorescence in resin, with JFX549 and JFX554 yielding best signal intensity and signal-to-background ratio during in-resin super-resolution microscopy. Since all red dyes possess an oxygen atom within their xanthene structure, our results indicate that this might be a crucial feature making them more tolerant to sample preparation for electron microscopy. Our work reports a rapid in-resin CLEM approach that combines fast and efficient labeling of SLE tags with EM-compatible fluorophores, and serve as benchmarks for experimental planning and future engineering of fluorophores for CLEM.

molecular biology↗

bioRxiv2023

Microbially-derived indole-3-acetate alleviates diet induced steatosis and inflammation in mice

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. There is growing evidence that dysbiosis of the intestinal microbiota and disruption of microbiota-host interactions contribute to the pathology of NAFLD. We previously demonstrated that gut microbiota derived tryptophan metabolite indole-3-acetate (I3A) was decreased in both cecum and liver of high-fat diet-fed mice and attenuated the expression of inflammatory cytokines in macrophages and TNF-a and fatty acid induced inflammatory responses in an aryl-hydrocarbon receptor (AhR) dependent manner in hepatocytes. In this study, we investigated the effect of orally administered I3A in a mouse model of diet induced NAFLD. Western diet (WD)-fed mice given sugar water (SW) with I3A showed dramatically decreased serum ALT, hepatic TG, liver steatosis, hepatocyte ballooning, lobular inflammation, and hepatic production of inflammatory cytokines, compared to WD-fed mice given only SW. Metagenomic analysis show that I3A administration did not significantly modify the intestinal microbiome, suggesting that I3As beneficial effects likely reflect the metabolites direct actions on the liver. Administration of I3A partially reversed WD induced alterations of liver metabolome and proteome, notably, decreasing expression of several enzymes in hepatic lipogenesis and {beta}- oxidation. Mechanistically, we also show that AMP-activated protein kinase (AMPK) mediates the anti-inflammatory effects of I3A in macrophages. The potency of I3A in alleviating liver steatosis and inflammation clearly demonstrates its potential as a therapeutic modality for preventing the progression of steatosis to NASH.

cell biology↗

bioRxiv2022

From vacuole to cytosol: Disruptive invasion triggers cytosolic release of Salmonella Paratyphi A and subsequent cytosolic motility favors evasion of xenophagy

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal S. enterica serovars like Paratyphi A (SPA) are human restricted and cause a severe systemic disease, while many S. enterica serovars like Typhimurium (STM) have broad host range, and in human hosts usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Several genes encoding Salmonella pathogenicity island (SPI) effector proteins are absent or pseudogenes in SPA. Expression of virulence and metabolism genes show differential expression compared to STM. The intracellular transcriptomic architecture and phenotypes during presence in epithelial cells were recently described. Surprisingly, induction of motility, flagella and chemotaxis genes showed distinct expression patterns in intracellular SPA vs. STM and led to cytosolic motility of SPA. This study applies single cell microscopic analyses approaches to investigate the triggers and cellular consequences of cytosolic motility. Live cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at the invasion site leads to increased membrane damage in the nascent SCV with subsequent cytosolic release. After release into the cytosol, motile bacteria showed same velocity as under culture conditions used for infection. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Our results reveal flagella-mediated cytosolic motility as possible xenophagy evasion mechanism that could drive disease progression and contributes to dissemination of invasion-primed SPA during systemic infection. ImportanceIntracellular pathogens are commonly adapted to life in host cells either in a pathogen-containing vacuole, or free in host cell cytosol. However, transitions between these lifestyles are possible and demand specific adaptations, especially to avoid recognition and killing by host cell-autonomous immune defense. Salmonella enterica serovar Paratyphi A (SPA) belongs to typhoidal Salmonella able to cause live-threatening systemic infections in human hosts. We observed that SPA invades host cells in a way that often results in damage of the nascent vacuole and release of SPA in host cell cytosol. Here, SPA deploy flagella-mediated motility for rapid locomotion within infected cells. We demonstrate on single cell level that flagella-mediated motility enables evasion of xenophagic capture and control by the host cells. SPA uses a novel form of intracellular motility to successfully colonize human host cells.

microbiology↗

bioRxiv2022

Intralumenal docking of Cx36 channels in the ER isolates mis-trafficked protein

The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ binding motif is critical for proper ER export of Cx36. Removing the PDZ binding motif of Cx36 results in ER retention and the formation of multi-membrane vesicles containing gap junction-like connexin aggregates. Using a combination of site directed mutagenesis and electron micrographs we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.

cell biology↗

bioRxiv2022

Single molecule analyses of Salmonella translocated effector proteins reveal targeting to and dynamics in host cell endomembranes

Bacterial pathogens deliver proteins in temporal and spatial coordinated manner to manipulate mammalian host cells. The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. The pathogen resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV). By Salmonella- induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector molecules translocated by the SPI2-encoded type III secretion system (SPI2-T3SS) into host cells. A subset of these effectors is associated with, or integral in SCV and SIF membranes. It remained to be determined how SPI2-T3SS effectors reach their subcellular destination, and how these effectors interact with endomembranes remodeled by Salmonella. We deployed self-labeling enzyme (SLE) tags as novel approach to label translocated effector proteins in living host cells, and analyzed their dynamics on single molecule level. We found that SPI2-T3SS effector proteins diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differed between various effector proteins investigated and was dependent on membrane architecture of SIF. In the early infection, we observed host endosomal vesicles associated with Salmonella effector proteins. Effector-positive vesicles continuously fused with SCV and SIF membranes, providing a route of effector delivery by SPI2-T3SS translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This novel mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

microbiology↗

bioRxiv2021

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to Intracellular S. Typhimurium

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portion of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared for the first time their intracellular trascriptomic architecture and cellular phenotypes during epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was common in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated and possibly function differently, during their intracellular phase. Moreover, we show that the induction of flagella genes by intracellular SPA leads to cytosolic motility, a conserved trait specific to SPA. To the best of our knowledge, this is the first report of a flagellum-dependent intracellular motility of any Salmonella serovar in living host cells. Importantly, we demonstrate that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. We propose that such changes prime SPA towards new cycles of host cell infection and contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host, during enteric fever. IMPORTANCESalmonella enterica is a ubiquitous, facultative intracellular animal and human pathogen. Although non-typhoidal Salmonella (NTS) and typhoidal Salmonella serovars belong to the same phylogenetic species and share many virulence factors, the disease they cause in humans is very different. While the underlying mechanisms for these differences are not fully understood, one possible reason expected to contribute to their different pathogenicity is a distinct expression pattern of genes involved in host-pathogen interactions. Here, we compared the global gene expression and the intracellular behavior, during epithelial cell infection of S. Paratyphi A (SPA) and S. Typhimurium (STM), as prototypical serovars of typhoidal and NTS, respectively. Interestingly, we identified different expression patterns in key virulence and metabolic pathways, together with intracellular motility and increased invasiveness of SPA, following exit from infected cells. We hypothesize that these differences are pivotal to the invasive and systemic disease developed following SPA infection in humans.

microbiology↗

bioRxiv2021

A chemotactic sensor controls Salmonella-host cell interaction

Intimate cell contact and subsequent type three secretion system-dependent cell invasion are key steps in host colonization of Salmonella. Adhesion to complex glycostructures at the apical membrane of polarized cells is mediated by the giant adhesin SiiE. This protein is secreted by a type 1 secretion system (T1SS) and needs to be retained at the bacterial surface to exert its adhesive function. Here, we show that SiiE surface expression was linked to the presence of L-aspartate sensed by the Salmonella-specific methyl-accepting chemotaxis protein CheM. Bacteria lacking CheM were attenuated for invasion of polarized cells, whereas increased invasion was seen with Salmonella exposed to the non-metabolizable aspartate analog -methyl-D, L-aspartate (MeAsp). While components of the chemotaxis phosphorelay or functional flagella were dispensable for the increased invasion, CheM directly interacted with proteins associated with the SiiE T1SS arguing for a novel non-canonical signaling mechanism. As a result, CheM attractant signaling caused a shift from secreted to surface-retained and adhesion-competent SiiE. Thus, CheM controls the virulence function of SiiE in a precise spatio-temporal fashion depending on the host micro-milieu.

microbiology↗

bioRxiv2021

Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while S. enterica serovar Typhimurium (STM) has a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies a set of single cell analyses approaches such as dual fluorescent protein reports, effector translocation, or correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.

microbiology↗

bioRxiv2020

Comprehensive single cell analyses of the nutritional environment of intracellular Salmonella enterica

The facultative intracellular pathogen Salmonella enterica Typhimurium (STM) resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). STM is able to obtain all nutrients required for rapid proliferation, although being separated from direct access to host cell metabolites. The formation of specific tubular membrane compartment, called Salmonella-induced filaments (SIFs) are known to provides bacterial nutrition by giving STM access to endocytosed material and enabling proliferation. Additionally, STM expresses a range of nutrient uptake system for growth in nutrient limited environments to overcome the nutrition depletion inside the host. By utilizing dual fluorescence reporters, we shed light on the nutritional environment of intracellular STM in various host cells and distinct intracellular niches. We showed that STM uses nutrients of the host cell and adapts uniquely to the different nutrient conditions. In addition, we provide further evidence for improved nutrient supply by SIF formation or presence in the cytosol of epithelial cells, and the correlation of nutrient supply to bacterial proliferation.

microbiology↗

bioRxiv2020

Single cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that resides in a specialized membrane-bound compartment termed Salmonella-containing vacuole (SCV). Essential for survival and proliferation in the SCV is Salmonella pathogenicity island II (SPI2) that encodes a type III secretion system (T3SS). The SPI2-T3SS and the effector translocation maintain SCV integrity and formation of specific tubular membrane compartments, called Salmonella-induced filaments (SIFs). The SCV/SIF continuum allows STM to bypass nutritional restriction in the intracellular environment by acquiring nutrients from the host cell. Phosphate is one of the most abundant elements in living organisms and in STM, inorganic phosphate (Pi) homeostasis is mediated by the two-component regulatory system PhoBR, resulting in expression of the high affinity phosphate transporter pstSCAB-phoU. Using fluorescent protein reporters, we investigate Pi availability for STM at single cell level over time within the intracellular habitats of different host cells. We observed that the pstSCAB-phoU encoded phosphate uptake system is essential for intracellular replication of STM because there is a Pi ion concentration less 10 M within the SCV. Additionally, the demand and consumption of Pi correlates with intracellular proliferation of STM and we identify a dependency of SPI2 activity and Pi starvation.

microbiology↗