Biology subjects

Rudel, T.

Publications and source records attributed to Rudel, T..

17 recordsLinked to original sources

bioRxiv2026

Monitoring Sphingomyelin Biosynthesis at Nanoscale Resolution by Expansion Microscopy

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is synthesized by two isoenzymes, sphingomyelin synthase 1 and 2, located in the Golgi and in the plasma membrane. Abnormal sphingomyelin synthesis is associated with infections and diseases such as diabetes and cancer. Measuring cellular sphingomyelin synthase activity fosters our understanding of how these enzymes are involved in pathological processes and can be crucial for the identification of therapeutic compounds modifying sphingomyelin biosynthesis. We have developed a novel fluorometric assay that enables microscopic detection of cellular sphingomyelin synthase activity. We show that sphingomyelin synthases use propargyl choline and -NH2-{omega}-N3-C6-ceramide to generate trifunctional sphingomyelin, a lipid derivative detectable with spatial resolution via Forster resonance energy transfer. By combining this assay with expansion microscopy, a super-resolution imaging technique, we measured the distribution of de novo synthesized trifunctional sphingomyelin in cells at nanoscale resolution, thereby directly demonstrating sphingomyelin biosynthesis at the Golgi and the plasma membrane. By monitoring sphingomyelin biosynthesis and degradation in cells infected with the obligate intracellular pathogen Chlamydia trachomatis, we dissected the complex sphingolipid metabolization of these bacteria with unprecedented resolution. By correlating spatial metabolic information with lipidomics, we provide a powerful tool for investigating cellular sphingomyelin metabolism. TeaserCombining functional lipids, expansion microscopy and FRET helps visualise sphingolipid metabolism at unprecedented resolution.

biochemistry↗

bioRxiv2026

Chlamydia trachomatis infection upregulates MicroRNA21 to deplete tumor suppressor PTEN

Infection with obligate intracellular Chlamydia trachomatis (Ct) has been associated with cervical and ovarian carcinoma in humans. The possible cause of the tumor-promoting effect of the infection is thought to be a manipulation of host signaling pathways, which are essential for the growth of the bacteria, and which at the same time can support tumor growth. The PI3K and MAPK signaling cascades are outstanding candidates for this concept, as they are persistently activated during infection and required for the growth of Chlamydia and many tumors. The mechanism by which Chlamydia activates these pre-transforming survival signals in the cells is not well understood. Here we show that Ct infection up-regulates the oncomiR, miR21 by activating the host transcription factor AP-1. This depletes the target gene of miR-21, the tumor suppressor PTEN that ensures the persistent activation of the PI3K pathway. Blocking miR21 adversely affects the growth and development of the pathogen. We show here that miR21 KO mice are less susceptible to infection with Ct compared to control mice. Our data thus provides direct in vivo evidence of the induction and dependency of this obligate human pathogen on tumor-promoting miR-21-induced signaling. ImportanceChlamydia trachomatis is an obligate intracellular pathogen that relies on host metabolism for survival, yet the mechanisms by which it sustains nutrient access remain incompletely understood. Here, we identify a conserved host regulatory axis in which infection induces miR-21 to deplete the tumor suppressor PTEN, thereby enabling persistent activation of PI3K signaling. This pathway promotes a tumor-like metabolic state that supports bacterial growth while protecting infected cells from apoptosis. We further demonstrate that the AP-1-miR-21-PTEN circuit is required for efficient chlamydial replication in vitro and in vivo, and that disruption of miR-21 significantly impairs bacterial propagation in epithelial tissues. These findings reveal how C. trachomatis hijacks a central oncogenic signaling network to remodel host cell physiology and highlight a mechanistic link between infection and cancer-associated pathways. Targeting this host-driven signaling axis may provide new strategies for controlling infection independent of traditional antibiotics.

microbiology↗

bioRxiv2026

Chlamydia trachomatis deploys sphingolipids for genome organisation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and a leading cause of sexually transmitted infections worldwide. During its biphasic developmental cycle, infectious, non-replicative elementary bodies alternate with replicative reticulate bodies within a membrane-bound intracellular niche known as the inclusion. C. trachomatis relies heavily on host-derived metabolites, including sphingolipids, which are essential for inclusion integrity, bacterial growth and production of infectious progeny. Here, using expansion microscopy, we uncover an unexpected localization of sphingolipid derivatives within the highly condensed DNA nucleoids of elementary bodies. These sphingolipids are released from nucleoids prior to DNA decondensation during the elementary-to-reticulate body transition, the earliest phenotypic event in the complex developmental cycle of these bacteria. Thereafter, nucleoids undergo a characteristic DNA decondensation process that we visualized by expansion microscopy. By combining super-resolution imaging with a FRET-based metabolic tracking approach and lipidomics, we identified sphingomyelin derived from the sphingolipid analogues as the sphingolipid species predominantly associated with the condensing nucleoids of elementary bodies. Notably, reticulate bodies arrested in their developmental stage fail to accumulate sphingomyelin, suggesting a role for this lipid in stage-specific DNA condensation. Together, our findings suggest an unanticipated role for sphingolipids in bacterial DNA organization and developmental regulation in C. trachomatis.

microbiology↗

bioRxiv2025

The role of type IV pilus in the interaction of Neisseria gonorrhoeae with a corneal epithelium tissue model

Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, can infect intact corneal epithelium, leading to inflammation, cornea perforation, and blindness. Models for studying this type of gonococcal infection are few and limited. We have tested cornea models based on an immortalized hTCEpi corneal epithelium cell line for infection research using four derivatives of the N. gonorrhoeae MS11 strain that differ in pilus expression. We show that bacterial adherence depends on the functional type IV pilus in the early stages of infection, and that the formation of bacterial microcolonies and biofilms on the model surface leads to tissue destruction. The infection induces a specific cytokine response characterized by an increase in the secretion of IL-8 and TNF-, but not of IL-6. The testing of trifluoperazine, a drug that induces pilus retraction, on infected corneal tissue models showed that the drug strongly diminished the number of adherent gonococci only when applied simultaneously with bacteria, and not when bacteria were allowed to form microcolonies on the tissue surface. Our work describes, for the first time, hTCEpi-based corneal epithelium tissue models as a useful tool for investigating N. gonorrhoeae infection, with potential for application in high-throughput studies and drug screening.

microbiology↗

bioRxiv2025

Minute-scale control of ubiquitin-mediated degradation reveals dynamics of bacterial secreted effector-functions

Precise temporal control of protein abundance is essential for dissecting dynamic cellular processes. While degron-based systems enable rapid protein depletion in eukaryotic cells, comparable tools are lacking for bacterial effectors delivered into host cells during infection. Here, we establish AIDE (Auxin-Inducible Degradation of Effectors), a host-directed degradation platform that harnesses the ubiquitin-proteasome system to selectively eliminate secreted bacterial proteins, including membrane-integrated effectors. By integrating a minimal auxin-inducible degron (AID) tag into effector genes, AIDE enables rapid, reversible, and spatially confined degradation while preserving native expression and secretion. Applied to Chlamydia trachomatis, AIDE revealed that the membrane-integrated deubiquitinase Cdu1 suppresses autophagy early and later promotes developmental transitions, whereas the integral membrane fusogen IncA is continuously required for inclusion integrity. This AIDE platform provides minute-scale, spatiotemporal control over bacterial effector activity, offering a broadly applicable framework for dissecting virulence mechanisms and host-pathogen interactions across diverse secretion-dependent pathogens.

microbiology↗

bioRxiv2025

Metabolic role of the LutACB operon in Neisseria gonorrhoeae resistance to neutrophil-mediated clearance

Neisseria gonorrhoeae, an obligate human pathogen, causes the sexually transmitted disease gonorrhoea and primarily colonizes the human genitourinary tract. An infection triggers a strong neutrophil-driven inflammatory response; however, viable gonococci are frequently recovered from neutrophil-rich patient exudates, indicating that these pathogens can survive neutrophil-mediated killing in vivo. To identify bacterial factors involved in this interaction, we screened a N. gonorrhoeae transposon library for mutants with reduced survival in polymorphonuclear leukocytes (PMNs). Among the identified candidates was NGFG_RS05085 (lutB), encoding an L-lactate oxidation FeS protein within the LutACB operon, which includes lutA (NGFG_RS05075), an FeS-binding protein, and lutC, the lactate utilisation protein C, (NGFG_RS05080). Deletion of lutA, lutB, or lutC did not significantly impair growth in standard culture medium or in minimal medium complemented with glucose, L-Lactate, or pyruvate. lutB and lutC, but not lutA, knockout significantly reduced bacterial survival within neutrophils compared to the wild-type strain. Polar effects of the mutations were ruled out by monitoring gene expression in deletion mutants. Metabolic profiling revealed disruption in nucleotide and glutathione synthesis, connecting the LutACB operon with sulfur metabolism, which could be required for a defence against neutrophil-derived reactive oxygen species (ROS). These findings highlight the important novel role of the LutACB operon, which connects lactate utilization and sulfur metabolism, and enables N. gonorrhoeae to withstand neutrophil-mediated oxidative stress during infection.

microbiology↗

bioRxiv2025

The Chlamydia trachomatis secreted effector protein CT181 binds to Mcl-1 to prolong neutrophil survival

Chlamydia trachomatis (C.t) infections can lead to severe complications due to the pathogens ability to evade the host immune response, often resulting in asymptomatic infections. The mechanisms underlying this immune subversion remain incompletely understood but likely involve specific bacterial effector proteins. Here, we identify CT181 as a novel effector that directly binds to Mcl-1, a key regulator of neutrophil survival. While a C.t. CT181 mutant exhibited only modest defects in epithelial cell replication and inclusion development, it was essential for C.t. survival in neutrophils, correlating with Mcl-1 stabilization. Using a murine infection model, we demonstrate that CT181 is required for C.t. colonization and cytokine production in vivo. Our findings establish CT181 as the first bacterial effector protein known to bind Mcl-1 to enhance neutrophil survival, revealing a critical strategy by which C.t. promotes immune dysregulation, facilitating bacterial persistence while driving C.t. pathogenesis.

microbiology↗

bioRxiv2024

Infection of human organoids supports an intestinal niche for Chlamydia trachomatis

Several reports suggest that intestinal tissue may be a natural niche for Chlamydia trachomatis infection and a reservoir for persistent infections in the human body. Due to the human specificity of the pathogen and the lack of suitable host models, there is limited knowledge on this topic. In our study, we modelled the course of the chlamydial infection in human primary gastrointestinal (GI) epithelial cells originating from patient-derived organoids. We show that GI cells are resistant to apical infection and C. trachomatis needs access to the basolateral membrane to establish an infection. Transmission electron microscopy analysis reveals the presence of both normal as well as aberrant chlamydial developmental forms in the infected cells, suggesting a possible cell-type specific nature of the infection. Furthermore, we show that the plasmid-encoded Pgp3 is an important virulence factor for the infection of human GI cells. This is the first report of C. trachomatis infection in human primary intestinal epithelial cells supporting a possible niche for chlamydial infection in the human intestinal tissue. Author summaryChlamydial infection has a high global prevalence and is a major health concern. Untreated infections may cause complications and lead to serious health problems, especially in women. Although the infection is usually localized to the genital tract, experiments performed in a mouse infection model as well as the accumulating clinical data suggest that the human gastrointestinal (GI) tract might represent a hidden infection niche and a source of reinfections. In our study, we used the advantages of the organoid technology to model the chlamydial infection in patient-derived primary GI epithelial cells. We were able to show that these cells are resistant to the infection, however, Chlamydia could utilize a basolateral entry route for efficient infection. Chlamydia form either normal or persistent-like developmental forms in these GI epithelial cells. We also showed the importance of the plasmid-mediated virulence in the infection of human GI cells. The results obtained in the GI infection model replicated phenotypes predicted and expected for Chlamydia human intestinal infection, and therefore support a role of the human GI tract as a potential niche for chlamydial infection.

microbiology↗

bioRxiv2024

The AusAB non-ribosomal peptide synthase in Staphylococcus aureus preferentially incorporates exogenous phenylalanine and tyrosine into the aureusimine natural products

Non-ribosomal peptide synthases (NRPS) are modular multidomain enzymes, responsible for the biosynthesis of various secondary metabolites, in a mRNA-template independent manner. They are predominantly present in bacteria and fungi, where they synthesize a variety of products, including antibiotics, siderophores, toxins and signalling molecules. The human pathogen Staphylococcus aureus possesses one single NRPS, AusA, highly conserved in all sequenced S. aureus strains. AusA incorporates the aromatic amino acids (AAA) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAA) valine and leucine into three cyclic dipeptides collectively called aureusimines: phevalin, tyrvalin and leuvalin. By using targeted metabolomics, we found that AusA preferentially incorporates phenylalanine and tyrosine from an exogenous source into aureusimines, whereas the source of valine can be either endo- or exogenous. Upon cultivation in a chemically defined medium (CDM) lacking phenylalanine, the amino acid was not incorporated into phevalin, despite de novo phenylalanine biosynthesis. Tyrosine production remained unaffected. Conversely, upon cultivation in medium lacking tyrosine, tyrvalin production was not detected, despite tyrosine de novo biosynthesis. Phevalin production, however, remained unaltered. By contrast, omission of valine in the culture medium not only resulted in de novo valine biosynthesis but also was accompanied by phevalin production. To our knowledge, this is the first report of a selective incorporation of AAAs by a bacterial NRPS, which provides useful basis for linking bacterial cell metabolic status to the biosynthesis of secondary metabolites. IMPORTANCEPeptide and protein synthesis are fundamental processes in nature, which are largely mediated by the ribosomal machinery. An alternative pathway for peptide synthesis is non-ribosomal mRNA-template independent synthesis, performed by so-called non-ribosomal peptide synthases (NRPS). NRPSs are multi-enzyme complexes, which serve the simultaneous role of template and biosynthetic machinery. They are mostly found in bacteria and fungi and are responsible for the biosynthesis of many pharmacologically significant products, including antibiotics, anticancer compounds or immunosuppressants. The human pathogen S. aureus possesses one such NRPS, AusA, which synthesizes three cyclic dipeptides termed "aureusimines" using the aromatic amino acids phenylalanine and tyrosine, and the branched-chain amino acid valine. Although the biological role of aureusimines remains unknown, AusA appears to play a role in the interaction of S. aureus with the host. In addition, owing to its minimal canonical NRPS structure and autonomous function (i.e. most NRPS pathways require the assembly of several NRPS proteins), AusA represents an excellent model system for studying such molecular assembly lines. Our observation is, to our knowledge, the first report of a NRPS incorporating phenylalanine and tyrosine only from exogenous sources (e.g. environment, culture medium), but not from de novo "self-made" pool. This opens up new avenues in understanding and modulating the function of NRPSs (e.g. for biotechnological purposes).

microbiology↗

bioRxiv2023

Deprivation of arginine and lysine interferes with growth of patient-derived tumor xenografts

In recent years, a novel treatment method for cancer emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the L-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the L-amino acids oxidase was described to oxidate the essential amino acid L-lysine and L-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of amino acid oxidase Aplysia Punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG which lead to deprivation of arginine was well tolerated.

cancer biology↗

bioRxiv2022

A central role of sibling sRNAs NgncR_162/163 in main metabolic pathways of Neisseria gonorrhoeae

Bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in very few cases has the role in metabolism been demonstrated. We performed here a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 and their impact on the metabolism of the major human pathogen Neisseria gonorrhoeae. These sRNA have previously been shown to control genes of the citric acid and methylcitrate cycle by post-transcriptional negative regulation. By transcriptome analysis we expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially the uptake of glycine, branched chain amino acids and phenylalanine. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and hence probably affect biosynthesis of nucleotides, vitamins and other amino acids via the supply of C1-units. Metabolic flux analysis demonstrated a bipartite metabolism with glucose degradation providing intermediates for anabolic pathways, while energy metabolism via the citric acid cycle is mainly driven by amino acids, which feed into the cycle. Thus, by combined RNA-seq and metabolomics we significantly extended the regulon of NgncR_162/163 and demonstrate their role in the regulation of central metabolic pathways of the gonococcus. ImportanceNeisseria gonorrhoeae is a major human pathogen which infects more than 100 million people every year. An alarming development is the emergence of gonococcal strains resistant against virtually all of the antibiotics used for their treatment. Despite the medical importance and the vanishing treatment options of gonococcal infections, the bacterial metabolism and its regulation is only ill defined until today. We investigate here the regulation of the gonococcal metabolism by two previously studied sRNAs, NgncR_162/163 using RNA-seq and metabolomics. The results provided in this study demonstrate the regulation of transport processes and metabolic pathways involved in the biosynthesis of nucleotides, vitamins and amino acids by NgncR_162/163. Combined transcriptome and metabolome analyses provide a thus far unreached depth in the regulation of metabolic pathways by the neisserial sibling sRNAs and may therefore also be suitable for functional analysis of a growing number of other bacterial metabolic sRNA regulators.

microbiology↗

bioRxiv2021

c-Myc plays a key role in IFN-γ induced persistence of Chlamydia trachomatis

Chlamydia trachomatis (Ctr) can persist over long periods of time within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-{gamma}) released by immune cells as a mechanism of immune defence. IFN-{gamma} activates the catabolic depletion of L-tryptophan (Trp) via indoleamine 2,3-dioxygenase (IDO), resulting in persistent Chlamydia. Here we show that IFN-{gamma} depletes c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Chlamydia from IFN-{gamma}-induced persistence in cultured cell lines, but also in human fallopian tube organoids. L-tryptophan concentrations control c-Myc levels via the PI3K-GSK3{beta} axis. Unbiased metabolic analysis revealed that Chlamydia infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Chlamydia from IFN-{gamma}-induced persistence. Thus, our results challenge the longstanding hypothesis of L-tryptophan depletion through IDO as the major mechanism of IFN-{gamma}-induced metabolic immune defence and significantly extends the understanding of the role IFN-{gamma} as a broad modulator of host cell metabolism.

microbiology↗

bioRxiv2020

Intracellular Staphylococcus aureus perturbs the host cell Ca2+-homeostasis to promote cell death

The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases ranging from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction and spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide shRNA screen and identified the calcium signaling pathway to be involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+-increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by decrease in endoplasmic reticulum Ca2+-concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+-increase. As a consequence, we observed that the cytoplasmic Ca2+-rise led to increase in mitochondrial Ca2+-concentration, the activation of calpains and caspases and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+-homeostasis and induces cytoplasmic Ca2+-overload, which results in both apoptotic and necrotic cell death in parallel or succession. ImportanceDespite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered as hide-out from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+-overload, which results in host cell death. Thus, this study firstly showed how an intracellular bacterium perturbs the host cell Ca2+-homeostasis.

microbiology↗

bioRxiv2020

Identification of a novel LysR-type transcriptional regulator in Staphylococcus aureus that is crucial for secondary tissue colonization during metastatic bloodstream infection

Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection into secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used a S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used a S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched chain amino-acid biosynthesis, a methionine sulfoxide reductase and a copper transporter as wells as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enable a rapid adaptation of S. aureus to the changing host microenvironment.

microbiology↗

bioRxiv2020

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4x to 10x expansion. Confocal and structured illumination microscopy (SIM) enabled imaging of sphingolipids and their interactions with proteins in the membrane of intracellular organelles with a spatial resolution of 10-20 nm. Because sphingolipids accumulated efficiently in pathogens we used sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allowed us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 {+/-} 7.7 nm.

microbiology↗

bioRxiv2020

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Intracellularity is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death by intracellular S. aureus after translocation into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. Our study suggests that staphopain A is utilized by S. aureus to mediate escape from the host cell and thus contributes to tissue destruction and dissemination of infection. Author SummaryStaphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus is not regarded as a professional intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

microbiology↗

bioRxiv2019

A central role of glutamine in Chlamydia infection

Obligate intracellular bacteria like Chlamydia trachomatis undergo a complex developmental cycle between infectious non-replicative (EBs) and non-infectious replicative (RBs) forms. EBs shortly after entering a host cell transform to RBs, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell it is currently unknown how the transition from EBs to RBs is initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine by the bacteria is critical to initiate EB-RB transition. These bacteria utilize glutamine to synthesize cell wall peptidoglycan which has recently been detected in the septa of replicating intracellular Chlamydia. The increased requirement for glutamine in infected cells is achieved by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine was effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limited growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 knockout mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infective strategies.

microbiology↗