Biology subjects

Rietsch, A.

Publications and source records attributed to Rietsch, A..

9 recordsLinked to original sources

bioRxiv2026

Spatial transcriptomics identifies the Pseudomonas aeruginosa major outer membrane protein OprF as a critical factor in blinding corneal infections

Pseudomonas aeruginosa is a globally recognized pathogen causing pulmonary, skin, and severe corneal infections (keratitis), with the potential to induce irreversible blindness if untreated. Spatial transcriptomic analysis of P. aeruginosa infected corneas identified elevated expression of the outer membrane proteins OprF and OprL and PA1414, which encodes the small RNA SicX in the corneal stroma compared with corneal epithelium. Comparative spatial transcriptomics analysis of corneas infected with an oprF transposon (TN) mutant showed reduced expression of the type III effector protein ExoT, which was absent in an oprF deficient mutant ({Delta}oprF) and in contrast to PA14, did not inhibit reactive oxygen species (ROS) production by neutrophils. Corneal infection with the {Delta}oprF mutant resulted in reduced corneal virulence and lower CFU compared to the parental PA14 strain. Collectively, our findings demonstrate a coordinated virulence program connecting OprF functionality with the release of ExoT and its ability to block ROS production and survive in infected corneas.

microbiology↗

bioRxiv2026

Pseudomonas aeruginosa Exotoxin A is not essential for corneal disease severity or bacterial survival

Pseudomonas aeruginosa produces multiple toxins and exoenzymes that contribute to its survival and ability to cause disease. As prior studies reported an important role for type III-secretion in the severity of corneal disease in P. aeruginosa keratitis, we examined if there is a requirement for the type II-secreted cytotoxin Exotoxin A (ToxA) in bacterial persistence and disease severity in infected murine corneas. Using independently generated {Delta}toxA mutants and complemented strains, we found that ToxA is expressed in vivo, but ToxA deletion did not significantly affect bacterial replication, neutrophil recruitment or disease severity. These findings contrast with an earlier study identifying a critical role for ToxA.

microbiology↗

bioRxiv2025

The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a {Delta}pscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a {Delta}exsD mutant constitutively on for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes ({Delta}pscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted ({Delta}pscU-L{Delta}exoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented {Delta}pscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to development of therapeutics targeting the T3SS system. IO_SCPLOWMPORTANCEC_SCPLOWWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations of in vivo/in situ models and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

microbiology↗

bioRxiv2024

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

microbiology↗

bioRxiv2024

The transcription factor ZNF469 regulates collagen production in liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the worlds population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

molecular biology↗

bioRxiv2023

A novel HERC4-dependent glue degrader targeting STING

Stimulator of interferon genes (STING) is a central component of the pathway sensing the presence of cytosolic nucleic acids, having a key role in type I interferon innate immune response. Localized at the endoplasmic reticulum (ER), STING becomes activated by cGAMP, which is generated by the intracellular DNA sensor cyclic GMP-AMP synthase (cGAS). Due to its critical role in physiological function and its involvement in a variety of diseases, STING has been a notable focus for drug discovery. Recent advances in drug discovery allow the targeting of proteins previously considered "un-druggable" by novel mechanism of actions. Molecular glue degraders are defined as the compounds leading targeted protein degradation (TPD) by creating novel ligase-substrate interactions. Here, we identified AK59 as a novel molecular glue degrader for STING. A genome-wide, CRISPR/Cas9 knockout screen showed that the compound-mediated degradation of STING by AK59 is compromised by the loss of HECT and RLD domain containing E3 ubiquitin protein ligase 4 (HERC4), ubiquitin-like modifier activating enzyme 5 (UBA5) and ubiquitin like modifier activating enzyme 6 (UBA6). While UBA5 and UBA6 could be the auxiliary factors for AK59 activity, our results indicate that HERC4 is the main E3 ligase for the observed degradation mechanism. Validation by individual CRISPR knockouts, co-immunoprecipitations, as well as proximity mediated reporter assays suggested that AK59 functions as a glue degrader by forming a novel interaction between STING and HERC4. Furthermore, our data reveals that AK59 was effective on the most common pathological STING mutations that cause STING-associated vasculopathy with onset in infancy (SAVI), suggesting a potential clinical application of this mechanism. Thus, these findings not only reveal a novel mechanism for compound-induced degradation of STING but also utilize HERC4 as potential E3 ligase that for TPD, enabling novel therapeutic applications.

molecular biology↗

bioRxiv2022

Neutrophil IL-1β secretion induced by ExoS expressing Pseudomonas aeruginosa is dependent on NLRP3 and Gasdermin D

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-mediated IL-1{beta} secretion and pyroptosis. Here we examined inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1{beta} secretion by neutrophils required the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT ({Delta}exoST) stimulated NLRC4 for IL-1{beta} secretion. While IL-1{beta} release from{Delta} exoST infected neutrophils was also NLRC4-dependent, this was redirected to NLRP3-dependence by PAO1 infection via the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches revealed that NLRP3, but not NLRC4, was essential for bacterial killing and limiting disease severity in a murine model of P. aeruginosa corneal infection. This reveals a novel role for ExoS ADPRT in regulating inflammasome subtype usage by neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

microbiology↗

bioRxiv2022

PopB-PcrV Interactions are Essential for Pore Formation in the Pseudomonas aeruginosa Type III Secretion System Translocon

The type III secretion system (T3SS) is a syringe-like virulence factor which delivers bacterial proteins directly into the cytoplasm of host cells. An essential component of the system is the translocon, which creates a pore in the host cell membrane through which proteins are injected. In Pseudomonas aeruginosa, the translocation pore is formed by proteins PopB and PopD and attaches to the T3SS needle via the needle tip protein PcrV. The pore is multimeric, but the exact stoichiometry and structure of the pore are unknown. We took a genetic approach to map contact points within the system by taking advantage of the fact that the translocator proteins of Pseudomonas aeruginosa and the related Aeromonas hydrophila T3SS are incompatible and cannot be freely exchanged. We created chimeric versions of P. aeruginosa PopB and A. hydrophila AopB to intentionally disrupt and restore protein-protein interactions. We identified a chimeric B-translocator that specifically breaks an interaction with the needle tip protein and interferes with the formation of the translocation pore. Breaking the interaction did not disrupt membrane insertion, arguing that the needle tip protein chaperones formation of the translocation pore.

microbiology↗

bioRxiv2020

The PopN gate-keeper complex acts on the ATPase PscN to regulate the T3SS secretion switch from early to middle substrates in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa. HighlightsO_LIT3SS substrates are secreted sequentially but information on the switches are missing C_LIO_LIInteraction of the T3SS ATPase with secreted proteins were investigated by different approaches C_LIO_LIMicroscale Thermophoresis revealed a lower affinity for chaperones alone compared to complexes C_LIO_LIThe Gate-keeper complex binds to the ATPase and increases its affinity for the needle complex C_LIO_LIA new role of the Gate-keeper complex is proposed, directly acting on the T3SS ATPase C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/224923v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1e6803corg.highwire.dtl.DTLVardef@1ed155aorg.highwire.dtl.DTLVardef@14faf6org.highwire.dtl.DTLVardef@1685ec8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗