7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Professor

Selective HDAC inhibition potentiates host defenses to prevent uropathogenic Escherichia coli infection

Microbial Pathogenicity

Introduction Uropathogenic E. coli (UPEC) can cause urinary tract infection (UTI) by manipulating multiple aspects of the host immune system, including epigenetic reprogramming of gene expression. Epigenetic mechanisms such as histone acetylation are a dynamically regulated process involved in many aspects of physiological and pathological immune responses. In particular, members of the histone deacetylase (HDAC) family of acetylation "erasers" are key regulators of inflammatory response during bacterial infection. Despite this association, the precise mechanistic understanding of how UPEC utilizes HDAC functions to enhance its survival and dampen host inflammatory responses remains elusive.GoalBy delving into the functional characterization of distinct HDAC genes using complementary approaches, we aspire to uncover novel mechanisms of UPEC infection in the urinary tract.MethodsTo expedite the identification of conserved HDACs that are commonly targeted during UPEC infection, we established the surrogate insect model Galleria mellonella as a cost-effective, ethically acceptable system. RNA was isolated from UPEC-infected larvae and HDAC expression was analyzed by RT-PCR. Selected HDAC genes exhibiting distinct expression patterns in UPEC-infected larvae, underwent silencing using siRNAs, chemical inhibitors, and CRISPR-Cas9 based gene editing in UPEC-infected human bladder epithelial cells (RT-112). Subsequently, we systematically examined the repercussions of silencing selected HDACs on both UPEC invasion and the host immune response.ResultsWe found that pronounced expression of selected HDACs during UPEC infection was associated with the increased mortality of larvae and infectivity in bladder epithelial cells. We report that loss of specific HDAC function led to reduced UPEC invasion in bladder epithelial cells. This is associated with increased expression of several immunity-related genes.ConclusionsOur study unveils a previously unknown infection mechanism of UPEC to manipulate host responses by targeting HDACs. The results project HDACs as a promising target for developing host-directed, non-antibiotic therapies for treating UTI.

Oral Presentation

Prof. Dr.

Discovery of pathogen-specific markers for novel antimicrobial targets through multi-omics investigation

Multidrug-resistant strains of Escherichia coli (EC) and Klebsiella pneumoniae (KP) pose a global health threat necessitating alternative treatment approaches. Understanding key pathogenic (PAT) features and their shared regulatory mechanisms, especially when compared to commensal (COM) counterparts, holds the promise of unveiling pathogen-specific markers for anti-virulence strategies. Employing bioinformatics, we constructed pangenomes encompassing 22,267 EC and 6,740 Klebsiella genomes. Focused on high-risk clonal lineages, we identified genes exclusive to PAT sequence types (ST) while absent in COM ones like EC ST10, Klebsiella variicola (KV), and Klebsiella quasipneumoniae. This set, termed the patho-core genome, was investigated for intriguing markers, subject to detailed analysis and phenotypic exploration. Further investigations involved transcriptomic and proteomic analyses in a urine-like medium for select isolates. The analysis revealed 273 unique markers for KP and 24 for PAT EC including the L-sorbose phosphotransferase system. Early stationary growth phase induction in nutrient broth supplemented with sorbose prompted diauxic growth in PAT EC STs. Omics investigations revealed co-regulation of metabolic pathways (e.g., purine and tryptophan) and induction of virulence-associated pathways (e.g., flagellar and capsule production). The integrative omics analysis of KP demonstrated similar regulatory profiles for KP, while KV profiles exhibited marked differences. Enhanced growth of KP coincided with the induction of various metabolic pathways, some aligning with the patho-core genome (e.g., cellobiose), and others displaying differential expression (e.g., citrate). Our study demonstrates that a comprehensive multi-omics approach facilitates the identification of potential targets by comparing PAT with COM bacteria. This highlights the importance of distinct genetic make-ups and regulatory mechanisms that contribute to overall bacterial fitness benefits. The induction of metabolic pathways, expanding nutrient utilization, emerged as a key mechanism. Future studies are warranted to explore the targetability and in vivo relevance of these findings.

PD. Dr.

Intermolecular interactions in the periplasmic chaperone SurA can be linked to its activity in vitro

The periplasmic chaperone SurA shuttles outer membrane proteins (OMPs) across the periplasm in many Gram-negative bacteria. Loss or proper SurA function is linked to a reduction of virulence and outer membrane integrity (1, 2). SurA has three distinct domains, the NC-core, and the peptidyl-prolyl-isomerase domains PPI1 and PPI2, which sample various conformations in solution. NC-core and PPI1 predominantly interact with substrates whereas PPI2 and the very N-terminus can be linked to an interaction with the beta-barrel assembly machinery in vitro (4-9).We established a novel SurA activity assay using a partially unfolded luciferase as a model substrate. This unfolded luciferase is catalytically inactive, upon refolding becomes active and can emit light in presence of ATP and Luciferin. Addition of SurA enhances its refolding. Molecular dynamics simulations in which we provided higher kinetic energy to overcome local minima in the SurA domain dynamics, revealed a to date unknown interaction between PPI2 and NC-core. To validate our simulations, we incubated SurA at elevated temperatures prior to the assay setup.We observed an altered behavior of SurA after heat treatment. Instead of assisting (chaperoning) the refolding of the Luciferase, SurA inhibited refolding (holdase). Mutational analysis of the PPI2 NC-core interaction surface revealed a connection between interactions between PPI2 and NC-core and holdase activity.In summary, we developed a novel assay to investigate the holdase activity of SurA and could show that interactions between the SurA NC-core and PPI2 domain regulate this holdase activity.Klein, K et al. 2019. Front. Microbiol. 10 (FEB): 100. https://doi.org/10.3389/fmicb.2019.00100.Justice, SS et al. 2006. Infect. Immun.74 (8): 4793–4800. https://doi.org/10.1128/IAI.00355-06.Calabrese, AN et al. 2020 Nat. Com. 11 (1): 2155. https://doi.org/10.1038/s41467-020-15702-1.Schiffrin, B et al. 2022 Com. Biol. 5 (1): 560. https://doi.org/10.1038/s42003-022-03502-w.Marx, DC et al 2020 Prot. Sci. 53 (9): 1689–99. https://doi.org/10.1002/pro.3924.Jia, M et al 2020 Biochem. 59 (35): 3235–46. https://doi.org/10.1021/acs.biochem.0c00507.

Antisense oligomers repress the genotoxic activity of colibactin producing E. coli

The genotoxin colibactin is a secondary metabolite produced by several Enterobacteriaceae that triggers interstrand crosslinking of DNA and thereby induces DNA damage in eukaryotic cells. The presence of colibactin-producing bacteria has been correlated to the promotion of colorectal cancer and urinary tract tumors. Further, a specific mutational signature is associated with the genotoxic activity of colibactin. Colibactin is encoded by a 54-kb gene cluster, referred to as pks (polyketide synthase) island, which consists of 19 clb genes (clbA-clbS).Here, we present a new strategy to repress the production of colibactin via targeted gene knockdown using antisense oligomers. Specifically, various peptide nucleic acid (PNAs) 10mers were designed to inhibit translation of clbA, clbP, clbQ, and clbR. The efficiency of PNAs to dampen colibactin production was evaluated at different levels. First, PNA-mediated decrease of target protein level was validated in vitro as well as in living bacterial cells. Second, we provide evidence that PNAs can decrease the in vitro crosslinking capacity of colibactin-producing bacteria. Third, cell culture infection experiments revealed a reduction of the colibactin-induced mammalian DNA damage response when bacteria were pretreated with specific PNAs.In summary, we identified three promising PNAs, two of them targeting clbA, involved in the activation of colibactin synthesis, and one targeting the key transcriptional activator ClbR. Further research is needed to evaluate off-target effects of the PNAs and to optimize their delivery to target infected mammalian cells and organoids.

Utilizing a novel reactive oxygen species-generating antimicrobial to potentiate antibiotics

The emerging antimicrobic resistance and decline in the discovery of new antibiotics requires drug development to consider alternative strategies. Silver-containing compounds became a promising alternative due to their multi-specific effects on inhibiting bacterial growth. We focus on the antimicrobial effects of AGXX, a novel antimicrobial composed of silver and ruthenium, which exerts its broad-spectrum effects through the production of highly cytotoxic reactive oxygen species (ROS) that inflict extensive macromolecular damage. Due to recent connections identified between ROS-stress and antibiotic activity, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. We investigated possible synergistic effects of AGXX on members of several antibiotic classes in 5 different bacterial pathogens. We discovered that combined treatment of sublethal concentrations of AGXX and aminoglycoside antibiotics exponentially enhanced their bactericidal activity and restored sensitivity in aminoglycoside resistant clinical isolates. To explore the underlying mechanism of the synergizing effects of AGXX on aminoglycosides, we utilized redox sensitive probes and found that the bactericidal effect of the combined AGXX/aminoglycosides treatment is mediated through elevated ROS production, which was attenuated with the addition of the ROS scavenger thiourea. We discovered that the increase in oxidative stress resulted in a disruption of iron homeostasis evidenced by a significant decrease in aconitase activity under combined AGXX/aminoglycoside treatment. Using spectroscopic methods and fluorescent microscopy, we further demonstrate that combined treatment of the two antimicrobials inflicted significant membrane damage, thus increasing membrane permeability. Our subsequent goal is to further delineate the mechanisms that underpin this synergistic effect by exploring how the metabolic processes and cellular targets disrupted by AGXX contributes to the synergistic effects with aminoglycosides. Our findings will potentially provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials.

Functional inhibition of the virulence factor ProA from Legionella pneumophila by novel zinc-binding compounds

The M4 zinc metalloprotease ProA is an important effector protein of the human lung pathogen Legionella pneumophila, which is known as the causative agent of Legionnaires' disease. Due to its versatile spectrum of substrates, ProA contributes to bacterial proliferation, immune evasion, and serious tissue destruction. Successful treatment of the atypical pneumonia is based on antibiotic therapies but cannot always prevent severe symptoms and irreversible lung damage. Additional application of specific inhibitors against virulence factors like ProA might be able to reduce life-threatening complications in patients. In order to identify promising candidates, inhibitory effects of ten novel-designed zinc-binding substances were initially evaluated in azocasein assays. The three best-performing compounds were chosen for subsequent analyses regarding versatile physiological functions of ProA during infection. It was demonstrated that a 20-fold molar excess compared to the enzyme leads to successful inhibition of human collagen IV degradation, cleavage of bacterial flagellin, immune evasion from TLR5-mediated immunity, and PMN inflammation in human lung tissue explants. Interestingly, biochemical assays revealed different tendencies in terms of inhibitor efficacy than studies in a host background. The present results not only indicate great potential of the selected zinc-binding compounds for ProA research but also demonstrate methods which are suitable to test potential drug compounds on different and important organizational levels during infection.

Oral Session
Novel Anti-Infective Measures

Appointment

Chair

Topics

Programme

Oral SessionNovel Anti-Infective Measures

Appointment

Chair

Topics

Programme

Oral Session
Novel Anti-Infective Measures