Biology subjects

Hirsch, A. K. H.

Publications and source records attributed to Hirsch, A. K. H..

4 recordsLinked to original sources

bioRxiv2025

Identification of DksA as a novel pro-inflammatory mediator of Pseudomonas aeruginosa under conditions mimicking chronic cystic fibrosis lung infection

Chronic infection with Pseudomonas aeruginosa is a major driver of airway inflammation, which plays a central role in the progression of cystic fibrosis (CF) lung disease. During long-term colonization, P. aeruginosa adapts to the CF lung by downregulating virulence factors and adopting a biofilm-associated, mucoid lifestyle. Despite the expected reduction in immune activation due to these adaptations, excessive inflammation persists, a paradox that remains poorly understood. Our objective was to identify novel bacterial mediators sustaining persistent inflammation by P. aeruginosa in the CF lung. To this end, we analyzed clinical P. aeruginosa CF isolates, cultured them in synthetic CF sputum medium, and exposed 3D lung epithelial cell cultures to the resulting cell-free supernatants. There was considerable variability in pro-inflammatory activity among the isolates, with a subset of the isolates inducing strong IL-8 secretion by the 3D cells despite low production of known virulence factors. Comparative proteomics analysis of the cell-free supernatants of pro- and anti-inflammatory isolates revealed several mediators not previously linked to inflammation. Thirteen of these candidate pro-inflammatory mediators were selected for further analysis. Using P. aeruginosa transposon mutants lacking the respective mediators, DksA (a transcription factor) was confirmed as an immunomodulatory mediator in the 3D lung model. Finally, analysis of existing transcriptomes of P. aeruginosa in CF sputum, revealed that dksA was found to be one of the most strongly expressed genes in this patient population, highlighting the relevance of our findings. In conclusion, we identified a novel P. aeruginosa mediator that may contribute to CF airway inflammation.

microbiology↗

bioRxiv2024

IspE Kinase as an Anti-infective Target: Role of a Hydrophobic Pocket in Inhibitor Binding

Enzymes of the methylerythritol phosphate (MEP) pathway are potential targets for antimicrobial drug discovery. Here we focus on 4-diphosphocytidyl-2-C-methyl-D-erythritol (IspE) kinase from the MEP pathway. We use biochemical and structural biology methods to investigate homologs from the pathogenic microorganisms; Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. We determined the X-ray structures of IspE-inhibitor complexes and studied selected inhibitors binding modes targeting the substrate pocket. The experimental results indicate the need for distinct inhibitor strategies due to structural differences among IspE homologs, particularly for A. baumannii IspE, which displays unique inhibitory profile due to a tighter hydrophobic subpocket in the substrate binding site. This study enhances our understanding of the MEP enzymes and sets the stage for structure-based drug design of selective inhibitors to combat pathogenic microorganisms.

biophysics↗

bioRxiv2022

Insights into the biosynthesis of icumazole unveiling a distinctive family of crotonyl-CoA carboxylase/reductase

Icumazoles are potent antifungal polyketides with intriguing structural features. Here, we present the polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid biosynthetic gene cluster of icumazoles. Surprisingly, an unusual non-terminal thioesterase domain divides the PKS/NRPS assembly line. The succeeding PKS modules potentially form a rare precursor 4-methyl-2-hexenoyl-ACP thus deviating from the previously proposed polyoxypeptin pathway. The 4-methyl-2-hexenoyl-ACP is further reductive carboxylated to 2-methylbutylmalonyl-ACP essential for icumazole biosynthesis by IcuL, representing a new type of crotonyl-CoA carboxylase/reductase (CCR). We characterize IcuL and its homologs TgaD and Leu10 in vitro, suggesting a stricter substrate specificity of this new family of CCRs than found in canonical ones. Intriguingly, we also find that TgaD unprecedently utilizes both NADPH and NADH as cofactors with similar efficiency, diverging from the NADPH-specific characteristic of canonical CCRs. Furthermore, a sequence similarity network-based bioinformatic survey reveals that the IcuL-like CCRs are evolutionarily separated from canonical CCRs.

biochemistry↗

bioRxiv2019

Membrane fluidity controls peptidoglycan synthesis and MreB movement.

The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions.

microbiology↗