Biology subjects

Kalscheuer, R.

Publications and source records attributed to Kalscheuer, R..

5 recordsLinked to original sources

bioRxiv2024

α-Aminooxyacetic acid derivatives acting as pro-drugs against Mycobacterium tuberculosis

Tuberculosis (TB), a significant cause of mortality globally, continues to claim 1.5 million lives each year. Despite recent advances in TB management, the emergence of multidrug-resistant strains of TB is exacerbating the treatment of TB. Therefore, there is an immediate necessity to uncover new anti-TB compounds with unprecedented targets. This study introduces novel antimycobacterial molecules that are based on -aminooxyacetic acid core structures. The lead compounds KSK-104 and KSK-106 displayed potent sub-micromolar antibacterial activity against Mycobacterium tuberculosis H37Rv and XDR clinical isolates, while exhibiting virtually no cytotoxicity against various human cells. Complementation experiments following whole genome sequencing of spontaneously resistant mutants generated against these bactericidal compounds suggested that they are pro-drugs that are intracellularly hydrolyzed by one or both of two specific amidohydrolases, Rv0552 and AmiC. Furthermore, proteomic and transcriptomic analyses of stressed cells and genetic interaction mapping employing transposon insertion sequencing suggest a "dirty drug" mechanism that involves the simultaneous attack of the various drug cleavage products on multiple intracellular targets. Our results suggest a primary role of the pyridoxal 5-phosphate (PLP) synthesis and salvage pathway and/or PLP-dependent enzymes, the oxidative stress network, and the largely uncharacterized Rv3092c-Rv3095 gene cluster in the mode of action.

microbiology↗

bioRxiv2023

The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113

Spread of antimicrobial resistances in the pathogen Mycobacterium tuberculosis remains a public health challenge. Thus, there is a continuous need for new therapeutic options with modes-of-action differing from current antibiotics. Previously, bioactivity-guided isolation identified the callyaerins, a class of hydrophobic cyclopeptides with an unusual (Z)-2,3-di-aminoacrylamide unit, as promising antitubercular agents. In this study, we investigated the molecular mechanisms underlying their antimycobacterial properties. Structure-activity relationship studies enabled the identification of the structural determinants relevant for their antibacterial activity. The antitubercular callyaerins are bacteriostatics selectively active against M. tuberculosis, including extensively drug-resistant (XDR) strains, with minimal cytotoxicity against human cells and a promising intracellular activity in a macrophage infection model. Via spontaneous resistance mutant screens and various chemical proteomics approaches, we showed that they act by direct targeting of the non-essential, M. tuberculosis-specific putative membrane protein Rv2113, thereby triggering a complex stress response in M. tuberculosis characterized by global downregulation of lipid biosynthesis, cell division, DNA repair and replication. Our study thus not only identifies Rv2113 as a new M. tuberculosis-specific target for antitubercular drugs, which should result in less harm of the microbiome and weaker resistance development in off-target pathogens. It furthermore demonstrates that also non-essential proteins may represent efficacious targets for antimycobacterial drugs.

microbiology↗

bioRxiv2022

The mycotoxin Beauvericin exhibits immunostimulatory effects on dendritic cells via activating the TLR4 signaling pathway

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFN{beta} production in Toll-like receptor 4 (Tlr4)-deficient BMDCs, whereas induction of these cytokines was not compromised in Tlr3/7/9 deficient BMDCs. This suggests that TLR4 might be the functional target of BEA on BMDCs. Consistently, in luciferase reporter assays BEA stimulation significantly promotes NF-{kappa}B activation in mTLR4/CD14/MD2 overexpressing but not control HEK-293 cells. RNA-sequencing analyses further confirmed that BEA induces transcriptional changes associated with the TLR4 signaling pathway. Together, these results identify TLR4 as a cellular BEA sensor and define BEA as a potent activator of BMDCs, implying that this compound can be exploited as a promising candidate structure for vaccine adjuvants or cancer immunotherapies.

immunology↗

bioRxiv2020

The household resistome - frequency of beta-lactamases, class 1 integron and antibiotic resistant bacteria in the domestic environment

The widespread of antibiotic resistance (ABR) among bacteria has become a global health concern for humans, animals and the environment. In this respect, beta-lactams and colistin are of particular interest due to the emergence of multidrug-resistant gram-negative bacteria. Households provide a habitat for bacteria originating from humans, animals, foods, contaminated clothes or other sources in which detergents and biocides are frequently used. Thus, bacteria carrying antibiotic resistance genes (ARGs) might be introduced into private households and may consequently be also released from households to the environment via domestic wastewater. Since data on ABR in the domestic environment is limited, this study aimed to determine the abundance and correlation of beta-lactamase, mobile colistin resistance and class 1 integron genes and to characterize phenotypic resistant strains in private households in Germany. Additionally, the persistence of ABR bacteria to laundering and automated dishwashing was assessed. Shower drains, washing machines and dishwashers were sampled and analyzed using quantitative real-time PCR. Resistant strains were isolated, followed by identification and antibiotic susceptibility testing using VITEK 2. The results show a significantly higher occurrence of ARGs in shower drains compared to washing machines and dishwashers. Several beta-lactamase genes co-occurred and resistance of bacterial isolates correlated positively with genotypic resistance. Laundering and automated dishwashing reduced ABR bacteria significantly and the efficacy increased with increasing duration and temperature. Overall, the domestic environment seems to represent a potential reservoir of beta-lactamase genes and beta-lactam resistant bacteria with shower drains as the dominant source of ABR. ImportanceThe abundance of ABR bacteria and ARGs is steadily increasing and has been comprehensively analyzed in natural environments, animals, foods or wastewater treatment plants. Despite of their connection to these environments, private households seem to be neglected. Therefore, the present study investigated shower drains, washing machines and dishwashers as possible sites of ARGs and ABR bacteria. The analysis of the domestic environment as a potential reservoir of resistant bacteria is crucial to determine whether households contribute to the spread of ABR or are a habitat where resistant bacteria from the environment, humans, food or water accumulate.

microbiology↗

bioRxiv2020

The molecular basis for a temperature-sensitive Mycobacterium smegmatis glgE mutant

BackgroundThe bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. MethodsWe reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. ResultsThe mutant strain accumulated the substrate for GlgE, -maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with -maltose-1-phosphate. The accumulation of -maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 {o}C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. ConclusionsWe have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. General SignificanceThis study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.

biochemistry↗