Biology subjects

Antelmann, H.

Publications and source records attributed to Antelmann, H..

7 recordsLinked to original sources

bioRxiv2026

The diadenosine tetraphosphate hydrolase YqeK controls fitness, biofilm formation, staphyloxanthin production and virulence in Staphylococcus aureus

Diadenosine tetraphosphate (Ap4A) is a nucleotide metabolite, which is degraded by the YqeK hydrolase in Staphylococcus aureus in vitro. In this study, we analyzed the phenotypes of the yqeK mutant under stress, antibiotics, biofilm and macrophage infection conditions to investigate the functions of Ap4A in S. aureus COL. Using nucleotide metabolomics, we confirmed that Ap4A levels are 105-fold higher in the yqeK mutant, accompanied by lower adenylate and guanylate nucleotide pools. The yqeK mutant showed a delayed growth in LB, TSB and RPMI medium, and decreased survival under lethal oxidative and quinone stress. Transcriptome analysis revealed the upregulation of the sspABC operon and the CodY, T-box Met and G-box regulons indicating increased amino acids and GTP biosynthesis, whereas the AgrA, Fur, PurR, and T-box Cys regulons were downregulated in the yqeK mutant. These gene expression changes could be restored to WT level in the yqeK complemented strain, resulting also in a DNA damage response as revealed by the induction of LexA regulon members and mobile genetic elements (pathogenicity island SAPI3 and prophage L54a). Moreover, the yqeK mutant showed enhanced biofilm formation, higher intracellular iron levels and lower staphyloxanthin levels. Using infection assays, we demonstrated a decreased survival of the yqeK mutant inside J774A.1 murine macrophages, supporting a link between Ap4A and pathogenicity regulation via Agr-controlled virulence factors in S. aureus. Future research should be directed to understand how Ap4A regulates nucleotide, iron and amino acid metabolism as well as biofilm and virulence phenotypes in S. aureus. IMPORTANCE: S. aureus is an important human pathogen, which can cause life-threatening infections especially in immunocompromised patients. Due to the prevalence of multidrug resistant strains, the search for new drug targets is an urgent goal. Diadenosine tetraphosphate (Ap4A) has been shown to contribute to stress responses, antibiotic resistance, biofilm development and virulence in bacteria. In this work, we showed that Ap4A is upregulated upon deletion of yqeK encoding the Ap4A hydrolase in S. aureus COL. Moreover, the yqeK mutant was impaired in growth and survival during oxidative stress and after infection of murine macrophages, indicating that Ap4A contributes to the host-pathogen interactions in S. aureus. Transcriptome analyses revealed alterations of the nucleotide, amino acid and iron metabolism as well as the downregulation of Agr-controlled cytotoxins, contributing to the lower virulence of the yqeK mutant. Altogether, our results provide leads for the design of inhibitors against YqeK to combat S. aureus infections.

microbiology↗

bioRxiv2026

A compact Druantia defense clears phage infections via single-stranded DNA recognition and directional duplex unwinding

Bacteria encode diverse anti-phage defense systems triggered by invader-specific molecular cues. Here, we report that the compact type III-A Druantia system recognizes exposed single-stranded DNA to drive phage clearance. Using representative systems from Escherichia coli, we show that the two encoded proteins, DruE and DruH, together clear restriction-sensitive or recombination-prone phages without affecting cell growth or viability. DruE dimerizes and engages DNA at exposed single-stranded regions to unwind DNA with 3'-to-5' directionality, resorting to unique molecular lock, wedge, and clamp elements that aid strand separation and processive translocation. DruH is a monomer in isolation and indirectly interacts with DruE and other host proteins under uninfected conditions, with an infection resulting in the dissociation of the complex. Taken together, our results reveal that exposed single-stranded DNA can trigger bacterial immunity through the directional helicase activity of type III-A Druantia. HighlightsO_LIThe E. coli Druantia III-A defense, comprising DruE and DruH, clears infecting phages C_LIO_LIDruE dimers bind exposed single-stranded DNA and unwind the upstream DNA duplex C_LIO_LI3'-to-5' DNA unwinding is aided by molecular lock, wedge, and clamp elements C_LIO_LIDruE interacts with DruH and host proteins, which are displaced upon infection C_LI

microbiology↗

bioRxiv2025

Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modelling was used to predict the structure of the SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric 3-4 loop adapted a helically wound conformation to elongate helix 4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix 4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the {beta}1/{beta}2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the {beta}1/{beta}2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved SaMhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus. IMPORTANCES. aureus is a major human pathogen, which can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance towards quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for regulation of uric acid resistance by UrtR family repressors (1). Our results contribute to a better understanding of antimicrobial resistance regulation, which can be exploited for future drug-design to eradicate multidrug-resistant S. aureus.

microbiology↗

bioRxiv2025

The co-localizing Zorya II, Druantia III, and ARMADA II defense systems on O-island 172 confer synergistic anti-phage defense in enterohemorrhagic Escherichia coli

In this study, we show that the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 {Delta}stx1/2 is highly resistant to phages. The genes encoding the three phage defense systems Zorya II, Druantia III, and ARMADA II were found to co-localize on O-island 172 (OI-172) in EDL933. The three phage defense systems co-occur in 426 E. coli strains comprising 66 distinct serotypes, including 277 O157:H7 isolates and 117 diverse non-pathogenic strains. Efficiency of plating (EOP) assays were used to explore the phage spectrum and synergy of the three defense systems and their contribution to the phage resistance of strain EDL933 {Delta}stx1/2. Using {Delta}zorABE, {Delta}druHE, and {Delta}armABCD single system deletion mutants in EDL933 {Delta}stx1/2, we showed that Druantia III and ARMADA II protect to different extents against a broad spectrum of phages, including the Drexlerviridae, Siphoviridae, Demerecviridae, Vequintavirinae and Autographiviridae, but not against hypermodified Tevenvirinae. Additionally, Zorya II caused strong protection only against Autographiviridae. Furthermore, EOP assays of combined system mutants revealed strong synergistic interactions of Druantia III and ARMADA II to provide more robust immunity against a similar phage spectrum than the additive protection of the individual systems. In contrast, Druantia III and Zorya II act only weakly synergistically against a few phages. Altogether, our results revealed that Druantia III and ARMADA II are responsible for most of the phage resistance of EDL933 {Delta}stx1/2, whereas Zorya II provides additional immunity against podoviruses. Future studies are underway to elucidate the molecular basis of the synergistic interactions between the Druantia III and ARMADA II defense systems. IMPORTANCEShiga toxin-producing E. coli (STEC) O157:H7 strains cause life-threatening diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. Currently, EHEC infections can be only treated symptomatically, since antibiotics are not recommended due to induction of the Shiga toxin. While phage therapy could offer a treatment option, we show that the EHEC strain EDL933 {Delta}stx1/2 is highly resistant to many phages. EOP assays of defense mutants in the host strain revealed that the co-occurring defense systems Zorya II, Druantia III, and ARMADA II on OI-172 confer most of the phage resistance in EHEC. Moreover, our data uncovered that Druantia III and ARMADA II act synergistically in the anti-phage defense in EDL933 {Delta}stx1/2, explaining the robust immunity against a broad spectrum of phages. These results support the idea that the design of novel inhibitors against the Druantia III and ARMADA II systems could be combined with phage therapies to efficiently eradicate EHEC.

microbiology↗

bioRxiv2022

The catalase contributes to microaerophilic H2O2 priming and the peroxiredoxins AhpC, Tpx and Bcp confer resistance to organic hydroperoxides in Staphylococcus aureus

Staphylococcus aureus is a major human pathogen, which has to cope with oxidative stress as part of the host innate immune defense under macrophage and neutrophil infections. In this study, we have investigated the role of the catalase KatA and the peroxiredoxins AhpC, Tpx, and Bcp for priming and resistance under oxidative stress in S. aureus during aerobic and microaerophilic growth. The results revealed that S. aureus is resistant to high doses of up-to 100 mM H2O2 during the aerobic growth. While KatA is essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributes to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, AhpC, Tpx and Bcp were shown to be required for detoxification of cumene hydroperoxide (CHP) and regeneration of the reduced state of the bacillithiol (BSH) redox potential during recovery from CHP stress in S. aureus. The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the basal level of EBSH (-291 mV) compared to that in microaerophilic cells (-310 mV). In contrast, S. aureus can be primed by sub-lethal doses of 100 {micro}M H2O2 during the microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased katA transcription and elevated KatA activity, whereas aerobic control cells showed already constitutive high KatA activity. Thus, KatA is the major player contributing to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus to oxidative stress under infections in different oxygen environments.

microbiology↗

bioRxiv2021

Allicin inhibits SARS-CoV-2 replication and abrogates the antiviral host response in the Calu-3 proteome

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 25OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.

microbiology↗

bioRxiv2019

Staphylococcus aureus uses the bacilliredoxin (BrxAB)/bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was proposed to function as BSSB reductase. Here, we investigated the role of the bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ypdA deletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (EBSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced EBSH upon recovery from oxidative stress. In addition, the ypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S-bacilithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.

microbiology↗