Biology subjects

Leichert, L. I.

Publications and source records attributed to Leichert, L. I..

9 recordsLinked to original sources

bioRxiv2026

A newly identified detoxification system protects uropathogenic Escherichia coli from reactive chlorine species

Neutrophils eliminate invading pathogens through the production of reactive oxygen and chlorine species (ROS/RCS), with hypochlorous acid (HOCl) representing the most abundant and bactericidal oxidant produced in this process. Compared to bacterial defenses against ROS, which are well studied, little is known about how pathogens respond to and counter RCS, including HOCl. Here, we identify and mechanistically characterize RcrB, a protein of the uncharacterized DUF417 protein family, for which no role in oxidative stress defense has been described yet. We report a previously unrecognized role as an RCS detoxification system that confers high-level resistance to uropathogenic Escherichia coli (UPEC). We show that RcrB is an inner membrane protein and strongly induced during RCS exposure and phagocytosis. Loss of RcrB results in profound HOCl hypersensitivity, accompanied by elevated macromolecular damage and severe metabolic perturbations, establishing RcrB as a central determinant of UPECs RCS stress resistance. Heterologous expression of RcrB in HOCl-sensitive intestinal E. coli strains is sufficient to restore resistance but requires functional glutathione biosynthesis. Quantitative HOCl trapping assays demonstrate that RcrB expression protects the bacterial population by significantly reducing extracellular HOCl, indicating active chemical quenching rather than passive membrane protection. Structure-function analysis of RcrB confirms this conclusion and demonstrate that conserved, redox-active amino acids facing the periplasm are essential for its detoxification activity. In summary, our study reveals a hitherto unknown bacterial strategy for mitigating RCS and reveal a distinct mechanism by which UPEC may survive the mammalian host defense. Significance StatementDuring infection, human immune cells such as neutrophils kill bacteria by releasing powerful oxidants, including hypochlorous acid (HOCl), the active ingredient of household bleach. How pathogenic bacteria survive this chemical attack is still poorly understood. This study identifies a membrane protein in uropathogenic Escherichia coli as a key defense factor that neutralizes HOCl before it can damage the cell. RcrB acts at the bacterial cell envelope, where it detoxifies HOCl in a glutathione-dependent manner to maintain a balanced redox homeostasis and cellular integrity, thereby even protect neighboring bacteria. These findings reveal a previously unrecognized frontline defense strategy that helps pathogens survive immune attack and may represent a new target for antimicrobial therapies.

microbiology↗

bioRxiv2024

Comprehensive elucidation of glutathione import in Escherichia coli

Glutathione is the major thiol-based antioxidant in a wide variety of biological systems, ranging from bacteria to eukaryotes. As a redox couple, consisting of reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), it is crucial for the maintenance of the cellular redox balance. Glutathione transport out of and into cellular compartments and the extracellular space is a determinant of the thiol-disulfide redox state of the organelles and bodily fluids in question, but is currently not well understood. Here we use the genetically-encoded, glutathione-measuring redox probe Grx1-roGFP2 to comprehensively elucidate the import of extracellular glutathione into the cytoplasm of the model organism Escherichia coli. The elimination of only two ATP-Binding Cassette (ABC) transporter systems, Gsi and Opp, completely abrogates glutathione import into E. colis cytoplasm, both in its reduced and oxidized form. The lack of only one of them, Gsi, completely prevents import of oxidized glutathione (GSSG), while the lack of the other, Opp, substantially retards the uptake of reduced glutathione (GSH).

biochemistry↗

bioRxiv2024

Auranofin induces disulfide bond-mimicking S-Au-S bonds in protein thiol pairs

Auranofin is an inhibitor of human thioredoxin reductase, clinically used in the treatment of rheumatoid arthritis. More recently, it has been shown to possess strong antibacterial activity. Despite the structural dissimilarity and the independent evolutionary origins of human thioredoxin reductase and its bacterial counterpart (TrxB), inhibition of bacterial thioredoxin reductase is often suggested to be a major factor in auranofins antibacterial mode of action. To test this hypothesis, we attempted to determine the mechanism of inhibition of auranofin for bacterial TrxB in the presence of thioredoxin, TrxBs natural substrate. However, the data obtained in these experiments was not consistent with a specific and exclusive interaction between TrxB and auranofin. Instead, it suggested that auranofin directly interacts with the cysteine thiols in thioredoxin, TrxBs substrate. Using the fluorescent redox protein roGFP2, we showed that auranofin does indeed directly interact with cysteine pairs in proteins, forming a thiol modification that is similar to, but clearly distinct from a disulfide bond. The Au:S stoichiometries of auranofin-treated roGFP2 and thioredoxin strongly suggest the presence of an S-Au-S bridge between two cysteines in those proteins. These S-Au-S bonds form independent of thioredoxin reductase at a rate that indicates their pertinence in auranofins antibacterial mode of action.

biochemistry↗

bioRxiv2023

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

The antirheumatic gold(I) compound auranofin has been suggested to exhibit bactericidal activity by disrupting the thiol homeostasis through direct inhibition of bacterial thioredoxin reductase (TrxB). While highly effective at killing Gram-positive bacteria, it lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. A change in the proteome of E. coli exposed to auranofin suggested that the effect of this gold compound is a combination of inactivation of thiol-containing enzymes, upregulation of proteins involved in basal metabolism, and the consequent induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking the proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. colis high tolerance to auranofin. To elucidate factors that could make Gram-negative bacteria less susceptible to auranofin, we tested E. coli cells lacking the efflux pump component TolC. These cells were more sensitive to auranofin treatment than the wild type, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested if E. colis thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. E. coli cells lacking components of the thioredoxin-system were also only marginally more susceptible to auranofin. However, E. coli strains lacking the low molecular weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. In line with their susceptibility, the kinetics of probe oxidation and the degree of oxidation promoted by auranofin is significantly higher in Gram-positive bacteria when compared to Gram-negative bacteria. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.

biochemistry↗

bioRxiv2023

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding in Escherichia coli

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues, that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a {Delta}dsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

biochemistry↗

bioRxiv2023

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5-untranslated region (5-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37{degrees}C. The trxA gene is transcribed into two mRNA isoforms, of which the short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25{degrees}C suggesting partially open structures available to the ribosome in the living cell. Upstream of katY we uncovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37{degrees}C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with temperature regulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37{degrees}C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature. Author summaryThe external conditions dramatically change when a bacterial pathogen enters a mammalian host. Sensing the new situation and rapidly responding to it is of critical importance for pathogens, like Yersinia pseudotuberculosis, since they often circulate between their environmental reservoirs and a warm-blooded host. Many virulence-related genes encode a temperature-sensitive mRNA element, a so-called RNA thermometer (RNAT), in the 5-end of their transcript. Melting of this structure at 37{degrees}C allows ribosome binding and translation initiation. The host immune system typically fights microbial pathogens by the production of reactive oxygen species (ROS). Here, we find that several ROS defense genes in Yersinia are upregulated at host body temperature to counteract the ROS attack. In particular, the massive RNAT-mediated upregulation of the catalase KatY confers protection against H2O2 at 37{degrees}C. Our study reveals a close regulatory link between temperature sensing and the oxidative stress response in a notorious food borne pathogen.

microbiology↗

bioRxiv2021

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidAs chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidAs chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N- chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCls chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

biochemistry↗

bioRxiv2020

Identification of mtROS-sensitive processes in activated CD4+ T cells

T lymphocytes are key components in adaptive immunity and their activation naturally involves mitochondrial-derived oxygen species (mtROS). In particular, H2O2 has been implicated as an important signaling molecule regulating major T cell functions. H2O2 targets the oxidation status of functional cysteine residues but knowledge if and where this happens in T cell signaling networks is widely missing. This study aimed to identify mtROS-sensitive processes in activated primary human CD4+ T cells. By using a thiol-specific redox proteomic approach we examined the oxidation state of 4784 cysteine-containing peptides of ex vivo stimulated T cells from healthy individuals. Upon activation, a shift in oxidation was observed at catalytic cysteine residues of peroxiredoxins (PRDX5 & PRDX6), and T cells were found to maintain their global thiol-redox homeostasis. In parallel, a distinct set of 88 cysteine residues were found to be differentially oxidized upon T cell activation suggesting novel functional thiol switches. In mitochondria, cysteine oxidations selectively modified regulators of respiration (NDUFA2, NDUFA8, and UQCRH) confirming electron leakage from electron transport complexes I and III. The majority of oxidations occurred outside mitochondria and enriched sensitive thiols at regulators of cytoskeleton dynamics (e.g. CYFIP2 and ARPC1B) and known immune functions including the non-receptor tyrosine phosphatase PTPN7. Conversely, cysteine reduction occurred predominantly at transcriptional regulators and sites that coordinate zinc-binding in zinc-finger motifs. Indeed, fluorescence microscopy revealed a colocalization of zinc-rich microenvironments and mitochondria in T cells suggesting mtROS-dependent zinc-release of identified transcriptional regulators including ZFP36, RPL37A and CRIP2. In conclusion, this study complements knowledge on the mtROS signaling network and suggests zinc-dependent thiol switches as a mechanism of how mtROS affects transcription and translation in T cells.

immunology↗

bioRxiv2019

N-chlorination mediates protective and immunomodulatory effects of oxidized human plasma proteins

Hypochlorous acid (HOCl), a powerful antimicrobial oxidant, is produced by neutrophils to fight infections. Here we show that N-chlorination, induced by HOCl concentrations encountered at sites of inflammation, converts blood plasma proteins into chaperone-like holdases that protect other proteins from aggregation. This chaperone-like conversion was reversible by antioxidants and was abrogated by prior methylation of basic amino acids. Furthermore, reversible N-chlorination of basic amino acid side chains is the major factor that converts plasma proteins into efficient activators of immune cells. Finally, HOCl-modified serum albumin was found to act as a pro-survival molecule that protects neutrophils from cell death induced by highly immunogenic foreign antigens. We propose that activation and enhanced persistence of neutrophils mediated by HOCl-modified plasma proteins, resulting in the increased and prolonged generation of ROS, including HOCl, constitutes a potentially detrimental positive feedback loop that can only be attenuated through the reversible nature of the modification involved.

biochemistry↗