Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in a recently discovered cytosine demethylation pathway. We present here the crystal structures of hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine (5mC) demethylation.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen that infects patients with cystic fibrosis, burn wounds, immunodeficiency, chronic obstructive pulmonary disorder (COPD), cancer, and severe infection requiring ventilation, such as COVID-19. P. aeruginosa is also a widely-used model bacterium for all biological areas. In addition to continued, intense efforts in understanding bacterial pathogenesis of P. aeruginosa including virulence factors (LPS, quorum sensing, two-component systems, 6 type secretion systems, outer membrane vesicles (OMVs), CRISPR-Cas and their regulation), rapid progress has been made in further studying host-pathogen interaction, particularly host immune networks involving autophagy, inflammasome, non-coding RNAs, cGAS, etc. Furthermore, numerous technologic advances, such as bioinformatics, metabolomics, scRNA-seq, nanoparticles, drug screening, and phage therapy, have been used to improve our understanding of P. aeruginosa pathogenesis and host defense. Nevertheless, much remains to be uncovered about interactions between P. aeruginosa and host immune responses, including mechanisms of drug resistance by known or unannotated bacterial virulence factors as well as mammalian cell signaling pathways. The widespread use of antibiotics and the slow development of effective antimicrobials present daunting challenges and necessitate new theoretical and practical platforms to screen and develop mechanism-tested novel drugs to treat intractable infections, especially those caused by multi-drug resistance strains. Benefited from has advancing in research tools and technology, dissecting this pathogen’s feature has entered into molecular and mechanistic details as well as dynamic and holistic views. Herein, we comprehensively review the progress and discuss the current status of P. aeruginosa biophysical traits, behaviors, virulence factors, invasive regulators, and host defense patterns against its infection, which point out new directions for future investigation and add to the design of novel and/or alternative therapeutics to combat this clinically significant pathogen.

Summary Thiol group oxidation of active and allosteric cysteines is a widespread regulatory post-translational protein modification. Pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus, use regulatory cysteine oxidation to respond to and overcome reactive oxygen species (ROS) encountered in the host environment. To obtain a proteome-wide view of oxidation-sensitive cysteines in these two pathogens, we employed a competitive activity-based protein profiling approach to globally quantify hydrogen peroxide (H2O2) reactivity with cysteines across bacterial proteomes. We identified ~200 proteins containing H2O2-sensitive cysteines, including metabolic enzymes, transcription factors, and uncharacterized proteins. Further biochemical and genetic studies identified an oxidation-responsive cysteine in the master quorum sensing regulator LasR, and redox-regulated activities for acetaldehyde dehydrogenase ExaC, arginine deiminase ArcA, and glyceraldehyde 3-phosphate dehydrogenase GAPDH. Taken together, our data indicate that pathogenic bacteria exhibit a complex, multi-layered response to ROS that includes the rapid adaption of metabolic pathways to oxidative stress challenge.

Sepsis is a severe and complicated syndrome symbolized by dysregulation of host inflammatory responses and organ failure with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. Here, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in Pseudomonas aeruginosa abdominal infection. Following intraperitoneal challenge with P. aeruginosa, atg7fl/fl mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared to wild type mice. The septic atg7fl/fl mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1β and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that P. aeruginosa flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis.