Biology subjects

Garsin, D. A.

Publications and source records attributed to Garsin, D. A..

5 recordsLinked to original sources

bioRxiv2026

Characterization of tRNA ligase function in pathogenic fungi and trypanosomes reveals the ligase domain as a promising drug target

The majority of eukaryotes encode some intron-containing pre-tRNAs. Splicing of these pre-tRNAs requires a dedicated tRNA splicing machinery. The fungal and trypanosome tRNA ligase, Trl1, and the human RNA ligase, RTCB, catalyze an essential step in tRNA splicing. However, Trl1 and RTCB are nonhomologous and biochemically and structurally distinct from each other. Therefore, Trl1 could serve as a broad-spectrum antifungal and anti-trypanosomal target. While the functions and requirements of the three catalytic Trl1 domains have been extensively characterized in the model yeast Saccharomyces cerevisiae, the roles of Trl1 orthologs in pathogenic fungi remain unexplored. Here, we validate Trl1 as one of the few promising novel drug targets for the development of antifungal therapeutics. Functional analyses of the three Trl1 domains show that only the "sealing" domain is essential for growth and viability in Candida albicans and Aspergillus fumigatus. In contrast, the two "healing" domains are dispensable in these pathogenic fungi, suggesting the presence of redundant healing enzymes, unlike in S. cerevisiae. These findings indicate that only the sealing domain is a good drug target. Our analysis also shows that the Mucor enzyme, which only contains the sealing domain, is essential. Using a Caenorhabditis elegans infection model of C. albicans, we further demonstrated that inhibiting Trl1 expression protects worms during an established infection. In contrast to these fungal pathogens, we show that all three domains of Trl1 are essential in Trypanosoma brucei. Our findings show that the essentiality of the Trl1 sealing is conserved in important human pathogens and provides an impetus for future drug development. SIGNIFICANCEFungal infections are an important cause of human disease and death and difficult to treat and there is an urgent need to develop additional drugs. Based on studies in yeast, one promising target for antifungal drug development is the tRNA splicing pathway. Human tRNA ligase is fundamentally distinct from the fungal one. To investigate the possibility of developing tRNA ligase-targeting drugs, we investigated the function of the catalytic domains of fungal tRNA ligase in different fungal pathogens. Surprisingly, only the first domain is essential in these pathogens and yeast is not a good model fungus. In contrast, all three domains of Trypanosome tRNA ligase are essential. These findings provide an impetus for future drug development.

molecular biology↗

bioRxiv2026

LIN-23 Affects C. elegans Pathogen and Stress Resistance by Modulating SKN-1 Activity

During pathogen infection, the C. elegans transcription factor SKN-1 is activated through the p38 MAPK cascade to protect against oxidative damage and promote host survival. SKN-1, the functional ortholog of the mammalian Nrf family of transcription factors, participates in various biological processes and is subject to complex regulation. In this study, we identify a previously unrecognized role for LIN-23 in regulating SKN-1 during adult stress conditions. LIN-23 is an F-box protein that functions as the substrate recognition component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex and has been implicated in diverse cellular processes, including cell cycle regulation, neurite outgrowth, and centrosome duplication. Although LIN-23 has previously been reported to negatively regulate SKN-1 in other contexts, our findings demonstrate that during pathogen exposure, LIN-23 acts as a positive regulator of SKN-1 activity. Specifically, loss of LIN-23 reduced SKN-1 activity and decreased survival of infected adult animals. Having established this novel relationship between LIN-23 and SKN-1, we investigated the mechanism by which LIN-23 regulates SKN-1 activity. Because SKN-1 activation occurs via the p38 MAPK signaling pathway, followed by nuclear localization, we examined whether LIN-23 influences these events, but observed no decrease in p38 MAPK phosphorylation or SKN-1 nuclear localization. Instead, we provide evidence that LIN-23 function is dependent on WDR-23, a well-established negative regulator of SKN-1. A model is proposed in which LIN-23 promotes SKN-1 activity by targeting nuclear WDR-23 for degradation. SUMMARYSKN-1 is a C. elegans transcription factor and the ortholog of mammalian Nrf proteins. Under oxidative stress conditions, including those induced by infection, SKN-1 plays a protective role. Since SKN-1 regulation is complex, understanding the mechanisms that modulate its activity is important for defining stress response pathways. The authors demonstrate that the F-box protein LIN-23 functions as a positive regulator of SKN-1. Their genetic analyses indicate that LIN-23 does not influence the canonical SKN-1 activation cascade. Instead, LIN-23 appears to regulate SKN-1 activity by modulating a negative regulator, WDR-23.

genetics↗

bioRxiv2023

Role of Ethanolamine Utilization and Bacterial Microcompartment Formation in Listeria monocytogenes Intracellular Infection

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host-cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium (S. Typhimurium). The EA utilization (eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant ({Delta}eutV) was equally deficient in intracellular replication compared to an EA metabolism mutant ({Delta}eutB), and this was consistent across Caco-2, RAW 264.7 and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro, and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that{Delta} eutB and{Delta} eutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.

microbiology↗

bioRxiv2023

Membrane Lipids Augment Cell Envelope Stress Signaling and Resistance to Antibiotics and Antimicrobial Peptides in Enterococcus faecalis

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

microbiology↗

bioRxiv2020

Cardiac Microlesions Form During Severe Bacteremic Enterococcus faecalis Infection

Enterococcus faecalis is a significant cause of hospital-acquired bacteremia. Herein, the discovery is reported that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those formed by Streptococcus pneumoniae during invasive pneumococcal disease (IPD). However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation. Rather, disulfide bond forming protein DsbA was found to be required for E. faecalis virulence in a C. elegans model and was necessary for efficient cardiac microlesion formation. Furthermore, E. faecalis promoted cardiomyocyte apoptotic and necroptotic cell death at sites of microlesion formation. Additionally, loss of DsbA caused an increase in pro-inflammatory cytokines unlike the wild-type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterium and the host response that are mechanistically involved. SUMMARYThis work presents the observation of cardiac microlesion formation during severe blood stream infection with Enterococcus faecalis in mice. Moreover, we identify the contribution of a novel enterococcal virulence determinant in modulating microlesion formation and the host immune response.

microbiology↗