Biology subjects

Pethe, K.

Publications and source records attributed to Pethe, K..

10 recordsLinked to original sources

bioRxiv2026

Growth-dependent tRNA Reprogramming and Codon Bias Link Translation to Metabolic State in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive commensal bacterium of the human gut microbiome and an opportunistic pathogen responsible for many hospital-acquired infections. Despite the clinical importance of E. faecalis, how gene and protein expression are coordinated with growth remains poorly defined. Here, we profiled transcript, protein, and tRNA pool dynamics across distinct phases of E. faecalis growth. Differences in protein abundance and corresponding mRNA levels suggested growth phase-dependent posttranscriptional regulation. Growth-associated genes exhibited biased synonymous codon usage, with ribosomal and glycolytic proteins enriched in low-abundance codons read by queuosine-modifiable tRNAs. Analysis of tRNA modification and tRNA isoacceptor abundance revealed growth phase-dependent changes, particularly in anticodon stem loop modifications that influence synonymous codon translation. Changes in queuosine levels preceded shifts in ribosomal proteins, suggesting a contribution to codon-biased translation. Collectively, these findings reveal growth phase-associated remodeling of the E. faecalis tRNA pool and support a model in which queuosine-dependent translational reprogramming shapes protein expression during bacterial growth. IMPORTANCEEnterococcus faecalis is a common cause of hospital-acquired infections. Despite its clinical importance, a comprehensive understanding of the organisms physiology and adaptation to environmental changes remains incomplete. Here, we characterized protein, transcript, and tRNA dynamics across bacterial growth phases, uncovering a role for post-transcriptional regulation marked by tRNA reprogramming and biased synonymous codon usage. These findings enhance our understanding of E. faecalis growth and support a model of translational reprogramming therein.

microbiology↗

bioRxiv2025

Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

Enterococcus faecalis is a common opportunistic pathogen, frequently isolated from chronic wounds, yet the mechanisms underlying its virulence and persistence in this niche remain incompletely understood. We previously showed that a subpopulation of E. faecalis can survive intracellularly for several days during murine wound infection and can replicate within macrophages, revealing an unexpected intracellular phase for this traditionally extracellular bacterium. Here, we identify the secreted metalloprotease gelatinase (GelE) and its regulator, the Fsr quorum sensing system, as key modulators of E. faecalis intracellular survival and replication. Mechanistically, Fsr quorum sensing is induced during intracellular replication, promoting GelE-dependent host cell lysis and bacterial egress. In the absence of active GelE, E. faecalis accumulates as large intracellular clusters, a phenotype observed consistently across GelE deficient wound isolates. In a mouse wound model, GelE deficient E. faecalis similarly exhibited higher intracellular numbers within wound infection-associated host cells. Together, our study uncovers GelE as a central effector that orchestrates the transition between intracellular and extracellular lifestyles of E. faecalis, providing a possible explanation for its persistence in chronic wound infection. Author summaryPathogenic bacteria are traditionally classified as either "intracellular" or "extracellular", but growing evidence suggests that many extracellular bacteria also adopt transient intracellular lifestyles that promote persistent and recurrent infection. Enterococcus faecalis, a leading cause of chronic wound infection, exemplifies this duality. We discovered that E. faecalis strains lacking the secreted protease gelatinase accumulate to high numbers inside host cells such as macrophages. Our data indicate that gelatinase facilitates bacterial escape following intracellular replication, regulating the transition between an intracellular and extracellular lifestyle. During infection, gelatinase-deficient bacteria remain hidden within various wound cell types, potentially evading immune clearance and antibiotic treatment. This work reveals a previously unrecognized role for gelatinase in controlling E. faecalis intracellular dynamics, highlighting a mechanism that may underline chronic and persistent infection.

microbiology↗

bioRxiv2025

Enterococcus faecium colonization and persistence in a model of diabetic wound infection

Chronic wound infections are a common comorbidity of diabetes mellitus and can progress to amputation if untreated, yet effective strategies to manage these infections are limited. Commensals such as Enterococcus faecium and Staphylococcus epidermidis can transition into opportunistic pathogens when host defenses are compromised, underscoring the complexity of chronic wound microbiology. E. faecium, particularly vancomycin-resistant strains, are an understudied, clinically important cause of chronic diabetic wound infections. Using a low-dose streptozocin-induced diabetic mouse model, we characterized E. faecium wound infection dynamics and identified differences in colonization and clearance compared to non-diabetic animals. At eight hours post infection (hpi), control mice exhibited higher E. faecium wound colony forming units (CFU) than diabetic mice, but cleared the infection more efficiently, resulting in similar CFU by 24 hpi. By contrast, diabetic mice showed impaired clearance, with elevated CFU persisting through 72 hpi. In mixed species infection with S. epidermidis, S. epidermidis CFU increased at 72 hpi while E. faecium CFU remained comparable to single species infection. Despite strong initial cytokine and neutrophil responses, E. faecium persisted in all wounds. Sustained neutrophil recruitment at 72 hpi occurred only in diabetic mice, whereas macrophage accumulation increased from 24 to 72 hpi in all wounds, including sterile controls. Histological analysis showed epithelial hyper-thickening in both groups, indicating that diabetes and E. faecium each contribute to impaired wound healing. This study establishes a diabetic mouse model of E. faecium wound infection and suggests that E. faecium modulates innate immune responses to persist in the wound bed.

microbiology↗

bioRxiv2025

A bioluminescence-based chemical screen identifies a bactericidal naphthalene scaffold targeting MmpL3 in Mycobacterium abscessus

Mycobacterium abscessus pulmonary disease (Mabs-PD) presents a significant and growing global health threat, particularly in individuals with underlying lung conditions like cystic fibrosis and chronic obstructive pulmonary disease. A key challenge in treating Mabs-PD is the lack of bactericidal antibiotics effective at therapeutically relevant concentrations, underscoring an urgent need for drug discovery. Targeting cell-wall synthesis is a promising approach, as evidenced by the success of broad-spectrum {beta}-lactam antibiotics and the frontline antituberculosis drug isoniazid. However, these agents exhibit limited efficacy against Mabs, often requiring concentrations unachievable in lung tissues. Here, we used a bioluminescence-based whole-cell assay optimized to identify drugs targeting both cell-wall synthesis and the oxidative phosphorylation pathway. Screening a small drug library against Mabs revealed multiple hits, including {beta}-lactam antibiotics, validating the effectiveness of this approach to identify cell wall-targeting agents. Among these, we identified a chemically tractable naphthalene scaffold with potent bactericidal activity. The optimized derivative GM47-1 targets MmpL3, disrupting cell wall integrity, inducing ATP leakage into the extracellular milieu, and uncoupling respiration, predominantly through the cytochrome bcc:aa3 branch. Further chemical optimization resulted in a new derivative exhibiting a nanomolar minimum inhibitory concentration, with potent activity against intracellular Mabs and in a zebrafish model of infection. This study offers a promising scaffold for future therapeutic development and highlights the utility of this approach as a rapid assay platform for identifying bactericidal compounds against Mabs.

microbiology↗

bioRxiv2025

The mycobacterial ATP burst is a lysis artifact and serves as an assay for drug-induced cell wall damage

Antibiotics targeting the mycobacterial cell wall are a cornerstone of tuberculosis treatment. Understanding how these drugs facilitate bacterial killing has important implications for identifying novel therapeutic targets and optimizing drug combinations. Previous studies on mycobacteria using the BacTiter-Glo luminescence assay have reported increased ATP levels after treatment with cell wall inhibitors such as isoniazid, a phenomenon referred to as an ATP burst. This phenomenon has been proposed to contribute to drug-induced killing. In this study, we demonstrate that the ATP burst is not a biological response but rather an experimental artifact resulting from enhanced cell lysis induced by cell wall-targeting drugs. The addition of a bead-beating lysis step prior to luminescence measurement abolishes the ATP burst, enabling a more reliable assessment of intracellular ATP levels with this assay. Our findings thus establish a direct link between the ATP burst and the canonical mechanism of action of cell wall inhibitors. We further demonstrate the utility of this approach as a functional readout for identifying compounds that disrupt the mycobacterial cell wall and for screening synergistic or antagonistic interactions with known cell wall-targeting agents. Together, our results elucidate the mechanistic basis of the ATP burst and provide a valuable tool for antimycobacterial drug discovery and development.

microbiology↗

bioRxiv2025

Fsr quorum sensing system restricts biofilm growth and activates inflammation in enterococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening biofilm-associated infection, yet the factors driving biofilm formation remain poorly understood. Here, we identified the Fsr quorum sensing (QS) system of Enterococcus faecalis as a potent negative regulator of IE pathogenesis. Using microfluidic and in vivo models, we show that Fsr is induced in late IE when bacteria become shielded from blood flow. Deleting Fsr altered biofilm metabolism and promoted robust biofilm growth and gentamicin tolerance in vivo. Furthermore, Fsr inactivation attenuated inflammation by disrupting IL-1{beta} cleavage and activation via the Fsr-regulated gelatinase (gelE), allowing biofilm to grow unchecked by the immune system. Consistent with our pre-clinical findings, analysis of two IE patient cohorts linked naturally occurring Fsr-deficient E. faecalis to prolonged bacteremia. Overall, our findings provide insights into the role of QS in biofilm growth, persistence, and immune evasion in enterococcal IE.

microbiology↗

bioRxiv2024

Dietary iron deficiency impairs effector function of memory T cells following influenza infection

The establishment of memory T cell responses is critical to protection against pathogens and is influenced by the conditions under which memory formation occurs. Iron is an essential micronutrient for multiple immunologic processes and nutritional deficiency is a common problem worldwide. Despite its prevalence, the impact of nutritional iron deficiency on the establishment of memory T cell responses is not fully understood. In this study we investigate the impact of nutritional iron deficiency on the generation, phenotype, and function of memory T cell responses using a murine model of dietary iron modulation in the context of influenza infection. Iron deficient mice have decreased systemic iron levels and develop significant anemia. Increased T cell expression of the transferrin receptor (CD71) is seen in iron deficient mice at baseline. During primary influenza infection, iron deficient mice experience increased weight loss and phenotypic evidence of impairments in T cell activation. Following recovery from infection, iron deficient mice generate increased influenza specific memory T cells which exhibit impaired ability to produce IFN{gamma}, most notably within the lung. Importantly, the ability to produce IFN{gamma} and TNF is not recovered by co-culture with iron replete dendritic cells, suggesting a T cell intrinsic alteration in functional memory formation. Altogether, these results isolate a critical effect of nutritional iron deficiency on T cell memory development and function.

immunology↗

bioRxiv2023

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis

Daptomycin is a last-line antibiotic commonly used to treat vancomycin resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the {Delta}mprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced glycolysis and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/551240v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12ff3adorg.highwire.dtl.DTLVardef@127044aorg.highwire.dtl.DTLVardef@8ab082org.highwire.dtl.DTLVardef@14a4afc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Toward a single dose cure for Buruli ulcer

A single dose of TELACEBEC (Q203), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks post treatment. Clinical use of Q203 could dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

microbiology↗

bioRxiv2019

Q203 containing fully intermittent oral regimens exhibited high sterilizing activity against Mycobacterium ulcerans in mice

Buruli ulcer (BU), caused by Mycobacterium ulcerans is currently treated by a daily combination of rifampin and either injectable streptomycin or oral clarithromycin. An intermittent oral regimen would facilitate the treatment supervision. We first evaluated the bactericidal activity of newer antimicrobials against M. ulcerans using a BU animal model. The imidazopyridine amine Q203 exhibited high bactericidal activity whereas tedizolid (oxazolidinone close to linezolid), selamectine and ivermectine (avermectine compound) and the benzothiazinone PBTZ169 were not active. Consequently, Q203 was evaluated for its bactericidal and sterilizing activities in combined intermittent regimens. Q203 given twice a week in combination with one of the other long half-life compounds, rifapentine or bedaquiline, sterilized the mice footpads in 8 weeks, i.e. after a total of only 16 doses, and prevented relapse during a period of 20 weeks after stopping the treatment. These results are very promising for future intermittent oral regimens which would greatly simplify BU treatments in the field.\n\nAuthor summaryThe current treatment of Buruli ulcer (BU), infection caused by Mycobacterium ulcerans is based on a daily antibiotic combination of rifampin associated with streptomycin or clarithromycin. A shorter or intermittent treatment without an injectable drug would clearly simplify the management on the field. We evaluated the bactericidal activity of several new antimicrobials drugs in a mice model of BU and found that the Q203 exhibited the highest bactericidal effect. We subsequently identified new antibiotic combinations containing Q203 with high sterilizing activity when administrated twice a week for 8 weeks.

microbiology↗