Biology subjects

Kremer, L.

Publications and source records attributed to Kremer, L..

13 recordsLinked to original sources

bioRxiv2026

Cryo-electron microscopy structure of Jabs, a bacteriophage infecting the multidrug-resistant pathogen Mycobacterium abscessus

Exploring bacteriophage structural diversity is essential for understanding phage biology and for advancing phage-based therapies. Here, we determine the cryo-electron microscopy structure of Jabs, providing, to our knowledge, the first high-resolution view of a phage infecting the multidrug-resistant human pathogen Mycobacterium abscessus. Although Jabs displays the canonical organization of a siphophage, its virion combines several unusual architectural features. The T=9 icosahedral capsid is assembled from two distinct major capsid proteins, with one forming the hexons and the other the pentons, revealing an unprecedented capsid assembly strategy among icosahedral phages. An extensive network of ~1,700 disulfide bonds stabilize individual structural components and covalently links the capsid, connector, tail, and adhesion device into a continuous assembly. At the distal end of the tail, an elaborate and conformationally dynamic adhesion device comprises multiple candidate receptor-binding proteins organized into complex multidomain architectures, including carbohydrate-binding modules and {beta}-sandwich hetero- and homotrimers resembling the receptor-binding proteins of phages infecting lactic acid bacteria. Together, these findings expand our understanding of phage structural diversity and provide a framework for investigating phage-host interactions and guiding the engineering of therapeutic phages.

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

AngleCam V2: Predicting leaf inclination angles across taxa from daytime and nighttime photos

O_LIUnderstanding how plants capture light and maintain their energy balance is crucial for predicting how ecosystems respond to environmental changes. By monitoring leaf inclination angle distributions (LIADs), we can gain insights into plant behaviour that directly influences ecosystem functioning. LIADs affect radiative transfer processes and reflectance signals, which are essential components of satellite-based vegetation monitoring. Despite their importance, scalable methods for continuously observing these dynamics across different plant species throughout day-night cycles are limited. C_LIO_LIWe present AngleCam V2, a deep learning model that estimates LIADs from both RGB and near-infrared (NIR) night-vision imagery. We compiled a dataset of over 4,500 images across 200 globally distributed species to facilitate generalization across taxa. Moreover, we developed a method to simulate pseudo-NIR imagery from RGB imagery to enable an efficient training of a deep learning model for tracking LIADs across day and night. The model is based on a vision transformer architecture with mixed-modality training using the RGB and the synthetic NIR images. C_LIO_LIAngleCam V2 achieved substantial improvements in generalization compared to AngleCam V1 (R2 = 0.62 vs 0.12 on the same holdout dataset). Phylogenetic analysis across 100 genera revealed no systematic taxonomic bias in prediction errors. Testing against leaf angle dynamics obtained from multitemporal terrestrial laser scanning demonstrated the reliable tracking of diurnal leaf movements (R2 = 0.61-0.75) and the successful detection of water limitation-induced changes over a 14-day monitoring period. C_LIO_LIThis method enables continuous monitoring of leaf angle dynamics using conventional cameras, enabling applications in ecosystem monitoring networks, plant stress detection, interpreting satellite vegetation signals, and citizen science platforms for global-scale understanding of plant structural responses. C_LI

ecology↗

bioRxiv2025

Regulation of the expression of nucS, a key component of mismatch repair system in mycobacteria

Alterations in the expression of the mismatch repair (MMR) system can lead to transient hypermutation, accelerating the emergence of adaptive mutations under stress conditions, such as antibiotic pressure. While most bacteria and eukaryotes rely on the MutS and MutL protein families for MMR, many archaeal and actinobacterial species, including the major human pathogen Mycobacterium tuberculosis, lack these components. Instead, they utilize NucS, a structurally and evolutionarily distinct enzyme driving a non-canonical MMR pathway. Given the role of MMR in mutation control, understanding how nucS expression is regulated could be essential for uncovering the molecular basis of antibiotic resistance development in mycobacteria. In this study, we defined the promoter region and transcription start site of the nucS gene in Mycobacterium smegmatis. We found that nucS expression is growth phase-dependent in both M. smegmatis and M. tuberculosis, significantly decreasing during the stationary phase. This downregulation mirrors that of canonical MMR components, aligning with replication activity. Additionally, we present evidence that the alternative sigma factor {sigma}B may negatively regulate nucS expression during the stationary phase. We also identified candidate compounds capable of modulating nucS expression, underscoring its responsiveness to environmental cues. These findings deepen our understanding of mycobacterial stress adaptation and provide a groundwork for further investigation into the molecular mechanisms underlying antibiotic resistance. Strikingly, our work reveals a case of double convergent evolution: both canonical (MutS/MutL) and non-canonical (NucS) pathways have independently evolved not only the same DNA repair function, but also similar regulatory frameworks for genome integrity preservation under stress conditions. IMPORTANCEBacteria like Mycobacterium tuberculosis, which causes tuberculosis, develop antibiotic resistance through mutations. Most organisms use a MutS/MutL-based system called mismatch repair to correct DNA replication errors and prevent harmful mutations. However, some bacteria, including mycobacteria, rely on a unique repair protein called NucS. This study explores how the gene encoding NucS is regulated in mycobacteria. We found that less NucS is produced when bacteria stop growing, which mirrors what happens with other DNA repair systems. We also discovered that a regulatory protein called SigB may help reduce its production during this phase. In addition, we identified chemical compounds that influence how much NucS bacteria produce, showing that its levels respond to environmental conditions. Importantly, the study reveals that different DNA repair systems--despite being unrelated--have evolved similar ways to protect bacteria under stress. These findings help scientists understand how mycobacteria adapt and could aid in fighting antibiotic resistance.

microbiology↗

bioRxiv2024

The diversity of clinical Mycobacterium abscessus isolates in morphology, glycopeptidolipids and infection rates in a macrophage model

Mycobacterium abscessus (Mab) colonies adopt smooth (S) or rough (R) morphotypes, which are linked to the presence or absence of glycopeptidolipids (GPL), respectively. Though clinically relevant, the association between GPL levels, morphotype and pathogenesis are poorly understood. To investigate the degree of correlation between Mab morphology, GPL levels, and infectivity, we generated isolates from Mab-positive sputum samples from cystic fibrosis patients. Isolated strains were categorised based on their morphology, GPL profile, and replication rate in macrophages. Our findings revealed that around 50% of isolates displayed mixed morphologies and GPL analysis confirmed a consistent relationship between GPL content and morphotype was only found in smooth isolates. Across morphotype groups, no differences were observed in vitro, yet using a high-content THP-1 cell ex vivo infection model, clinical R strains were observed to replicate at higher levels. Moreover, the proportion of infected macrophages was notably higher among clinical R strains compared to their S counterparts at 72 hours post-infection. Clinical variants also infected at significantly higher rates compared to laboratory strains, highlighting the limited translatability of lab strain infection data to clinical contexts. Our study confirmed the general correlation between morphotype and GPL levels in smooth strains yet unveiled more variability within morphotype groups than previously recognised, particularly during intracellular infection. As the rough morphotype is of highest clinical concern, these findings contribute to the expanding knowledge base surrounding Mab infections, offering insights that can steer diagnostic methodologies, and treatment approaches.

microbiology↗

bioRxiv2023

Mycobacterium trehalose polyphleates are required for mycobacteriophage infection

Mycobacteriophages are good model systems for understanding their bacterial hosts and show promise as therapeutic agents for nontuberculous mycobacterium infections. However, little is known about phage recognition of Mycobacterium cell surfaces, or mechanisms of phage resistance. We show here that surface-exposed trehalose polyphleates (TPPs) are required for infection of Mycobacterium abscessus and Mycobacterium smegmatis by clinically useful phages BPs and Muddy, and that TPP loss leads to defects in adsorption, infection, and confers resistance. Transposon mutagenesis indicates that TPP loss is the primary mechanism for phage resistance. Spontaneous phage resistance occurs through TPP loss, and some M. abscessus clinical isolates are phage-insensitive due to TPP absence. Both BPs and Muddy become TPP-independent through single amino acid substitutions in their tail spike proteins, and M. abscessus mutants resistant to TPP-independent phages reveal additional resistance mechanisms. Clinical use of BPs and Muddy TPP-independent mutants should preempt phage resistance caused by TPP loss.

microbiology↗

bioRxiv2021

C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

microbiology↗

bioRxiv2021

Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

Certain aminoacyl-tRNA synthetases developed a proofreading mechanism to ensure aminoacylation of tRNAs with cognate amino acids. Epetraborole (EPT) was identified as an inhibitor of the leucyl-tRNA synthetase (LeuRS) editing site in Mycobacterium abscessus. EPT displayed enhanced activity against M. abscessus over Mycobacterium tuberculosis. Crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar Kd. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed the non-proteinogenic amino acid norvaline, leucine residues in proteins were replaced by norvaline, inducing expression of GroEL chaperonins and Clp proteases. In vitro data revealed that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. The combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection.

microbiology↗

bioRxiv2020

Birnavirus Ribonucleoprotein Assembly

The interaction of the structural VP3 polypeptide of infectious bursal disease virus (IBDV) with virus-encoded dsRNA is essential both for the assembly of ribonucleoprotein complexes responsible for genome transcription and replication and for the evasion of hosts antiviral responses. Surface plasmon resonance analysis allowed us to determine the kinetic constants of the VP3-dsRNA interaction as well as to map the VP3 dsRNA bipartite dsRNA binding domain (dsRBD), uncovering the specific role of the previously described Patch1 and Patch2 dsRB subdomains. Here we show that the Patch1 domain plays a primary binding function while Patch2 exerts a subordinate role stabilizing VP3-dsRNA complexes. The use of a set of VP3 mutant versions facilitated the identification of K99 and K106 within Patch1 as the essential residues for the formation of VP3-dsRNA complexes. Furthermore, replacement of either one of these two residues by aspartic acid completely thwarts both evasion from hosts sensors and virus replication. Data presented here allow us to propose a VP3-dsRNA interaction model that should help to further elucidate the mechanics of IBDV morphogenesis and genome packaging as well as to better understand how VP3 counteracts recognition of virus-encoded dsRNA by specialized hosts sensors.

microbiology↗

bioRxiv2020

Adaptive mitochondrial regulation of the proteasome

The proteasome is the main proteolytic system for targeted protein degradation in the cell. Its function is fine-tuned according to cellular needs. Regulation of proteasome function by mitochondrial metabolism, however, is unknown. Here, we demonstrate that mitochondrial dysfunction reduces the assembly and activity of the 26S proteasome in the absence of oxidative stress. Impaired respiratory complex I function leads to metabolic reprogramming of the Krebs cycle and deficiency in aspartate. Aspartate supplementation activates assembly and activity of 26S proteasomes via transcriptional activation of the proteasome assembly factors p28 and Rpn6. This metabolic adaptation of 26S proteasome function involves sensing of aspartate via the mTORC1 pathway. Metformin treatment of primary human cells similarly reduced assembly and activity of 26S proteasome complexes, which was fully reversible and rescued by supplementation of aspartate or pyruvate. Of note, respiratory dysfunction conferred resistance towards the proteasome inhibitor Bortezomib. Our study uncovers a fundamental novel mechanism of how mitochondrial metabolism adaptively adjusts protein degradation by the proteasome. It thus unravels unexpected consequences of defective mitochondrial metabolism in disease or drug-targeted mitochondrial reprogramming for proteasomal protein degradation in the cell. As metabolic inhibition of proteasome function can be alleviated by treatment with aspartate or pyruvate, our results also have therapeutic implications.

cell biology↗

bioRxiv2019

Chronic infection of adult zebrafish with rough or smooth variant Mycobacterium abscessus causes necrotising inflammation and differential activation of host immunity

Infections caused by Mycobacterium abscessus are increasing in prevalence within patient groups with respiratory comorbidities. Initial colonisation by the smooth colony M. abscessus (S) can be followed by an irreversible genetic switch into a highly inflammatory rough colony M. abscessus (R), often associated with a decline in pulmonary function. Our understanding of the role of adaptive immunity in M. abscessus pathogenesis is largely unknown. Here, we have used intraperitoneal infection of adult zebrafish to model M. abscessus pathogenesis in the context of fully functioning host immunity. We find infection with the R variant penetrates host organs causing an inflammatory immune response leading to necrotic granuloma formation within 2 weeks. The R bacilli are targeted by T cell-mediated immunity and burden is constrained. Strikingly, the S variant colonises host internal surfaces at high loads and is met with a robust innate immune response but little T cell-mediated immunity. Invasive granuloma formation is delayed in S variant infection compared to R variant infection upon which T cell-mediated immunity is required to control infection. In mixed infections, the S variant outcompetes the R variant. We also find the R variant activates host immunity to the detriment of S variant M. abscessus in mixed infections. These findings demonstrate the applicability of the adult zebrafish to model persistent M. abscessus infection and provide insight into the immunopathogenesis of chronic M. abscessus infection.

microbiology↗

bioRxiv2019

Glycosylation-dependent enhanced cell-binding and infectivity through DC-SIGN in the West African Ebola virus Makona

Since its discovery in Zaire in 1976, most ebolavirus outbreaks described occurred mainly in remote and poorly communicated areas of Central Africa and affected a limited number of individuals. Nonetheless, the Ebola epidemic that began in West Africa at the end of 2013 spread rapidly and reached an unprecedented scale. This epidemic was caused by the Makona variant of Zaire ebolavirus (EBOV). Monitoring of the EBOV Makona evolution throughout the epidemic identified the A82V substitution in the EBOV glycoprotein (GP) at the beginning of the epidemic, which correlated with its rapid spread. The Makona GP-V82 variant exhibits slightly higher human and primate cell infectivity. Host factors responsible for the enhanced transmission of EBOV Makona have yet to be identified. Here we show that the GP A82V substitution increases EBOV avidity for its DC-SIGN lectin receptor, which enhances cell-binding and infectivity of dendritic cells and macrophages, the primary cell subsets infected during the initial stages of the disease. Using a pseudotype lentivirus system, we identified two GP N-linked glycosylation responsible of the augmented Makona GP-V82 cell infection. Crystal structures indicated how the GP A82V substitution drives exposure of a key glycan and facilitates lectin recognition. Thus, DC-SIGN might be an important host determinant for the unique dissemination of EBOV during the 2013-2016 epidemic, which likely promoted host-to-host transmission by enhancing viral infection of dendritic cells in the skin and mucosa. This study also reveals that variations in virus envelope glycosylations can be a common pathway for virus adaptation to human transmission.\n\nSynopsisThe Zaire ebolavirus (EBOV) Makona, responsible of the largest outbreak of Ebola virus disease (EVD) in West Africa from 2013-2016, displayed a glycosylation-dependent enhancement of binding and subsequent infectivity in cells expressing the DC-SIGN surface lectin.

microbiology↗

bioRxiv2017

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking

Fluorescent protein based pH sensors are a useful tool for measuring protein trafficking through pH changes associated with endo-and exocytosis. However, commonly used pH sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering the ability to focus on specific trafficking pools of proteins. We developed a family of excitation-ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen activating protein, and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged {beta}2-adrenergic receptor (B2AR) at a single vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

cell biology↗