Biology subjects

Nielsen, K. L.

Publications and source records attributed to Nielsen, K. L..

5 recordsLinked to original sources

bioRxiv2026

Resistance to antibacterial peptide nucleic acids through altered ribosome function

When used as antibacterial agents, Peptide Nucleic Acid (PNAs) are generally designed to base-pair with complementary sequences of an essential mRNA and block translation initiation. Although bacterial susceptibility to peptide-conjugated PNAs is strongly influenced by cellular uptake, intracellular determinants of PNA activity remain poorly understood. Here, we identify a ribosome-centered mechanism of resistance to antibacterial PNAs in Escherichia coli. The rpsLI82N mutation increased the minimum inhibitory concentration (MIC) of an argininerich cell penetrating peptide-conjugated PNA targeting acpP eightfold. The effect of rpsLI82N was additive with mutations that reduce PNA entry into bacterial cells PNA resistance conferred by rpsLI82N was independent of carrier peptide and also applied when naked PNA was tested in an envelope-permeable strain. Similarly, rpsLI82N-associated resistance was independent of the targeted mRNA, as it applied to PNAs targeting either the Shine-Dalgarno or AUG region of acpP or ftsZ mRNA. Several additional substitutions within residues 74-82 of ribosomal protein uS12 conferred PNA resistance. Because resistance occurred among both error-restrictive and ribosomal-ambiguity alleles, it did not correlate with the classical decoding-fidelity phenotypes measured by stop-codon readthrough. Proteomic analysis revealed widespread changes in proteins under post-transcriptional control in rpsLI82N cells. The effect on selected sRNA-regulated genes correlated with the location of the sRNA-binding site: repression was less efficient when binding overlapped the translation-initiation region whereas it was more efficient when regulatory sites were located outside this region. In parallel, rpsLI82N increased the 30S and 50S ribosomal fractions and reduced the 70S fraction. Both loss of KsgA, which disrupts 30S maturation, and treatment with kasugamycin, which perturbs translation initiation, increased PNA resistance. We propose that rpsLI82N alters uS12-dependent 30S assembly and initiation-complex dynamics, thereby changing the effective occupancy of mRNA translation-initiation regions. This limits access of PNAs and sRNAs to overlapping sequences, resulting in PNA resistance and reduced sRNA-mediated repression. Conversely, a longer-lived 30S initiation state may facilitate sRNA binding to flanking regions and strengthen repression. These findings identify the ribosome-mRNA interface as an intracellular determinant of antibacterial PNA susceptibility.

molecular biology↗

bioRxiv2026

An activity-resistance tradeoff constrains enzyme evolution

The presence of self-resistance genes in antibiotic-producing organisms poses a paradox: how can resistance evolve before the antibiotic exists, and how can an antibiotic producer arise without first evolving resistance? Here we examine the evolutionary origins of self-resistance to mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). The MPA biosynthetic gene cluster (BGC) includes a resistant IMPDH-B. Homologs of IMPDH-B occur not only in MPA producers but also in many non-producing fungi, where remnants of the MPA BGC remain detectable. The phylogeny of IMPDH-B is incongruent with the fungal species tree, consistent with multiple horizontal gene transfer events between Aspergillus and Sordariomycetes. We characterized eleven extant IMPDH-Bs, five from MPA producers and six from nonproducers, along with seven resurrected ancestral enzymes (Anc1-Anc7). MPA resistance appeared between Anc2 and Anc3 and coincided with a loss of catalytic efficiency. Across both ancestral and extant enzymes, MPA resistance correlated strongly with reduced activity, revealing a robust activity-resistance trade-off that has persisted for millions of years. Unexpectedly, both the IMPDH-Bs and ancestral enzymes Anc3-Anc7 were also resistant to ribavirin-5'-monophosphate (RVP), an IMP-competitive inhibitor. Because MPA and RVP bind to similar enzyme conformations, the activity-resistance trade-off likely reflects a design constraint imposed by the need to maintain resistance to multiple inhibitors. Intriguingly, although Anc1 and Anc2 are equally sensitive to MPA, Anc2 shows reduced susceptibility to RVP. This pattern suggests that pre-existing resistance to another IMPDH inhibitor may have created a permissive background for the later evolution of MPA biosynthesis. SignificanceAntibiotic producers must be resistant to the toxins that they produce, but how such self-resistance develops is a mystery. The mycophenolic acid (MPA) biosynthetic gene cluster (BGC) encodes a resistant variant of the MPA target IMPDH (IMPDH-B). Many fungi retain IMPDH-B although they have lost the ability to produce MPA. The IMPDH-B and species phylogenies are incongruent, suggesting evolution of the BGC was complicated. MPA resistance correlates with low catalytic efficiency in modern and ancestral IMPDHs, revealing a robust design constraint tradeoff. Surprisingly, IMPDH-Bs are also resistant to an IMP-competitive inhibitor (RVP). RVP resistance appears to have emerged before MPA resistance. Perhaps resistance to RVP created a background that permitted the genesis of a new toxin.

evolutionary biology↗

bioRxiv2025

The Role of Genetic Variation in Shaping Phenotypic Responses to Diet in Aging Drosophila melanogaster

Nutrition plays a central role in healthy living, however, extensive variability in individual responses to dietary interventions complicates our understanding of its effects. Here we present a comprehensive study utilizing the Drosophila Genetic Reference Panel (DGRP), investigating how genetic variation influences responses to diet and aging. Quantitative genetic analyses of the impact of dietary restriction on lifespan, locomotor activity, dry weight, and heat knockdown time were performed. Locomotor activity, dry weight and heat knockdown time were measured on the same individual flies. We found significant genotype-by-diet interaction (GDI) and genotype-by-age interaction (GAI) for all traits. Therefore, environmental factors play a crucial role in shaping trait variation at different ages and diets, and/or distinct genetic variation influences these traits at different ages and diets. Our genome wide association study also identified a quantitative trait locus for age-dependent dietary response. The observed GDI and GAI indicates that susceptibility to environmental influences changes as organisms age, which could have significant implications for dietary recommendations and interventions aimed at promoting healthy aging in humans. The identification of associations between DNA sequence variation and age-dependent dietary responses opens new avenues for research into the genetic mechanisms underlying these interactions.

genetics↗

bioRxiv2024

The antimycotic 5-fluorocytosine is a virulence inhibitor of uropathogenic Escherichia coli and eradicates biofilm-embedded bacteria synergizing with β-lactams

ObjectivesBiofilm increases bacterial antibiotic tolerance, posing a challenge for treating biofilm-associated infections in clinical settings. 5-fluorocytosine (5-FC), an FDA-approved antifungal and antitumor drug, has been shown to inhibit virulence factor production and biofilm formation in Gram-negative bacteria. This work aims to determine whether 5-FC antivirulence and antibiofilm activity are preserved in clinical E. coli isolates and to test possible synergies with antibiotics in treating preformed biofilm. Methods5-FC ability to inhibit biofilm formation, disrupt mature biofilm, and modulate virulence determinants was tested on uropathogenic E. coli (UPEC) clinical strains using the Crystal Violet-based biofilm adhesion and minimum biofilm eradication concentration assays. Gene expression was measured by RT-qPCR. The effect on bacterial viability within preformed biofilm was monitored using fluorescein diacetate and by determining colony-forming units in biofilm treated with or without 5-FC and specific antibiotics. Bladder epithelial cell cultures were used to assess the effect of 5-FC on UPEC adherence and cytotoxicity. Results5-FC inhibited biofilm formation in all tested UPEC strains. Gene expression analysis suggested that 5-FC concurrently inhibits the expression of genes encoding curli fibers, a major adhesion factor in E. coli, as well as other virulence determinants such as secreted toxins and type I and P fimbriae. Accordingly, 5-FC reduced UPEC adherence to epithelial cells and promoted host cell survival. Although 5-FC could not disrupt preformed biofilm, combining 5-FC with {beta}-lactams drastically reduced the viability of biofilm-resident bacteria. Conclusions5-FC shows antibiofilm and antivirulence activity against uropathogenic E. coli strains by reducing the expression of several virulence factors and the overall UPEC pathogenic potential. In combination with {beta}-lactams, 5-FC eradicates bacteria within mature biofilms, which are known to be highly refractory to antibiotic treatment. Our data suggest that 5-FC is an excellent candidate for preventing and treating bacterial infections associated with recalcitrant biofilms.

microbiology↗

bioRxiv2018

Extreme genetic signatures of local adaptation during plant colonization

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with the recent colonization of Japan by Lotus japonicus. We carefully track the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We characterize the loss of diversity during this process and identify genomic regions with extreme genetic differentiation. Next, we perform population structure-corrected association mapping of phenotypic traits measured in a common garden and discover a number of genome-wide significant associations. Contrasting these analyses, we find that there is a strong concordance between phenotypic variation and extreme differentiation for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection by local adaptation during the colonization process and point to associated candidate genes.

evolutionary biology↗