Biology subjects

Parab, L.

Publications and source records attributed to Parab, L..

6 recordsLinked to original sources

bioRxiv2026

A humanized Galleria mellonella model reveals prophage-mediated breakdown of colonization resistance against Salmonella

BackgroundColonisation resistance provided by the gut microbiota is a critical barrier to pathogen invasion, yet its study in vivo is constrained by the complexity and cost of vertebrate models. Here, we developed a humanised Galleria mellonella infection model by inoculating wax moth larvae with complex human faecal microbiota. 16S rRNA gene sequencing confirmed stable, reproducible establishment of a diverse human-associated community across larvae over four days. ResultsHumanised larvae exhibited colonisation resistance against Salmonella enterica serovar Typhimurium, with mortality reduced to 20% compared to 90% in non-colonised controls. To test whether prophages could overcome this barrier, we infected larvae with isogenic S. Tm strains differing in the presence of prophage P22. Infection with the P22-carrying strain resulted in a threefold higher larval mortality (60% vs. 20%), increased pathogen load, and a significant reduction in the abundance of resident E. coli. Free P22 virions were detected early after infection, indicating extensive prophage activity. Notably, P22 can neither adsorb nor lyse resident E. coli, indicating that prophage-mediated invasion success did not rely on direct lysis. Instead, using high-throughput metabolic profiling paired with whole genome sequencing of three replicate lineages, we found that phage activation intensified resource-partitioning, accelerating functional metabolic adaptations in E. coli that significantly reduced the niche-overlap between the invading pathogen and the commensal E. coli. ConclusionOur findings establish the first humanised G. mellonella model supporting complex human microbiota and provide a novel non-lytic mechanism by which prophages influence species interactions. This scalable, low-cost model offers a new platform to dissect pathogen-phage-microbiota interactions relevant to human gut ecology.

microbiology↗

bioRxiv2025

Engineering Escherichia coli FAD synthetase for the biosynthesis of FAD nucleotide analogues results in aminoglycoside antibiotic tolerance

Cofactors such as flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide, coenzyme A, and S-adenosylmethionine cofactors share a common adenosine moiety that is generally not considered to contribute directly to catalysis. In FAD biosynthesis, the adenosine group is installed by the flavin mononucleotide adenylyltransferase (FMNAT) domain of the bifunctional FAD synthetase using FMN and ATP. This study explores the nucleotide selectivity of the Escherichia coli FMNAT domain and physiological effects of FAD nucleotide analogues synthesized with cellular nucleotides other than ATP. First, we engineer the FAD synthetase enzyme at an active site loop to produce FAD nucleotide analogues. Chromosomal substitution of the wild-type E. coli FAD synthetase with the mutated version results in the intracellular synthesis of these noncanonical FAD nucleotide analogues and confers tolerance to aminoglycoside antibiotics. Upon investigating their potential downstream metabolic roles, we find that their biosynthesis perturbs cellular metabolism and they could bind to a range of E. coli enzymes. These findings validate the FMNAT domain loop as a viable site for cofactor engineering, with potential for synthetic biology applications. Finally, the observed correlation between FAD analogue production and increased tolerance to aminoglycosides reveals novel metabolic mechanisms underlying antibiotic sensitivity.

microbiology↗

bioRxiv2024

Empirical evidence of a role for insertion sequences in the repair of DNA breaks in bacterial genomes

Insertion Sequences (ISs) are mobile pieces of DNA that are widespread in bacterial genomes. IS movements typically involve (i) excision of the IS element, (ii) cutting of the target site DNA, and (iii) IS element insertion. This process generates a new copy of the IS element, as well as a short duplication at the target site. It has been noted that, when observing extant IS element copies in a genome, occasionally no Target Site Duplication (TSD) is readily identifiable. This has been attributed to degeneration of the TSD at some point after the insertion event. Here, we provide evidence that some IS movement events - namely, those that occur in association with large-scale genome rearrangements - occur without generating TSDs. In support of this hypothesis, we provide two direct, empirical observations of such IS transposition events: an IS481 movement occurring with a large duplication in Pseudomonas fluorescens SBW25, and an IS5/IS1182 movement plus a large deletion in Escherichia coli C. Additionally, we use sequencing data from the Lenski long-term evolution experiment to provide a further 14 examples of IS150 movements in E. coli B that are associated with large deletions and do not carry TSDs. Overall, our results indicate that some IS elements can insert into, and thus repair, existing DNA breaks in bacterial genomes.

evolutionary biology↗

bioRxiv2023

Chloramphenicol reduces phage resistance evolution by suppressing bacterial cell surface mutants

Bacteriophages infect Gram-negative bacteria by attaching to molecules present on the bacterial outer membrane, often lipopolysaccharides (LPS). Modification of LPS can lead to resistance to phage infection. In addition, LPS modifications can impact antibiotic susceptibility, allowing for phage-antibiotic synergism. The evolutionary mechanism(s) behind such synergistic interactions remain largely unclear. Here, we show that the presence of antibiotics can affect the evolution of resistance to phage infection, using phage {Phi}X174 and Escherichia coli C. We use a collection of 34 E. coli C LPS strains, each of which is resistant to {Phi}X174, and has either a "rough" or "deep rough" LPS phenotype. Growth of the bacterial strains with the deep rough phenotype is inhibited at low concentrations of chloramphenicol (and, to a much lesser degree, gentamicin). Treating E. coli C wildtype with {Phi}X174 and chloramphenicol eliminates the emergence of mutants with the deep rough phenotype, and thereby slows the evolution of resistance to phage infection. At slightly lower chloramphenicol concentrations, phage resistance rates are similar to those observed at high concentrations; yet, we show that the diversity of possible mutants is much larger than at higher chloramphenicol concentrations. These data suggest that specific antibiotic concentrations can lead to synergistic phage-antibiotic interactions that disappear at higher antibiotic concentrations. Overall, we show that the change in survival of various {Phi}X174-resistant E. coli C mutants in the presence of antibiotics can explain the observed phage-antibiotic synergism. Classification: Biological Sciences, Evolution

evolutionary biology↗

bioRxiv2022

E. coli-{varphi}X174 genotype to phenotype map reveals flexibility and diversity in LPS structures

Phage therapy is a promising method for the treatment of multi-drug-resistant bacterial infections. However, its long-term efficacy depends on understanding the evolutionary effects of the treatment. Current knowledge of such evolutionary effects is lacking, even in well-studied systems. We used the bacterium Escherichia coli C and its bacteriophage {Phi}X174, which infects cells using host lipopolysaccharide (LPS) molecules. We first generated 31 bacterial mutants resistant to {Phi}X174 infection. Based on the genes disrupted by these mutations, we predicted that these E. coli C mutants collectively produce eight unique LPS structures. We then developed a series of evolution experiments to select for {Phi}X174 mutants capable of infecting the resistant strains. During phage adaptation, we distinguished two types of phage resistance: one that was easily overcome by {Phi}X174 with few mutational steps ("easy" resistance), and one that was more difficult to overcome ("hard" resistance). We found that increasing the diversity of the host and phage populations could accelerate the adaptation of phage {Phi}X174 to overcome the hard resistance phenotype. From these experiments, we isolated 16 {Phi}X174 mutants that, together, can infect all 31 initially resistant E. coli C mutants. Upon determining the infectivity profiles of these 16 evolved phages, we uncovered 14 distinct profiles. Given that only eight profiles are anticipated if the LPS predictions are correct, our findings highlight that the current understanding of LPS biology is insufficient to accurately forecast the evolutionary outcomes of bacterial populations infected by phage.

evolutionary biology↗

bioRxiv2020

Transcription factor binding dynamics shape noise across biological processes

Noise in expression of individual genes gives rise to variations in activity of cellular pathways and generates heterogeneity in cellular phenotypes. Phenotypic heterogeneity has important implications for antibiotic persistence, mutation penetrance, cancer growth and therapy resistance. Specific molecular features such as presence of the TATA box sequence and promoter nucleosome occupancy have been associated with noise. However, the relative importance of these features in noise regulation has not yet been assessed. In addition, how well these features can predict noise also remains unclear. Here through an integrated statistical model of gene expression noise in yeast we found that the number of regulating transcription factors (TFs) was a key predictor of noise. With an increase in the number of regulatory TFs, we observed a rise in the number of TFs that bound cooperatively. In addition, increased number of TFs meant more overlaps in TF binding sites that caused competition between TFs for binding to the same region of the promoter. Through modeling of TF binding to promoter and applying stochastic simulations, we demonstrated that competition and cooperation among TFs could increase noise. Thus, our work uncovers a process of noise regulation that arises out of the dynamics of gene regulation and is not dependent on any specific transcription factor or specific promoter sequence.

systems biology↗