Biology subjects

Rakonjac, J.

Publications and source records attributed to Rakonjac, J..

6 recordsLinked to original sources

bioRxiv2024

Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin in Escherichia coli.

Antibiotic combinations are a promising strategy to counteract the global problem of increasing antibiotic resistance. We have previously demonstrated furazolidone-vancomycin synergy against Gram-negative pathogens. Here, we selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination to which the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one of, or both furazolidone and vancomycin, but in all cases were associated with a decrease in furazolidone-vancomycin synergy. Among a variety of gene mutations identified in this screen, we investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, and found them to act predominantly through decreasing the activity of the NfsA and NfsB nitroreductases, which have FMN (flavin mononucleotide) or FAD (flavin adenine dinucleotide) as a prosthetic group. We further show that the ribB/ribE mutants isolated in our screen are riboflavin semi-auxotrophs. Riboflavin supplementation restored the normal growth of the ribB/ribE mutants but not the furazolidone sensitivity.

microbiology↗

bioRxiv2022

Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ff has seen an extraordinary range of applications, including in phage display and nanotechnology. However, the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. Using cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods, we have determined the first structure of a filamentous virus, including the filament tips. Structure combined with mutagenesis was employed to identify domains of the phage that are important in bacterial attack and for release of new phage progeny. These data allow new models to be proposed for the phage lifecycle and will undoubtedly enable the development of novel biotechnological applications.

microbiology↗

bioRxiv2021

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 [A] resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

biophysics↗

bioRxiv2021

Correlated transcriptional responses provide insights into the synergy mechanisms of the furazolidone, vancomycin and sodium deoxycholate triple combination in Escherichia coli

Effective therapeutic options are urgently needed to tackle antibiotic resistance. Furazolidone (FZ), vancomycin (VAN), and sodium deoxycholate (DOC) show promise as their combination can synergistically inhibit the growth of, and kill, multidrug-resistant Gram-negative bacteria that are classified as critical priority by the World Health Organization. Here, we investigated the mechanisms of action and synergy of this drug combination using a transcriptomics approach in the model bacterium Escherichia coli. We show that FZ and DOC elicit highly similar gene perturbations indicative of iron starvation, decreased respiration and metabolism, and translational stress. In contrast, VAN induced envelope stress responses, in agreement with its known role in peptidoglycan synthesis inhibition. FZ induced the SOS response consistent with its DNA damaging effects, but we demonstrate that using FZ in combination with the other two compounds enables use of lower dosages and largely mitigates its mutagenic effects. Based on the gene expression changes identified, we propose a synergy mechanism where the combined effects of FZ, VAN, and DOC amplify damage to Gram-negative bacteria while simultaneously suppressing antibiotic resistance mechanisms. ImportanceSynergistic combinations of existing antibacterials against multidrug-resistant "superbugs" are an alternative strategy to costly and arduous development of novel antibacterial molecules. The synergistic combination of nitrofurans, vancomycin and sodium deoxycholate shows promise in inhibiting and killing multidrug-resistant Gram-negative bacteria. We examined the mechanism of action and synergy of these three antibacterials, and proposed a mechanistic basis for their synergy. Our results highlight much needed mechanistic information necessary to advance this combination as a potential therapy.

microbiology↗

bioRxiv2019

In vitro synergy between Sodium Deoxycholate and Furazolidone against Enterobacteria via Inhibition of Multidrug Efflux Pumps

Antimicrobial combinations have been proven to be a promising approach in the confrontation with multi-drug resistant bacterial pathogens, owing to enhancement of antibacterial efficacy, deceleration of resistance development rate and mitigation of side effects by lowering the doses of two drugs. In the present study, we report that combination of furazolidone (FZ) and other nitrofurans with a secondary bile salt, Sodium Deoxycholate (DOC), generates a profound synergistic effect on growth inhibition and lethality in enterobacteria, including Escherichia coli, Salmonella, Citrobacter gillenii and Klebsiella pneumoniae. Taking E. coli as the model organism to study the mechanism of DOC-FZ synergy, we found that the synergistic effect involves FZ-mediated inhibition of efflux pumps that normally remove DOC from bacterial cells. We further show that the FZ-mediated nitric oxide production contributes to the synergistic effect. This is to our knowledge the first report of nitrofuran-DOC synergy against Gram-negative bacteria.

microbiology↗

bioRxiv2018

Butyrivibrio hungatei MB2003 competes effectively for soluble sugars released by Butyrivibrio proteoclasticus B316T from growth on xylan or pectin

Rumen bacterial species belonging to the genera Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four distinct species are recognized which have very similar substrate utilization profiles, but little is known about how these microorganisms are able to co-exist in the rumen. To investigate this question, Butyrivibrio hungatei (MB2003) and Butyrivibrio proteoclasticus (B316T) were grown alone or in co-culture on the insoluble substrates, xylan or pectin, and their growth, release of sugars, fermentation end products and transcriptomes were examined. In single cultures, B316T was able to degrade and grow well on xylan and pectin, while B. hungatei MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Co-cultures of B316T grown with MB2003 revealed that MB2003 showed almost equivalent growth to B316T when either xylan or pectin were supplied as substrates. The effect of co-culture on the transcriptomes of B316T and MB2003 was very marked; B316T transcription was largely unaffected by the presence MB2003, but MB2003 expressed a wide range of genes encoding carbohydrate degradation/metabolism and oligosaccharide transport/assimilation in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of the primary solubilization of xylan and pectin, while MB2003 competes effectively as a scavenger for the released soluble sugars to enable its growth and maintenance in the rumen.\n\nIMPORTANCEFeeding a global population of nine billion people and climate change are the primary challenges facing agriculture today. Determining the roles of rumen microbes involved in plant polysaccharide breakdown is fundamental to understanding digestion and maximizing productivity of ruminant livestock. Butyrivibrio are abundant rumen bacteria and are a substantial source of polysaccharide-degrading enzymes with biotechnological applications for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fibre and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes related to their enzymatic machinery by which these bacteria work in concert to degrade these different forms of polysaccharides contributes to our understanding of carbon flow in the rumen.

microbiology↗