Biology subjects

Totsika, M.

Publications and source records attributed to Totsika, M..

15 recordsLinked to original sources

bioRxiv2025

Transcriptional profiling of Pseudomonas aeruginosa biofilm life cycle stages reveals dispersal-specific biomarkers

Bacteria exhibit two lifestyles: planktonic free-floating individual cells or sessile multicellular aggregates known as biofilms. The biofilm lifecycle is characterised by three distinct stages: attachment, maturation and dispersal. Distinct adaptations occur in each stage, determining cellular behaviours such as surface attachment or synthesis and degradation of extracellular matrix components. Characterising stage-specific bacterial profiles therefore represents a valuable strategy for the development of novel antibiofilm therapies. Here, we used the model biofilm-forming bacterium Pseudomonas aeruginosa PAO1 to characterise the transcriptional profiles of each stage of the biofilm life cycle: attachment, biofilm maturation and spontaneous dispersal in closed cultures. We report that surface attachment was accompanied by the upregulation of genes comprising the Pil-Chp mechanosensory system, whereas biofilm maturation was characterised by the upregulation of genes involved in Pel polysaccharide synthesis, siaD and PA4396 diguanylate cyclases as well as pipA, fimX and PA5442. In contrast, dispersing cells upregulated genes responsible for the biosynthesis of alginate, rhamnolipid, and extracellular nucleases (eddA, eddB), as well as the transcriptional regulator of dispersal amrZ. Additionally, genes encoding the spontaneous dispersal molecule cis-2-decenoic acid (dspS and dspI), canonical phosphodiesterases (nbdA and rbdA), four non-canonical HD-GYP phosphodiesterases and seven other c-di-GMP-related enzymes were also upregulated during dispersal. Our comprehensive analysis of transcriptional changes across biofilm stages therefore provides benchmarking stage-specific transcriptional profiles for P. aeruginosa biofilms in closed culture systems. Furthermore, it allowed the identification of a subset of fourteen genes as transcriptional biomarkers of dispersal, which were used to build reporter plasmids as tools to determine the onset of dispersal. ImportanceBiofilm infections by P. aeruginosa are a major medical challenge due to the increased tolerance to antimicrobials displayed by bacteria living in sessile communities, which is reduced during spontaneous biofilm dispersal. Attachment, biofilm maturation and dispersal represent the main stages of a dynamic process known as the biofilm lifecycle. However, the global regulatory responses governing transitions between these stages remain understudied. Here, we combine live microscopy and biomass quantification to track the progression of P. aeruginosa cultures through the three main stages of the biofilm lifecycle. We show that cells from each stage recapitulate canonical, stage-specific transcriptional responses and identify a set of biomarkers associated with the onset of dispersal. These biomarkers may offer a practical tool for rapidly screening dispersal-inducing compounds, aiding in the discovery of the next generation of antibiofilm therapeutics.

genomics↗

bioRxiv2025

Nitric oxide donor sodium nitroprusside serves as a source of iron supporting Pseudomonas aeruginosa growth and biofilm formation

Biofilm dispersal agents, like nitric oxide (NO), restore antimicrobial effectiveness against biofilm infections by inducing bacteria to shift from a biofilm to a planktonic state, thereby overcoming the antimicrobial tolerance typically associated with biofilms. Sodium nitroprusside (SNP) is a widely used NO donor for investigating the molecular mechanisms underlying NO-mediated biofilm dispersal in the nosocomial pathogen Pseudomonas aeruginosa. However, the biofilm effects of SNP are variable depending on the in vitro experimental conditions, with some studies reporting enhanced growth in both planktonic and biofilm forms instead of dispersal. These discrepancies suggest that SNP affects P. aeruginosa biofilm-residing cells beyond the release of NO. In this study, we compared SNP with another NO donor, Spermine NONOate, to systematically contrast their effects on biofilm and planktonic cultures of P. aeruginosa. We found that SNP, but not Spermine NONOate, increased the biomass of P. aeruginosa biofilms in microplate cultures. This effect was also observed when biofilms were supplemented with iron. Additionally, supplementation with SNP rescued the planktonic growth of P. aeruginosa in iron-depleted media, similar to FeSO4 supplementation, suggesting that SNP may serve as an iron source. Our findings suggest that SNPs potential as an NO agent used for biofilm dispersal may be confounded by its role in promoting both biofilm and planktonic growth through its iron centre. Our study cautions investigators using SNP for studying NO-mediated biofilm dispersal.

microbiology↗

bioRxiv2025

Disruptions in Outer Membrane-Peptidoglycan Interactions Enhance Bile Salt Resistance in O-antigen-Producing E. coli

Bile salts (BS) are derived from cholesterol in the liver and act as antimicrobial agents in the intestines by disrupting bacterial cell membranes and inducing oxidative stress. The gut bacterium E. coli is naturally resistant to BS, including the model strain K12 that produces a truncated LPS without O-antigen (OAg). Paradoxically, restoring a wild-type like LPS with OAg sensitises E. coli K12 to exogenous BS. In this study, we investigate this phenomenon. We show that mutations causing truncation of the LPS core oligosaccharide render these strains even more susceptible to BS, similar to the mutant strain MG1655-S{Delta}waaL defective in OAg ligase, primarily due to the accumulation of the lipid-linked intermediate UndPP-OAg. Through the characterisation of BS-resistant suppressor mutants of MG1655-S{Delta}waaL, we identify key genetic disruptions involved in resistance. Notably, we observed the highest BS resistance in strains with a weaker connection between the outer membrane (OM) and peptidoglycan (PG), including strains lacking the major OM-anchored, PG-binding proteins OmpA or Lpp, or expressing versions of these that lack PG-binding. Our data suggest that BS-induced stress in OAg-producing E. coli is due to the spatial constraints between OM and PG, and that mutations disrupting OM-PG interactions alleviate this stress, enhancing BS resistance. These findings provide new insights into a major challenge E. coli faces in the gut environment where it needs to produce OAg for stable colonisation and resists BS. E. coli can only survive BS exposure by fine-tuning the connectivity between its cell envelope layers, which highlights a potential target for modulating bacterial responses to BS in the gut. Author summaryEnteric bacteria residing in the human gut must withstand the host-derived antimicrobial agents, bile salts (BS), but the underlying resistance mechanisms are not fully elucidated. This study investigates the BS resistance mechanisms in O-antigen (OAg)-producing Escherichia coli K-12. We show that truncation of lipopolysaccharide (LPS) core oligosaccharides or restoration of OAg production increases BS sensitivity due to the accumulation of UndPP-OAg intermediates. By analysing suppressor mutants, we identify key genetic disruptions, particularly affecting the level of outer membrane-peptidoglycan (OM-PG) interactions involving OmpA and Lpp, which confer heightened BS resistance. Our findings highlight how BS-induced stress is linked to spatial constraints between the OM and PG layer, offering new insights into bacterial adaptation to BS stress. This research may provide new targets for therapeutic interventions to modulate gut microbial responses to BS.

microbiology↗

bioRxiv2025

Optimal membrane fluidity modulates biofilm formation in Escherichia coli

IMPACT STATEMENTMembrane fluidity plays a crucial role in bacterial fitness and adaptation to cope rapid environmental changes. While high membrane fluidity promotes robust biofilm formation in Klebsiella pneumoniae, studies in several other species, including Salmonella enterica, suggest that biofilm formation is associated with reduced fluidity. This paradox may reflect the complex relationship between lipid composition and biofilm formation. Our findings demonstrated that both low and high extremes of lipid fluidity restrict biofilm formation. We propose that the required fluidity for biofilm growth, relative to that required for planktonic growth, may differ between species and are readily adjusted during lifestyle transitions.

microbiology↗

bioRxiv2024

Tolerance Mechanisms in Polysaccharide Biosynthesis: Implications for Undecaprenol Phosphate Recycling in Escherichia coli and Shigella flexneri

Bacterial polysaccharide synthesis is catalysed on the universal lipid carrier, undecaprenol phosphate (UndP). The cellular UndP pool is shared by other polysaccharide synthesis pathways and in peptidoglycan (PG) biogenesis. Disruptions in cytosolic polysaccharide synthesis steps are detrimental to bacterial survival due to affecting UndP recycling. In contrast, bacteria can survive disruptions in the periplasmic steps, suggesting a tolerance mechanism to mitigate UndP sequestration. Here we investigated tolerance mechanisms to disruptions of polymerases that are involved in UndP-releasing steps in two related polysaccharide synthesis pathways: the enterobacterial common antigen (ECA) and the O antigen (OAg), in Escherichia coli and Shigella flexneri. Our study reveals that polysaccharide polymerisation is crucial for efficient UndP recycling. In E. coli K-12, cell survival upon disruptions in OAg polymerase is dependent on a functional ECA synthesis pathway and vice versa. This is because disruptions in OAg synthesis leads to the redirection of the shared lipid-linked sugar substrate UndPP-GlcNAc towards increased ECA production. Conversely, in S. flexneri, the OAg polymerase is essential due to its limited ECA production, which inadequately redirects UndP flow to support cell survival. We propose a model whereby sharing the initial sugar intermediate UndPP-GlcNAc between the ECA and OAg synthesis pathways allows UndP to be redirected towards ECA production, mitigating sequestration issues caused by disruptions in the OAg pathway. These findings suggest an evolutionary buffering mechanism that enhances bacterial survival when UndP sequestration occurs due to stalled polysaccharide biosynthesis, which may allow polysaccharide diversity in the species to increase over time. ImportanceEnzymes involved in bacterial polysaccharide biosynthesis have substrate specificity to ensure the correct polysaccharide is produced at the appropriate place and time. However, this specificity poses a challenge for the diversification of polysaccharide structure and hence function, as the acquisition of a novel oligosaccharide RU would likely disrupt the synthesis pathways, leading to sequestration of the essential universal lipid carrier UndP and ultimately cause cell death. We investigated how cells tolerate disruptions in polysaccharide synthesis pathways and provide evidence that suggests that sharing a common substrate between the synthesis pathways of two common enteric bacterial surface polysaccharides (ECA and OAg), can redirect the flow of UndP. Our study provides insights into the mechanism of how bacteria alleviate sequestration issues, thereby enhancing cell survival which may allow them additional capacity for polysaccharide diversification.

microbiology↗

bioRxiv2023

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg based on serological typing and sequence analysis is important in epidemiology and surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs) described, the genetic basis of the assembly of these RUs remains poorly understood and represents a major limitation in our understanding of the gene functions in the polysaccharide biosynthesis. Here we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT) controlled system to allocate the functional order of the subsequent GT in a two-fold manner. First by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs, and second by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GTs involved in the assembly of S. flexneri backbone OAg RU, RfbG is responsible for both the committed step of OAg synthesis, as well as the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria. ImportanceThe genetic basis of enzymatic assembly of structurally diverse OAg repeating units in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to assigning glycosyltransferase (GT) functions and the order in which they act in the assembly of the OAg RU in their native background strain with reasonable confidence. We demonstrated with this approach the functional allocation of GTs for Shigella flexneri O antigen assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

microbiology↗

bioRxiv2023

O antigen biogenesis sensitises Escherichia coli K-12 to bile salts, a likely cause for how it lost its O antigen

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies reported the presence of independent mutations that abolish OAg biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for loss of OAg during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS mostly contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-carried OAg intermediates in the bacterial periplasm interfere with peptidoglycan synthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for. Hence our work provides a likely explanation for the long-held mystery of how E. coli K-12 lost its OAg production and opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria. Significance statementEscherichia coli K-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. The reason why it is devoid of O antigen remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation of E. coli K-12 strains in bile salt containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which paves a new research direction into the interplay of different cell envelope component synthesis pathways.

microbiology↗

bioRxiv2023

Extensive diversity in Escherichia coli Group 3 capsules is driven by recombination and plasmid transfer from multiple species

Bacterial capsules provide protection against environmental challenges and host immunity. Historically, Escherichia coli K serotyping scheme, which relies on the hypervariable capsules, has identified around 80 K forms that fall into four distinct groups. Based on recent work by us and others, we predicted that E. coli capsular diversity is grossly underestimated. We exploited Group 3 capsule gene clusters, the best genetically defined capsule group in E. coli, to analyse publicly available E. coli sequences for overlooked capsular diversity within the species. We report the discovery of seven novel Group 3 clusters that fall into two distinct subgroups (3A and 3B). The majority of the 3B capsule clusters were found on plasmids, contrary to the defining feature of Group 3 capsule genes localising at the serA locus on the E. coli chromosome. Other new Group 3 capsule clusters were derived from ancestral sequences through recombination events between shared genes found within the serotype variable central region 2. Intriguingly, flanking regions 1 and 3, known to be conserved areas among capsule clusters, showed considerable intra-subgroup variation in clusters from the 3B subgroup, containing genes of shared ancestry with other Enterobacteriaceae species. Variation of Group 3 kps clusters within dominant E. coli lineages, including multi-drug-resistant pathogenic lineages, further supports that E. coli capsules are undergoing rigorous change. Given the pivotal role of capsular polysaccharides in phage predation, our findings raise attention to the need of monitoring kps evolutionary dynamics in pathogenic E. coli in supporting phage therapy. IMPORTANCECapsular polysaccharides protect pathogenic bacteria against environmental challenges, host immunity, and phage predations. The historical Escherichia coli K typing scheme, which relies on the hypervariable capsular polysaccharide, has identified around 80 different K forms that fall into four distinct groups. Taking advantage of the supposedly compact and genetically well-defined Group 3 gene clusters, we analysed published E. coli sequences to identify seven new gene clusters and revealed an unexpected capsular diversity. Genetic analysis revealed that Group 3 gene clusters shared closely related serotype-specific region 2 and were diversified through recombination events and plasmid transfer between multiple Enterobacteriaceae species. Overall, capsular polysaccharides in E. coli are undergoing rigorous change. Given the pivotal role capsules play in phage interactions, this work highlighted the need to monitor the evolutionary dynamics of capsules in pathogenic E. coli for effective phage therapy.

microbiology↗

bioRxiv2023

Bacterial suppressor-of-copper-sensitivity (Scs) proteins exhibit diverse thiol-disulfide oxidoreductase cellular functions

Disulfide bond (Dsb) proteins catalyse oxidative protein folding governing bacterial survival and virulence. Dsb systems in Escherichia coli K-12 are well-studied, yet what determines dithiol oxidase or disulfide reductase activity remains unknown. Past studies suggest oligomerisation of periplasmic thiol oxidoreductases dictates the direction of thiol catalytic activity. Here, we studied three suppressor-of-copper-sensitivity C (ScsC) Dsb-like proteins known to exist in the reduced state and bind to copper. These proteins adopt different quaternary structures: Salmonella enterica ScsC (StScsC) is monomeric, while ScsC from Proteus mirabilis (PmScsC) and Caulobacter crescentus (CcScsC) are trimeric. When expressed in the model organism E. coli K-12, we showed that all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences. Interestingly, while ScsC reductase function was supported by the canonical E. coli DsbD reductase, oxidase activity depended on environmental oxidation. However, an engineered monomeric PmScsC synergises with E. coli DsbB to gain dithiol oxidase activity at the expense of reductase function. Thus, oligomerisation could be one mechanism by which ScsC proteins avoid interactions with the periplasmic thiol oxidase pathway. This tightly controls their re-oxidation and maintains ScsC proteins in the reduced state required for binding and sequestering toxic levels of cellular copper.

microbiology↗

bioRxiv2022

Bladder-draining lymph nodes support germinal centre B cell responses during urinary tract infection in mice

Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multi-drug resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we investigated whether host adaptive immune responses can influence infection outcomes. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with an MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1-/- C57BL/6J mice harboured larger bacterial burdens than wild-type counterparts, consistent with a role for T and/or B cells in optimal control of UTI. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11chi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal centre (GC) B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice, and although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model, that Gram-negative bacterial UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.

immunology↗

bioRxiv2022

Cysteine dependent conformation heterogeneity of Shigella flexneri autotransporter IcsA and implications in its function

Shigella IcsA is a versatile surface virulence factor required for both early and late pathogenesis stages, extracellularly to intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing of the host stimuli by Shigella Type III secretion system (T3SS). Here, we report that the single cysteine residue (C130) near the N-terminus of IcsA passenger has a role in IcsA adhesin activity. We also show that the IcsA passenger (IcsAp) exists in multiple conformations, and the conformation populations are influenced by a central pair of cysteine residues (C375 and C379), which is not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues alters the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA paired cysteine substitution mutants were expressed in a{triangleup} ipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovers a novel molecular switch empowered by a centrally localised, short-spaced cysteine pair in the Type V autotransporter IcsA that ensures conformational heterogeneity to aid IcsA evasion of host immunity. ImportanceShigella species are the leading cause of diarrheal related death globally by causing bacillary dysentery. The surface virulence factor IcsA which is essential for Shigella pathogenesis is a unique multi-functional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we show that the three cysteine residues in IcsA contribute to the proteins distinct functions. The N terminus cysteine residue within the IcsA passenger domain plays a role in adhesin function, while a centrally localised cysteine pair provides conformational heterogeneity resulting in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserves biological function in distinct IcsA conformations for cell adhesion, actin-based motility and autophagy escape, providing a potential strategy by which Shigella evade host immunity targeting of this essential virulence factor.

microbiology↗

bioRxiv2022

Loss of β-ketoacyl acyl carrier protein synthase III activity restores multidrug-resistant Escherichia coli sensitivity to previously ineffective antibiotics

Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, nine out of 12 are Gram-negative, with four being of Critical Priority. One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial Type II Fatty Acid Synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic susceptible and antibiotic resistant clinical isolates. The fabH gene encodes the {beta}-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multi-drug resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens. IMPORTANCEGram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor towards antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids and their synthesis constitutes an attractive antimicrobial target as it follows distinct pathways in prokaryotes and eukaryotes. Herein, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are re-sensitised to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

microbiology↗

bioRxiv2021

Fine tuning the mechanism of Antigen 43 self-association to modulate aggregation levels of Escherichia coli pathogens

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the tuning of bacterial aggregation.

biochemistry↗

bioRxiv2020

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics.

Antibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is often stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors of DsbA block bacterial oxidative protein folding and were previously identified by biophysical and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher throughput format, can provide a robust and target-specific platform for the evaluation of DsbA inhibitors. Our pipeline could also be used in fragment and compound screening for the identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In conclusion, the establishment of accurate, high-throughput microbiological assays for antivirulence drug identification and early preclinical development, is a significant first step towards their translation into effective therapeutics. ImportanceThe safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, where common infections and minor injuries are once again lethal. The paucity in new antibiotic discovery of the past decades has compounded the problem of increasing antibiotic resistance, to the point that it now constitutes a global health crisis that demands global action. There is currently an urgent need for new antibacterial drugs with new targets and modes of action. To address this, research and development efforts into antivirulence drugs, such as DsbA inhibitors, have been ramping up globally. However, the development of microbiological assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. Here, we present a high-throughput cell-based screening and evaluation pipeline, which could significantly advance development of DsbA inhibitor as antivirulence therapeutics.

microbiology↗

bioRxiv2019

Nitroxide functionalized antibiotics are promising eradication agents against Staphylococcus aureus biofilms.

Treatment of Staphylococcus aureus biofilm-related infections represents an important medical challenge worldwide, as biofilms, even of drug-susceptible S. aureus strains, are highly refectory to conventional antibiotic therapy. Nitroxides were recently shown to induce dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Co-administration of the nitroxide CTEMPO with ciprofloxacin significantly improved the antibiotics biofilm-eradication activity against S. aureus. Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced ciprofloxacins minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter into cells localising at the surface and base of the biofilm structure. No toxicity was observed for the nitroxide CTEMPO and the nitroxide-antibiotic hybrids against human cells. Taken together, our results show that nitroxides can mediate dispersal of Gram-positive biofilms and that dual-acting biofilm-eradication antibiotics could provide broad-spectrum therapies for the treatment of biofilm-related infections.

microbiology↗