Biology subjects

Kritikos, G.

Publications and source records attributed to Kritikos, G..

7 recordsLinked to original sources

bioRxiv2024

Systematic mapping of antibiotic cross-resistance and collateral sensitivity with chemical genetics

By acquiring or evolving resistance to one antibiotic, bacteria can become resistant to a second one, due to shared underlying mechanisms. This is called cross-resistance (XR) and further limits therapeutic choices. The opposite scenario, in which initial resistance leads to sensitivity to a second antibiotic, is termed collateral sensitivity (CS) and can inform cycling or combinatorial treatments. Despite their clinical relevance, our current knowledge of such interactions is limited, mostly due to experimental constraints in their assessment and lack of understanding of the underlying mechanisms. To fill this gap, we used published chemical genetic data on the impact of all Escherichia coli non-essential genes on resistance/sensitivity to 40 antibiotics, and devised a metric that robustly discriminates between known XR and CS antibiotic interactions. This metric, based on chemical genetic profile (dis)similarity between two drugs, allowed us to infer 404 XR and 267 CS interactions, thereby expanding the number of known interactions by more than 3-fold - including reclassifying 116 previously reported interactions. We benchmarked our results by validating 55 out of 59 inferred interactions via experimental evolution. By identifying mutants driving XR and CS interactions in chemical genetics, we recapitulated known and uncovered previously unknown mechanisms, and demonstrated that a given drug pair can exhibit both interactions depending on the resistance mechanism. Finally, we applied CS drug pairs in combination to reduce antibiotic resistance development in vitro. Altogether, our approach provides a systematic framework to map XR/CS interactions and their mechanisms, paving the way for the development of rationally-designed antibiotic combination treatments.

systems biology↗

bioRxiv2023

ChemGAPP; A Package for Chemical Genomics Analysis and Phenotypic Profiling.

High-throughput chemical genomic screens produce informative datasets, providing valuable insights into unknown gene function on a genome-wide level. However, there is currently no comprehensive analytic package publicly available. We developed and benchmarked ChemGAPP to bridge this gap. ChemGAPP allows integration of various steps in a streamlined and user-friendly format, including rigorous quality control measures to curate screening data. ChemGAPP provides three sub-packages for different chemical-genomic screens: ChemGAPP Big for handling large-scale high-throughput screens; ChemGAPP Small, designed for small-scale screen analysis and ChemGAPP GI for genetic interaction screen analysis. ChemGAPP is available at https://github.com/HannahMDoherty/ChemGAPP.

bioinformatics↗

bioRxiv2020

Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 [A], in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. ImportanceM48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the LPS biogenesis machinery. Here we present the structure of BepA and the results of a structure guided mutagenesis strategy, which reveal the key residues required for activity.

microbiology↗

bioRxiv2020

Phage proteins block and trigger retron toxin/antitoxin systems

Bacteria carry dozens of Toxin/Antitoxin systems (TAs) in their chromosomes. Upon growth, the antitoxin is co-expressed and neutralizes the toxin. TAs can be activated and inhibit growth, but when and how this occurs has largely remained enigmatic, hindering our understanding of their physiological roles. We developed TIC/TAC (Toxin Inhibition/Activation Conjugation), a high-throughput reverse genetics approach, to systematically identify molecular blockers and triggers of TAs. By applying TIC/TAC to a tripartite TA, the retron-Sen2 of Salmonella Typhimurium, we have identified multiple blockers and triggers of phage origin. We demonstrate that diverse phage functionalities are sensed by the DNA-part of the antitoxin and ultimately activate the retron toxin. Phage-origin proteins can circumvent activation by directly blocking the toxin. Some identified triggers and blockers also act on an E. coli retron-TA, Eco9. We propose that retron-TAs act as abortive-infection anti-phage defense systems, and delineate mechanistic principles by which phages trigger or block them.

microbiology↗

bioRxiv2019

Structure-function analysis of the Escherichia coli β-barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 [A]. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.

microbiology↗

bioRxiv2019

Biofilm inhibitor taurolithocholic acid alters colony morphology, specialized metabolism, and virulence of Pseudomonas aeruginosa

Biofilm inhibition by exogenous molecules has been an attractive strategy for the development of novel therapeutics. We investigated the biofilm inhibitor taurolithocholic acid (TLCA) and its effects on the specialized metabolism, virulence and biofilm formation of the clinically relevant bacterium Pseudomonas aeruginosa strain PA14. Our study shows that TLCA alters specialized metabolism, thereby affecting P. aeruginosa colony biofilm physiology. We observed an upregulation of metabolites correlated to virulence such as the siderophore pyochelin. A wax moth virulence assay confirmed that treatment with TLCA increases virulence of P. aeruginosa. Based on our results, we believe that future endeavors to identify biofilm inhibitors must consider how a putative lead is altering the specialized metabolism of a bacterial community to prevent pathogens from entering a highly virulent state.

microbiology↗

bioRxiv2019

The outer membrane lipoprotein NlpI nucleates hydrolases within peptidoglycan multi-enzyme complexes in Escherichia coli

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of PG synthases and hydrolases. In Escherichia coli, the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in the outer membrane. However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form multimeric complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and cell division biosynthetic complexes based on its localization and genetic interactions. In line with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that PG regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.\n\nSignificanceThe activity of PG hydrolases may cause lysis of the bacterial cell if left unregulated. Hence, the cell must have ways of regulating and coordinating their activities. Our current understanding of how this occurs is incomplete. In this work, we present the outer membrane (OM) anchored lipoprotein, NlpI, as a scaffold of peptidoglycan hydrolases. We propose that NlpI facilitates the formation of multi-enzyme complexes and that, along with other regulators, it coordinates a safe enlargement and separation of the PG layer in E. coli.

microbiology↗