Biology subjects

Frankel, G.

Publications and source records attributed to Frankel, G..

26 records · Page 2Linked to original sources

bioRxiv2024

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF revealed that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol modified -helical barrel domain. Unexpectedly, we identified chromosomal TraT homologues from multiple Gram-negative phyla which formed numerous deep-branching lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences largely cluster into two separate lineages that have more recently evolved, incorporating TraT from Enterobacterales IncF and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

microbiology↗

bioRxiv2023

Re-engineering of TNFα-NF-κB signalling dynamics in cancer cells using pathogenic E. coli effectors

Re-engineering NF-{kappa}B signalling towards enhancing beneficial outcomes such as tumour cell elimination, while minimising inflammatory damage, is a potential therapeutic avenue. In this study, we explored the ability of bacterial effectors injected into host cells by the type III secretion system to regulate NF-{kappa}B translocation dynamics. We used the enteropathogenic Escherichia coli effectors Tir (NF-{kappa}B activator), NleC (NF-{kappa}B protease) and NleE (TAB2/3 methyltransferase), to manipulate NF-{kappa}B translocation and cancer cell survival. We discovered that while these effectors have either limited or no cytotoxicity alone, they greatly enhanced caspase-8-dependent pancreatic cancer cell death in the presence of TNF. Single cell analysis revealed that the sub-population of cells showing high NF-{kappa}B activation is less susceptible to cell death caused by NleC or NleE but instead is more susceptible to Tir. A combination of Tir, NleE and TNF eliminated 95% cancer cells with limited NF-{kappa}B activation, potentially due to NleE-dependent blockage of the immediate pro-survival NF-{kappa}B activation without inhibiting Tirs long-term NF-{kappa}B activation that promotes cell death. This work demonstrates that effector combinations could be used to re-engineer stress responses towards favourable outcomes.

cell biology↗

bioRxiv2023

The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of seven TraN sequence types, which are grouped into four structural types, we named TraN, TraN{beta}, TraN{gamma}, TraN{delta}. Moreover, we have shown specific pairing between TraN and OmpW, TraN{beta} and OmpK36 of Klebsiella pneumoniae, TraN{gamma} and OmpA and TraN{delta} and OmpF. In this study we found that although structurally similar, TraN encoded by the pSLT plasmid (TraN2) binds OmpW in both Escherichia coli and Citrobacter rodentium while TraN encoded by the R100-1 plasmid (TraN1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraN{beta}-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraN{beta} can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. ImportanceConjugation plays a central role in the spread of antimicrobial resistance genes amongst bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilisation (MPS), mediated by tight interactions between the plasmid encoded outer membrane protein (OMP) TraN in the donor (of which there are seven sequence types grouped into the four structural isoforms , {beta}, {gamma}, {delta}) and an OMP receptor in the recipient (OmpW, OmpK36, OmpA and OmpF, respectively). In this study we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

microbiology↗

bioRxiv2022

Emergent regulation of ant foraging frequency through a computationally inexpensive forager movement rule

1Ant colonies regulate foraging in response to their collective hunger, yet the mechanism behind this distributed regulation remains unclear. Previously, by imaging food flow within ant colonies we showed that the frequency of foraging events declines linearly with colony satiation ([1]). Our analysis implied that as a forager distributes food in the nest, two factors affect her decision to exit for another foraging trip: her current food load and its rate of change. Sensing these variables can be attributed to the foragers individual cognitive ability. Here, new analyses of the foragers trajectories within the nest imply a different way to achieve the observed regulation. Instead of an explicit decision to exit, foragers merely tend toward the depth of the nest when their food load is high and toward the nest exit when it is low. Thus, the colony shapes the foragers trajectory by controlling her unloading rate, while she senses only her current food load. Using an agent-based model and mathematical analysis, we show that this simple mechanism robustly yields emergent regulation of foraging frequency. These findings demonstrate how the embedding of individuals in physical space can reduce their cognitive demands without compromising their computational role in the group.

animal behavior and cognition↗

bioRxiv2022

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. In a murine pneumonia model, KP mutants harbouring L3 insertions have a competitive disadvantage relative to a strain expressing wild-type OmpK36 in the absence of antibiotics. This explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies. Author summaryRapidly rising rates of antibiotic resistance among Klebsiella pneumoniae (KP) necessitate a comprehensive understanding of the diversity, spread and clinical impact of resistance mutations. In KP, mutations in outer membrane porins play an important role in mediating resistance to carbapenems, a key class of antibiotics. Here we show that resistance mutations in the extracellular loop 3 (L3) region of the OmpK36 porin are found at high prevalence among clinical genomes and we characterise their diversity and impact on resistance and virulence. They include amino acid insertions of Aspartate (D), Glycine-Aspartate (GD) and Threonine-Aspartate (TD), which act by decreasing the pore size and restricting entry of carbapenems into the bacterial cell. We show that these L3 insertions are associated with large clonal expansions of resistant lineages and impose only a low fitness cost. Critically, strains harbouring L3 insertions remain susceptible to novel drugs, including beta lactam-beta lactamase inhibitor combinations. This study highlights the importance of monitoring the emergence and spread of strains with OmpK36 L3 insertions for the control of resistant KP infections and provides crucial data for drug development and treatment strategies.

microbiology↗

bioRxiv2022

Recurrent emergence of carbapenem resistance in Klebsiella pneumoniae mediated by an inhibitory ompK36 mRNA secondary structure

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

microbiology↗

bioRxiv2020

OmpK36 and TraN facilitate conjugal transfer of the Klebsiella pneumoniae carbapenem resistance plasmid pKpQIL

We investigated the mechanism of conjugal transfer of the endemic Klebsiella pneumoniae carbapenem resistance plasmid, pKpQIL. Transfer efficiency of this plasmid was found to be dependent on the expression of the major outer membrane porin, OmpK36, in recipient cells. We also found that conjugal uptake is reduced in recipients expressing an OmpK36 isoform associated with the globally pervasive K. pneumoniae ST258 clade (OmpK36ST258). This reduction was attributed to a glycine-aspartate insertion in loop 3 of OmpK36ST258, which constricts the pore by 26%. Deletion of finO, which encodes an RNA-binding protein, derepressed transfer of pKpQIL and enabled visualisation of the conjugation pilus and OmpK36-dependent conjugation in real time. While deletion of traN abolished pKpQIL conjugation, substituting traN in pKpQIL with its homologue from R100-1 circumvented OmpK36 dependency. These results suggest that OmpK36 in recipient K. pneumoniae and the pKpQIL-encoded TraN in donor bacteria cooperate to facilitate plasmid transfer. This is the first report since 1998 to suggest a novel recipient cell receptor for IncF plasmid transfer and supports the idea that TraN mediates receptor specificity for plasmids belonging to this incompatibility group.

microbiology↗

bioRxiv2018

More than 16,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells

SignificanceLegionella pneumophila is a bacterial pathogen causing outbreaks of a lethal pneumonia. The genus Legionella comprises 65 species for which aquatic amoebae are the natural reservoirs. Using functional and comparative genomics to deconstruct the entire bacterial genus we reveal the surprising parallel evolutionary trajectories that have led to the emergence of human pathogenic Legionella. An unexpectedly large and unique repository of secreted proteins (>16,000) containing eukaryotic-like proteins acquired from all domains of life (plant, animal, fungal, archaea) is contrasting with a highly conserved type 4 secretion system. This study reveals an unprecedented environmental reservoir of bacterial virulence factors, and provides a new understanding of how reshuffling and gene-acquisition from environmental eukaryotic hosts, may allow for the emergence of human pathogens.\n\nAbstractThe bacterial genus Legionella comprises 65 species among, which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 16,000 secreted proteins encoding 137 different eukaryotic-like domains and more than 200 eukaryotic-like proteins is paired with a highly conserved T4SS. Specifically, we show that eukaryotic Rho and Rab GTPase domains are found nearly exclusively in eukaryotes and Legionella species. Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F/U-box and SET domains were present in >70% of all species suggesting that manipulation of host signal transduction, protein turnover and chromatin modification pathways, respectively are fundamental intracellular replication strategies for Legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by inter-domain gene transfer. Furthermore we revealed the surprising extent to which legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts and provides a new understanding of how dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens.

genomics↗