Biology subjects

Kelly, M. L.

Publications and source records attributed to Kelly, M. L..

4 recordsLinked to original sources

bioRxiv2023

A tale of two phage tails: Engineering the host range of bacteriophages infecting Clostridioides difficile

Clostridioides difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea across the globe. Although treatable with a restricted number of antibiotics, the emergence of resistant variants and high relapse rates necessitate alternative countermeasures. Phage therapy represents an attractive option. However, its implementation is handicapped by the narrow host specificity of the C. difficile bacteriophages isolated to date. One strategy to rationally expand phage host range would be to make appropriate modifications to the phage receptor binding protein (RBP). Here, we identify the tail fibre as the RBP of two Myoviridae phages, {Phi}CD1801 and {Phi}CD2301, which were previously isolated and propagated using the C. difficile strains CD1801 (RT078) and CD2301 (RT014), respectively. Contrary to studies into reprogramming the host ranges of phage of other bacterial other species, exchanging the tail fibre genes (tcf/tfp) alone between the two phage was insufficient to change host specificity. Rather, alterations to host range were dependent their exchange together with a putative chaperone encoded by hyp, localised adjacent to the tail fibre gene. Capitalising on this discovery, CRISPR/Cas9 was used to change the host range of one phage to that of the other by swapping the respective tcf/tfp and hyp genes. Significantly, one of the resulting mutants, surpassed both parental phages in terms of host range and efficiency of infection. This is the first time that genome engineering has successfully expanded the host range of a C. difficile phage, a prerequisite for implementing phage for the treatment of CDI. ImportanceAlternatives to antibiotics for treating Clostridioides difficile infection (CDI) are urgently required. Phage therapy presents an attractive option as it has the potential to clear the infection with minimal microbiome disruption and eliminate the possibility of recurrence. However, the C. difficile bacteriophages isolated to date have highly restricted host ranges. Moreover, rational strategies to alter specificity have till now been precluded as the identity of the phage receptor binding proteins involved was largely unknown. Here, we demonstrated that tail fibre proteins and an associated putative chaperone determine the host range of two Myoviridae phage. This enabled the alteration of specificity through CRISPR-mediated genome editing and the creation of a phage derivative with a host range and infection efficiency exceeding that of the parental phages. This is the first time that the host range of a C. difficile phage has been successfully expanded through rational genome engineering.

microbiology↗

bioRxiv2022

RNA conformational propensities determine cellular activity

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes 1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend not only on the strength of the contacts, but also on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities impact cellular activity. Here, we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

biophysics↗

bioRxiv2020

Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

Identifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

biochemistry↗

bioRxiv2019

Stabilizing non-native excited states as a therapeutic strategy for targeting RNA

Many regulatory RNAs undergo changes in their structure from the dominant ground-state (GS) toward short-lived low-abundance excited-states (ES) that reorganize local elements of secondary structure. ESs are increasingly observed in vitro and implicated in the folding and biological activities of regulatory RNAs and as targets for developing therapeutics. However, whether these ESs also form with comparable abundance within the complex cellular environment remains unknown. Here, we developed an approach for assessing the relative stability and abundance of RNA ESs within the functional cellular context. The approach uses point substitution mutations to increase the population of an inactive ES relative to the active GS. The cellular activity of such ES-stabilizing mutants then provides an indirect measure of any residual population of the active GS within the functional cellular context. Compensatory rescue mutations that restore the GS are used to control for changes in cellular activity arising due to changes in sequence. The approach is applied to probe ESs in two highly conserved and functionally important regulatory RNAs from HIV-1: the transactivation response element (TAR) and the Rev response element (RRE). For both RNAs, ES-stabilizing mutations inhibited cellular activity to a degree that correlates with the extent to which they stabilize the ES relative to the GS in vitro. These results indicate that the non-native ESs of TAR and RRE likely form in cells with abundances comparable to those measured in vitro and their targeted stabilization provides a new avenue for developing anti-HIV therapeutics.

biochemistry↗