Biology subjects

Starrs, L.

Publications and source records attributed to Starrs, L..

6 recordsLinked to original sources

bioRxiv2025

Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a

The trans cleavage activity of type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas12a system has been widely used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers, and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions of the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficiency. Overall this study provides a blueprint for future rational engineering of Cas12a nucleases for their trans cleavage activities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/684486v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@6de4aeorg.highwire.dtl.DTLVardef@c06f37org.highwire.dtl.DTLVardef@1c5c1d0org.highwire.dtl.DTLVardef@375002_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

CRISPR-Cas12a REC2 - NUC interactions drive target-strand cleavage and constrain trans cleavage.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis, after which it remains catalytically active and non-specifically cleaves ssDNA in trans. Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied NUC loop, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/644851v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@18c702aorg.highwire.dtl.DTLVardef@11816d1org.highwire.dtl.DTLVardef@822316org.highwire.dtl.DTLVardef@1cda6bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

GBP1 promotes killing of multidrug-resistant Acinetobacter baumannii and promotes host protection via interferon signalling and inflammasome activation.

Multidrug resistant (MDR) Acinetobacter baumannii are of major concern worldwide due to their resistance to last resort carbapenem and polymyxin antibiotics. To develop an effective treatment strategy, it is critical to better understand how an A. baumannii MDR bacterium interacts with its mammalian host. Pattern-recognition receptors sense microbes, and activate the inflammasome pathway, leading to pro-inflammatory cytokine production and programmed cell death. Here, we found that MDR A. baumannii activate the NLRP3 inflammasome complex predominantly via the non-canonical caspase-11-dependent pathway. We show that caspase-11-deficient mice are protected from a virulent MDR A. baumannii strain by maintaining a balance between protective and deleterious inflammation via IL-1. Caspase-11-deficient mice also compromise between effector cell recruitment, phagocytosis, and programmed cell death in the lung during infection. Importantly, we found that cytosolic immunity - mediated by guanylate-binding protein 1 (GBP1) and type I interferon signalling - orchestrates caspase-11-dependent inflammasome activation. This exerts a bactericidal activity against carbapenem- and colistin-resistant, lipooligosaccharide (LOS)- deficient bacteria. Together, our results suggest that developing therapeutic strategies targeting GBP1 might pave the way as a host-directed therapy to overcome multidrug resistance in A. baumannii infection.

microbiology↗

bioRxiv2022

Differential activation of NLRP3 inflammasome by Acinetobacter baumannii strains

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the hosts innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death pathway in primary macrophages and mice. We found a striking variability in survival outcomes of mice and bacterial dissemination in organs among three ATCC A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found a stark contrast in activation of the NLRP3 inflammasome pathway, the non-canonical caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokines IL-1{beta} and IL-18 between A. baumannii strains. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with differential virulence to investigate the innate immune response to A. baumannii infection.

microbiology↗

bioRxiv2020

Exploring heteroaromatic rings as a replacement for the labile amide of antiplasmodial pantothenamides

The Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although we show here that the two compounds fail to suppress proliferation of P. berghei in vivo, pharmacokinetic and contact time data presented provide a benchmark for the compound profile required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

pharmacology and toxicology↗

bioRxiv2019

Host porphobilinogen deaminase deficiency confers malaria resistance in Plasmodium chabaudi but not in Plasmodium falciparum or Plasmodium berghei during intraerythrocytic growth

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel nonsense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited approximately 50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was approximately 10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection. IMPORTANCEThe causative agent of malaria, Plasmodium, adopts a parasitic lifestyle during erythrocyte infection, and as such relies on host cell factors for its survival and growth. Host-encoded mutations that alter the availability of these factors confer disease resistance, including several well-known genetic erythrocyte abnormalities that have arisen due to the historical evolutionary pressure of malaria. This study identified in mice a novel malaria resistance-conferring host mutation in the heme biosynthesis enzyme, porphobilinogen deaminase (PBGD), and compared the relative requirements by Plasmodium for the host versus parasite-encoded forms of PBGD in both in vivo and in vitro settings. The findings demonstrated that parasite PBGD was dispensable, but that the host enzyme was important specifically during in vivo infection by P. chabaudi, and collectively suggest that Plasmodium requires a certain threshold of the enzyme to sustain its intraerythrocytic growth. Plasmodium may therefore be vulnerable to other interventions that limit host PBGD activity.

microbiology↗