Biology subjects

Gibson, P. S.

Publications and source records attributed to Gibson, P. S..

4 recordsLinked to original sources

bioRxiv2024

Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae

Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to the synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways that are essential under fluoroquinolone stress and thus might represent novel targets to revitalize the use of this class of antibiotics, we performed genome-wide CRISPRi-seq screens to determine antibiotic-gene essentiality signatures. As expected, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, such as recA, recJ, recF, recO, rexAB and ruvAB. Surprisingly, we also found that specific downregulation of the gene encoding the histidine kinase liaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that deletion of liaS causes hyperphosphorylation of LiaR and subsequent upregulation of the LiaR regulon. RNA-seq was used to refine the LiaR regulon, highlighting the role of the heat-shock response and the pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin. This synergistic antibiotic combination restored sensitivity in fluoroquinolone-resistant strains in vitro. Importantly, bacitracin/levofloxacin combination therapy was also effective in vivo and improved the treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. Together, the approaches and findings presented here provides a starting point for identification and validation of potent combination therapies that could be used in the clinic to treat antibiotic-resistant pneumococcal infections.

microbiology↗

bioRxiv2024

PneumoBrowse 2: An integrated visual platform for curated genome annotation and multiomics data analysis of Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations, and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018; a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations, and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/606308v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1a38ce5org.highwire.dtl.DTLVardef@773428org.highwire.dtl.DTLVardef@1368aorg.highwire.dtl.DTLVardef@1a39f0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacteriums poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. Significance statementWhile GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

microbiology↗

bioRxiv2021

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to {beta}-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

microbiology↗