Biology subjects

de Bakker, V.

Publications and source records attributed to de Bakker, V..

2 recordsLinked to original sources

bioRxiv2021

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the {beta}-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

microbiology↗

bioRxiv2020

Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h post-infection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

microbiology↗