Successful therapeutic intervention in new mouse models of frizzled 2-associated congenital malformations.

Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.

Shared biophysical mechanisms determine early biofilm architecture development across different bacterial species.

Bacterial biofilms are among the most abundant multicellular structures on Earth and play essential roles in a wide range of ecological, medical, and industrial processes. However, general principles that govern the emergence of biofilm architecture across different species remain unknown. Here, we combine experiments, simulations, and statistical analysis to identify shared biophysical mechanisms that determine early biofilm architecture development at the single-cell level, for the species Vibrio cholerae, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa grown as microcolonies in flow chambers. Our data-driven analysis reveals that despite the many molecular differences between these species, the biofilm architecture differences can be described by only 2 control parameters: cellular aspect ratio and cell density. Further experiments using single-species mutants for which the cell aspect ratio and the cell density are systematically varied, and mechanistic simulations show that tuning these 2 control parameters reproduces biofilm architectures of different species. Altogether, our results show that biofilm microcolony architecture is determined by mechanical cell-cell interactions, which are conserved across different species.

Identification of novel genetic factors that regulate c-di-AMP production in Staphylococcus aureus using a riboswitch-based biosensor.

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR (cdaR) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen Staphylococcus aureus. In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in S. aureus. We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant S. aureus strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance.

Targeting the phosphatidylglycerol lipid: An amphiphilic dendrimer as a promising antibacterial candidate.

The rapid emergence and spread of multidrug-resistant bacterial pathogens require the development of antibacterial agents that are robustly effective while inducing no toxicity or resistance development. In this context, we designed and synthesized amphiphilic dendrimers as antibacterial candidates. We report the promising potent antibacterial activity shown by the amphiphilic dendrimer AD1b, composed of a long hydrophobic alkyl chain and a tertiary amine-terminated poly(amidoamine) dendron, against a panel of Gram-negative bacteria, including multidrug-resistant Escherichia coli and Acinetobacter baumannii. AD1b exhibited effective activity against drug-resistant bacterial infections in vivo. Mechanistic studies revealed that AD1b targeted the membrane phospholipids phosphatidylglycerol (PG) and cardiolipin (CL), leading to the disruption of the bacterial membrane and proton motive force, metabolic disturbance, leakage of cellular components, and, ultimately, cell death. Together, AD1b that specifically interacts with PG/CL in bacterial membranes supports the use of small amphiphilic dendrimers as a promising strategy to target drug-resistant bacterial pathogens and addresses the global antibiotic crisis.

Quantitative image analysis of microbial communities with BiofilmQ.

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.