Biology subjects

Palmer, K.

Publications and source records attributed to Palmer, K..

21 records · Page 2Linked to original sources

bioRxiv2019

Phosphatidylcholine biosynthesis in Mitis group streptococci via a host metabolite scavenging pathway

The Mitis group streptococci include the major human pathogen Streptococcus pneumoniae and the opportunistic pathogens S. mitis and S. oralis which are human oral cavity colonizers and agents of bacteremia and infective endocarditis in immunocompromised patients. Bacterial membrane lipids play crucial roles in microbe-host interactions, yet for many pathogens, the composition of the membrane is poorly understood. In this study, we characterized the lipidomes of selected species of Mitis group streptococci and investigated the mechanistic basis for biosynthesis of the phospholipid phosphatidylcholine (PC). PC is a major lipid in eukaryotic cellular membranes, but it is considered to be comparatively rare in bacterial taxa. Using liquid chromatography/mass spectrometry (LC/MS) in conjunction with stable isotope tracing, we determined that Mitis group streptococci synthesize PC via the rare host metabolite scavenging pathway, the glycerophosphocholine (GPC) pathway, which is largely uncharacterized in bacteria. Our work demonstrates that Mitis group streptococci including S. pneumoniae remodel their membrane in response to the major human metabolites GPC and lysoPC.\n\nImportanceWe lack fundamental information about the composition of the cellular membrane even for the best studied pathogens of critical significance for human health. The Mitis group streptococci are closely linked to humans in health and disease, yet their membrane biology is poorly understood. Here, we demonstrate that these streptococci scavenge major human metabolites and use them to synthesize the membrane phospholipid phosphatidylcholine. Our work is significant because it identifies a mechanism by which the major human pathogen S. pneumoniae and the primary human oral colonizers S. mitis and S. oralis remodel their membrane in response to host metabolites.

microbiology↗

bioRxiv2018

Spontaneous resistance to phage NPV1 confers increased daptomycin susceptibility in Enterococcus faecalis OG1RF

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the human gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis. In this report, we identified genetic determinants of E. faecalis resistance to {phi}NPV1. We found that loss-of-function mutations in epaR confer {phi}NPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the modification or loss of an extracellular polymer in strains with inactivated epaR. Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibilities. Our results demonstrate that in vitro spontaneous resistance to {phi}NPV1 comes at a cost in E. faecalis OG1RF.

microbiology↗

bioRxiv2017

A semi-lethal CRISPR-Cas system permits DNA acquisition in Enterococcus faecalis

Antibiotic resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack CRISPR-Cas genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be transiently maintained. Here, we investigate the basis for this \"CRISPR tolerance.\" We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not up-regulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient non-lethality and use this knowledge to develop a robust CRISPR-assisted genome editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.\n\nImportanceCRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently non-lethal. We uncover novel phenotypes associated with this \"CRISPR tolerance\" and, after determining its genetic basis, develop a genome editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.

microbiology↗