Lipid discovery enabled by sequence statistics and machine learning.

Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that Streptococcus agalactiae MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococcal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in Enterococcus , diglucosyl-diacylglycerol (Glc2-DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.

Formation and function of the meningeal arachnoid barrier around the developing mouse brain.

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-ß-catenin signaling and that constitutively active ß-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development.

Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with Infection.

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS that were isolated from blood cultures in patients with invasive infections and oral swabs of healthy volunteers and then determined the best-performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP was within a previously undescribed gene that was conserved across the majority of VGS species. Using the growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlighted the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. IMPORTANCE The viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases, such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole-genome sequencing for a suite of downstream analyses. We focused on a diverse sample of Streptococcus oralis genomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene, nrdM, which shared homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and was highly conserved among VGS. This work increased our knowledge of VGS genomics and indicated that differences in virulence potential among S. oralis isolates were, at least in part, genetically determined.

Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis.

Bacterial membrane lipids are critical for membrane bilayer formation, cell division, protein localization, stress responses, and pathogenesis. Despite their critical roles, membrane lipids have not been fully elucidated for many pathogens. Here, we report the discovery of a novel cationic glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is synthesized in high abundance by the bacterium Streptococcus agalactiae (Group B Streptococcus, GBS). To our knowledge, Lys-Glc-DAG is more positively charged than any other known lipids. Lys-Glc-DAG carries 2 positive net charges per molecule, distinct from the widely described lysylated phospholipid lysyl-phosphatidylglycerol (Lys-PG) that carries one positive net charge due to the presence of a negatively charged phosphate moiety. We use normal phase liquid chromatography (NPLC) coupled with electrospray ionization (ESI) high-resolution tandem mass spectrometry (HRMS/MS) and genetic approaches to determine that Lys-Glc-DAG is synthesized by the enzyme MprF in GBS, which covalently modifies the neutral glycolipid Glc-DAG with the cationic amino acid lysine. GBS is a leading cause of neonatal meningitis, which requires traversal of the endothelial blood-brain barrier (BBB). We demonstrate that GBS strains lacking mprF exhibit a significant decrease in the ability to invade BBB endothelial cells. Further, mice challenged with a GBSΔmprF mutant developed bacteremia comparably to wild-type (WT) infected mice yet had less recovered bacteria from brain tissue and a lower incidence of meningitis. Thus, our data suggest that Lys-Glc-DAG may contribute to bacterial uptake into host cells and disease progression. Importantly, our discovery provides a platform for further study of cationic lipids at the host-pathogen interface.

Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum.

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

Streptococcus pneumoniae, S. mitis, and S. oralis Produce a Phosphatidylglycerol-Dependent, ltaS-Independent Glycerophosphate-Linked Glycolipid.

Lipoteichoic acid (LTA) is a Gram-positive bacterial cell surface polymer that participates in host-microbe interactions. It was previously reported that the major human pathogen Streptococcus pneumoniae and the closely related oral commensals S. mitis and S. oralis produce type IV LTAs. Herein, using liquid chromatography/mass spectrometry-based lipidomic analysis, we found that in addition to type IV LTA biosynthetic precursors, S. mitis, S. oralis, and S. pneumoniae also produce glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a biosynthetic precursor of type I LTA. cdsA and pgsA mutants produce DHDAG but lack (Gro-P)-DHDAG, indicating that the Gro-P moiety is derived from phosphatidylglycerol (PG), whose biosynthesis requires these genes. S. mitis, but not S. pneumoniae or S. oralis, encodes an ortholog of the PG-dependent type I LTA synthase, ltaS By heterologous expression analyses, we confirmed that S. mitisltaS confers poly(Gro-P) synthesis in both Escherichia coli and Staphylococcus aureus and that S. mitisltaS can rescue the growth defect of an S. aureusltaS mutant. However, we do not detect a poly(Gro-P) polymer in S. mitis using an anti-type I LTA antibody. Moreover, Gro-P-linked DHDAG is still synthesized by an S. mitisltaS mutant, demonstrating that S. mitis LtaS does not catalyze Gro-P transfer to DHDAG. Finally, an S. mitisltaS mutant has increased sensitivity to human serum, demonstrating that ltaS confers a beneficial but currently undefined function in S. mitis Overall, our results demonstrate that S. mitis, S. pneumoniae, and S. oralis produce a Gro-P-linked glycolipid via a PG-dependent, ltaS-independent mechanism.IMPORTANCE The cell wall is a critical structural component of bacterial cells that confers important physiological functions. For pathogens, it is a site of host-pathogen interactions. In this work, we analyze the glycolipids synthesized by the mitis group streptococcal species, S. pneumoniae, S. oralis, and S. mitis We find that all produce the glycolipid, glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a precursor for the cell wall polymer type I lipoteichoic acid in other bacteria. We investigate whether the known enzyme for type I LTA synthesis, LtaS, plays a role in synthesizing this molecule in S. mitis Our results indicate that a novel mechanism is responsible. Our results are significant because they identify a novel feature of S. pneumoniae, S. oralis, and S. mitis glycolipid biology.

Phosphatidylcholine Biosynthesis in Mitis Group Streptococci via Host Metabolite Scavenging.

The mitis group streptococci include the major human pathogen Streptococcus pneumoniae and the opportunistic pathogens Streptococcus mitis and Streptococcus 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; for many pathogens, however, 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 in conjunction with stable isotope tracing, we determined that mitis group streptococci synthesize PC via a 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 membranes in response to the major human metabolites GPC and lysophosphatidylcholine.IMPORTANCE We 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, but their membrane biology is poorly understood. Here, we demonstrate that these streptococci scavenge major human metabolites and use them to synthesize the membrane phospholipid PC. 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 membranes in response to host metabolites.

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 µg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.