When the Host Encounters the Cell Wall and Vice Versa.

Peptidoglycan (PGN) and associated surface structures such as secondary polymers and capsules have a central role in the physiology of bacteria. The exoskeletal PGN heteropolymer is the major determinant of cell shape and allows bacteria to withstand cytoplasmic turgor pressure. Thus, its assembly, expansion, and remodeling during cell growth and division need to be highly regulated to avoid compromising cell survival. Similarly, regulation of the assembly impacts bacterial cell shape; distinct shapes enhance fitness in different ecological niches, such as the host. Because bacterial cell wall components, in particular PGN, are exposed to the environment and unique to bacteria, these have been coopted during evolution by eukaryotes to detect bacteria. Furthermore, the essential role of the cell wall in bacterial survival has made PGN an important signaling molecule in the dialog between host and microbes and a target of many host responses. Millions of years of coevolution have resulted in a pivotal role for PGN fragments in shaping host physiology and in establishing a long-lasting symbiosis between microbes and the host. Thus, perturbations of this dialog can lead to pathologies such as chronic inflammatory diseases. Similarly, pathogens have devised sophisticated strategies to manipulate the system to enhance their survival and growth.

Spatial regulation of protein A in Staphylococcus aureus.

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.

Streptococcus gordonii Type I Lipoteichoic Acid Contributes to Surface Protein Biogenesis.

Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii, we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii Similarly to Streptococcus suis, S. gordonii produced a complex type I LTA, decorated with multiple d-alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions.IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii, LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

Salt-Induced Stress Stimulates a Lipoteichoic Acid-Specific Three-Component Glycosylation System in Staphylococcus aureus.

Lipoteichoic acid (LTA) in Staphylococcus aureus is a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, including d-alanine and glycosyl groups, to alter cellular function. While the genetic determinants of d-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation in S. aureus We demonstrate here that S. aureus utilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und) N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-type S. aureus LTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenous d-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCE The role of glycosylation in the structure and function of Staphylococcus aureus lipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role of N-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As the dlt and glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreased d-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

Extraction and Analysis of Bacterial Teichoic Acids.

Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide gel electrophoresis (PAGE) method for both WTA and LTA. To extract crude WTA, the peptidoglycan sacculus is first isolated and WTA is then liberated by hydrolysis. LTA is extracted by 1-butanol and pre-treated with lipase to prevent aggregation and improve single-band resolution by PAGE. Crude extracts of both TAs are then subjected to PAGE followed by Alcian blue and silver staining. These protocols are easily adoptable by laboratories interested in rapidly analyzing TAs and can be used determine the relative abundance, relative polymer length and whether TAs are glycosylated. More detailed TA structural and compositional information can be obtained using the described purification protocols by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) analysis.

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans.

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD(+) consumers to resynthesize NAD(+), resulting in a reduction in global NAD(+) bioavailability. We manipulate NAD(+) levels to demonstrate that a minor deficit in NAD(+) availability is incompatible with a normal pace of gonad development. The NAD(+) deficit compromises NAD(+) consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD(+) biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD(+) salvage biosynthesis for the purposes of inhibiting tumor growth.