Lipoteichoic Acid Synthesis Research Articles

Lipoteichoic acid synthesis and function in gram-positive bacteria.

Lipoteichoic acid (LTA) is an important cell wall polymer found in gram-positive bacteria. Although the exact role of LTA is unknown, mutants display significant growth and physiological defects. Additionally, modification of the LTA backbone structure can provide protection against cationic antimicrobial peptides. This review provides an overview of the different LTA types and their chemical structures and synthesis pathways. The occurrence and mechanisms of LTA modifications with D-alanyl, glycosyl, and phosphocholine residues will be discussed along with their functions. Similarities between the production of type I LTA and osmoregulated periplasmic glucans in gram-negative bacteria are highlighted, indicating that LTA should perhaps be compared to these polymers rather than lipopolysaccharide, as is presently the case. Lastly, current efforts to use LTAs as vaccine candidates, synthesis proteins as novel antimicrobial targets, and LTA mutant strains as improved probiotics are highlighted.

Development of an integration mutagenesis system in Lactobacillus gasseri

Lactobacillus gasseri ATCC 33323 is a member of the acidophilus-complex group, microbes of human origin with significant potential for impacting human health based on niche-specific traits. In order to facilitate functional analysis of this important species, a upp-based counterselective chromosomal integration system was established and employed for targeting the lipoteichoic acid (LTA) synthesis gene, ltaS, in L. gasseri ATCC 33323. The ltaS gene encodes a phosphoglycerol transferase responsible for building the glycerol chain of LTA. No isogenic mutant bearing the deletion genotype was recovered, but an integration knockout mutant was generated with insertion inactivation at the ltaS locus. The ltaS deficient derivative exhibited an altered cellular morphology and significantly reduced ability to adhere to Caco-2 intestinal cell monolayers, relative to the wild-type parent strain.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus.

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. In Staphylococcus aureus it consists of a polyglycerolphosphate-chain that is retained within the membrane via a glycolipid. Using an immunofluorescence approach, we show here that the LTA polymer is not surface exposed in S. aureus, as it can only be detected after digestion of the peptidoglycan layer. S. aureus mutants lacking LTA are enlarged and show aberrant positioning of septa, suggesting a link between LTA synthesis and the cell division process. Using a bacterial two-hybrid approach, we show that the three key LTA synthesis proteins, YpfP and LtaA, involved in glycolipid production, and LtaS, required for LTA backbone synthesis, interact with one another. All three proteins also interacted with numerous cell division and peptidoglycan synthesis proteins, suggesting the formation of a multi-enzyme complex and providing further evidence for the co-ordination of these processes. When assessed by fluorescence microscopy, YpfP and LtaA fluorescent protein fusions localized to the membrane while the LtaS enzyme accumulated at the cell division site. These data support a model whereby LTA backbone synthesis proceeds in S. aureus at the division site in co-ordination with cell division, while glycolipid synthesis takes place throughout the membrane.

Listeria monocytogenes MDR transporters are involved in LTA synthesis and triggering of innate immunity during infection

Multi-drug resistance (MDR) transporters are known eponymously for their ability to confer resistance to various antimicrobial drugs. However, it is likely that this is not their primary function and that MDR transporters evolved originally to play additional roles in bacterial physiology. In Listeria monocytogenes a set of MDR transporters was identified to mediate activation of innate immune responses during mammalian cell infection. This phenotype was shown to be dependent on c-di-AMP secretion, but the physiological processes underlying this phenomenon were not completely resolved. Here we describe a genetic approach taken to screen for L. monocytogenes genes or physiological pathways involved in MDR transporter-dependent triggering of the type I interferon response. We found that disruption of L. monocytogenes lipoteichoic acid (LTA) synthesis results in enhanced triggering of type I interferon responses in infected macrophage cells yet does not impact bacterial intracellular growth. This innate immune response required the MDR transporters and could be recapitulated by exposing macrophage cells to culture supernatants derived from LTA mutant bacteria. Notably, we found that the MDR transporters themselves are required for full production of LTA, an observation that links MDR transporters to LTA synthesis for the first time. In light of our findings, we propose that the MDR transporters play a role in regulating LTA synthesis, possibly via c-di-AMP efflux, a physiological function in cell wall maintenance that triggers the host innate immune system.

Trehalose diesters, lipoteichoic acids and α-GalCer: using chemistry to understand immunology

It is now widely recognised that glycolipids can play an important role in the activation and modulation of the immune system. Seminal findings in this field included the discovery of ‘endotoxin’ from Gram-negative bacteria, lipoteichoic acids from Gram-positive bacteria and ‘cord factor’ or trehalose dimycolates from mycobacteria. In this mini-review, we present our latest developments on deciphering how glycolipids modulate the immune response through the synthesis and biological evaluation of trehalose glycolipids, lipoteichoic acids and derivatives of α-GalCer. By doing so, we illustrate how chemistry can interface with immunology to aid in the understanding of disease, and show that synthesis can provide a powerful molecular tool for studying the role of glycolipids in biological systems.

Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity

A strictly anaerobic Gram-stain-variable but positive by structure, non-spore-forming bacterium designated Lachnospiraceae bacterium ACC2 strain DSM 24645(T) was isolated from human subgingival dental plaque. Bacterial cells were 4-40 µm long non-motile rods, often swollen and forming curved filaments up to 200 µm. Cells contained intracellular, poorly crystalline, nanometre-sized iron- and sulfur-rich particles. The micro-organism was able to grow on yeast extract, trypticase peptone, milk, some sugars and organic acids. The major metabolic end-products of glucose fermentation were butyrate, lactate, isovalerate and acetate. The growth temperature and pH ranges were 30-42 °C and 4.9-7.5, respectively. Major fatty acids were C14 : 0, C14 : 0 DMA (dimethyl aldehyde), C16 : 0, C16 : 1ω7c DMA. The whole-cell hydrolysate contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. The DNA G+C content was calculated to be 55.05 mol% from the whole-genome sequence and 55.3 mol% as determined by HPLC. There were no predicted genes responsible for biosynthesis of respiratory lipoquinones, mycolic acids and lipopolysaccharides. Genes associated with synthesis of teichoic and lipoteichoic acids, diaminopimelic acid, polar lipids and polyamines were present. According to the 16S rRNA gene sequence phylogeny, strain DSM 24645(T) formed, together with several uncultured oral clones, a separate branch within the family Lachnospiraceae, with the highest sequence similarity to the type strain of Moryella indoligenes at 94.2 %. Based on distinct phenotypic and genotypic characteristics, we suggest that strain DSM 24645(T) represents a novel species in a new genus, for which the name Stomatobaculum longum gen. nov., sp. nov. is proposed. The type strain of Stomatobaculum longum is DSM 24645(T) ( = HM-480(T); deposited in BEI Resources, an NIH collection managed by the ATCC).

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

The current epidemic of infections caused by antibiotic-resistant gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with defects in lipoteichoic acid synthesis

Lipoteichoic acid (LTA) is an important cell envelope component of Gram-positive bacteria. Bacillus subtilis has four homologous genes for LTA synthesis: ltaS (yflE), yfnI, yqgS and yvgJ. The products LtaS (YflE), YfnI and YqgS are bona fide LTA synthetases, whereas YvgJ functions only as an LTA primase. To clarify whether defects in LTA on the cell envelope trigger extracytoplasmic function (ECF) sigma factors, mRNA levels of the autoregulated ECF sigma factors in cells with singly and multiply deleted alleles of the ltaS homologues were examined by real-time RT-PCR. This revealed that sigM and sigX were induced in cells with a null allele of ΔltaS and ΔyfnI, respectively, and that no ECF sigma factor was induced in cells with a single null allele of ΔyqgS or ΔyvgJ. In cells with double null alleles (ΔltaS and ΔyfnI), sigW and ylaC were induced in addition to sigM and sigX. Cells with triple null alleles (ΔltaS ΔyfnI and ΔyqgS) showed a pattern of induction similar to that of the double null. In cells with quadruple null alleles, sigV and sigY were newly induced. Cells with ΔltaS had approximately 1/4 the diglucosyldiacylglycerol and over 10 times the CDP-diacylglycerol of wild-type cells. Compensatory elevation of the mRNA level of other homologues was observed (in ΔltaS cells the level of yfnI was elevated; in ΔyfnI cells that of yqgS and yvgJ was elevated; both were even higher in ΔltaS ΔyfnI cells). In ΔltaS cells, the mRNA level of yfnI was corroborated to be regulated by σ(M), which is activated in the null mutant cells. In ΔyfnI cells, the mRNA levels of yqgS and yvgJ reverted to less than those of wild-type when a defective sigX allele was introduced. Since sigX was activated in cells with ΔyfnI, this suggests that the induction of yqgS and yvgJ is dependent on σ(X). The LTAs produced by the four ltaS homologues seem to play distinct physiological roles to maintain the full function of LTA on the B. subtilis cell envelope.

Synthesis of Lipoteichoic Acids in Bacillus anthracis

Lipoteichoic acid (LTA), a glycerol phosphate polymer, is a component of the envelope of Gram-positive bacteria that has hitherto not been identified in Bacillus anthracis, the causative agent of anthrax. LTA synthesis in Staphylococcus aureus and other microbes is catalyzed by the product of the ltaS gene, a membrane protein that polymerizes polyglycerol phosphate from phosphatidyl glycerol. Here we identified four ltaS homologues, designated ltaS1 to -4, in the genome of Bacillus anthracis. Polyglycerol phosphate-specific monoclonal antibodies were used to detect LTA in the envelope of B. anthracis strain Sterne (pXO1(+) pXO2(-)) vegetative forms. B. anthracis mutants lacking ltaS1, ltaS2, ltaS3, or ltaS4 did not display defects in growth or LTA synthesis. In contrast, B. anthracis strains lacking both ltaS1 and ltaS2 were unable to synthesize LTA and exhibited reduced viability, altered envelope morphology, aberrant separation of vegetative forms, and decreased sporulation efficiency. Expression of ltaS1 or ltaS2 alone in B. anthracis as well as in other microbes was sufficient for polyglycerol phosphate synthesis. Thus, similar to S. aureus, B. anthracis employs LtaS enzymes to synthesize LTA, an envelope component that promotes bacterial growth and cell division.

Biosynthesis of Teichoic Acids inStreptococcus pneumoniaeand Closely Related Species: Lessons from Genomes

The cell wall of Streptococcus pneumoniae contains an unusually complex wall teichoic acid (WTA), which has identical repeating units as the membrane-anchored lipoteichoic acid (LTA). Both polymers share a common cytoplasmic pathway of precursor synthesis, but several TA enzymes have remained elusive. Bioinformatic analysis of the genome of various pneumococcal strains, including choline-independent mutant strains, has allowed us to identify the missing TA genes. We present here the deduced complete pathways of WTA and LTA synthesis in S. pneumoniae and point to the variations occurring in different pneumococcal strains and in closely related species such as Streptococcus oralis and Streptococcus mitis.

Lipoteichoic Acid in Streptomyces hygroscopicus: Structural Model and Immunomodulatory Activities

Gram positive bacteria produce cell envelope macroamphiphile glycopolymers, i.e. lipoteichoic acids or lipoglycans, whose functions and biosynthesis are not yet fully understood. We report for the first time a detailed structure of lipoteichoic acid isolated from a Streptomyces species, i.e. Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T. Chemical, MS and NMR analyses revealed a polyglycerolphosphate backbone substituted with α-glucosaminyl and α-N-acetyl-glucosaminyl residues but devoid of any amino-acid substituent. This structure is very close, if not identical, to that of the wall teichoic acid of this organism. These data not only contribute to the growing recognition that lipoteichoic acid is a cell envelope component of Gram positive Actinobacteria but also strongly support the recently proposed hypothesis of an overlap between the pathways of lipoteichoic acid and wall teichoic acid synthesis in these bacteria. S. hygroscopicus lipoteichoic acid induced signalling by human innate immune receptor TLR2, confirming its role as a microbe-associated molecular pattern. Its activity was partially dependant on TLR1, TLR6 and CD14. Moreover, it stimulated TNF-α and IL-6 production by a human macrophage cell line to an extent similar to that of Staphylococcus aureus lipoteichoic acid. These results provide new clues on lipoteichoic acid structure/function relationships, most particularly on the role of the polyglycerolphosphate backbone substituents.

ChemInform Abstract: Chemical Synthesis of Bacterial Lipoteichoic Acids: An Insight on Its Biological Significance.

AbstractReview: ca. 80 refs.

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.

Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.

The <i>Bacillus subtilis</i> essential gene <i>dgkB</i> is dispensable in mutants with defective lipoteichoic acid synthesis

The dgkB gene is essential for the growth of Bacillus subtilis. It encodes a diacylglycerol (DG) kinase that converts DG to phosphatidic acid to reintroduce it into the phospholipid synthesis pathway. Repression of the dgkB gene placed under a regulatable promoter causes accumulation of DG and leads to lethality. DG is formed as a byproduct of the synthesis of lipoteichoic acid (LTA), a polyanionic component of the cell envelope. B. subtilis synthesizes LTA by polymerizing the glycerophosphate moiety of phosphatidylglycerol (PG) onto a glucolipid membrane anchor, and releasing the DG moiety of PG. B. subtilis has four genes homologous to Staphylococcus aureus ltaS, which encodes LTA synthase. Disruption of either or both of two genes, yflE and yfnI, whose products show higher homology with S. aureus LtaS among the four homologues, suppressed the lethality caused by dgkB repression. In cells with dgkB repression, DG was accumulated to 43 ± 3% of total lipids, about three times the content of wild type cells (13 ± 1%). Disruption of yfnI in the dgkB-repressed cells reduced the DG content to 15 ± 2%, but yflE-disruption did not (42 ± 1%); this was probably due to efficient LTA synthesis by YfnI in the yflE-disrupted cells. Further introduction of a disrupted allele of ugtP, encoding glucolipid synthase that consumes DG as a substrate, partially lowered the colony forming capacity in strains with yflE-disruption. A disrupted dgkB allele was successfully introduced into strains disrupted for either or both of yflE and yfnI, indicating that the essential gene dgkB is dispensable in mutants defective in LTA synthesis.

Chemical synthesis of bacterial lipoteichoic acids: An insight on its biological significance

During infections caused by Gram-negative bacteria, lipopolysaccharide (LPS, endotoxin) has a dominant role leading to fulminant pro-inflammatory reactions in the host. As there is no LPS in Gram-positive bacteria, other microbial cell wall components have been identified to be the causative agent for the pro-inflammatory activity since also Gram-positive bacterial infections lead to comparable clinical symptoms and reactions. On search for the "Gram-positive endotoxin" a widely accepted hypothesis has been raised in that the lipoteichoic acids (LTAs) serve as pathogen-associated molecular patterns (PAMPs) during Gram-positive sepsis, although the amount necessary for a pro-inflammatory in vitro response is several orders of magnitude higher than that for LPS. Therefore, LTA cannot be considered to be "the (endo)toxin of Gram-positive bacteria". Although LPS and LTA show structural relatedness (amphiphilic, negatively charged glycophospholipids), they are structurally quite different from each other and one might expect that they are also recognized by different receptors of the innate immune system, the so called toll-like receptors 4 and 2 (TLR4 and TLR2), respectively. Based on their chemical structure, the LTAs were classified into four types (type I-IV) of which we have carefully investigated the LTA of Staphylococcus aureus (type I), Lactococcus garvieae (type II) and Streptococcus pneumoniae (type IV). Hence, these LTAs have been synthesized in our group and biologically evaluated with respect to their potency to activate cytokines in transiently TLR2/CD14-transfected human endothelial kidney cells (HEK 293) or human macrophages and whole blood cells. Although LTA of type I and IV are structurally quite different, especially in their hydrophilic moiety, they originally were believed to interact with the same receptor (TLR2). Hence, the chemical syntheses leading to structurally defined, non-contaminated stimuli have a major impact on the outcome and interpretation of these biological studies of the innate immune system. With this material, it became evident that synthetic LTA from S. aureus and S. pneumoniae are not recognized by TLR2. Instead, another receptor of the innate immune system, the lectin pathway of the complement, known since many years to interact with LTA in quite a specific way, has gained increasing attractivity. With the help of synthetic LTA we obtained first evidences that this receptor is indeed the pathogen recognition receptor (PRR) for LTA.

Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes.

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaSBS, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn2+-dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaSBS, YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc2-DAG glycolipid. Western blot analysis of LTA produced by ltaSBS, yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaSBS produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis.

In Vitro Analysis of the Staphylococcus aureus Lipoteichoic Acid Synthase Enzyme Using Fluorescently Labeled Lipids

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. The key enzyme responsible for polyglycerolphosphate lipoteichoic acid synthesis in the Gram-positive pathogen Staphylococcus aureus is the membrane-embedded lipoteichoic acid synthase enzyme, LtaS. It is presumed that LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. Here we describe an in vitro assay for this new class of enzyme using PG with a fluorescently labeled fatty acid chain (NBD-PG) as the substrate and the recombinant soluble C-terminal enzymatic domain of LtaS (eLtaS). Thin-layer chromatography and mass spectrometry analysis of the lipid reaction products revealed that eLtaS is sufficient to cleave the glycerolphosphate head group from NBD-PG, resulting in the formation of NBD-diacylglycerol. An excess of soluble glycerolphosphate could not compete with the hydrolysis of the fluorescently labeled PG lipid substrate, in contrast to the addition of unlabeled PG. This indicates that the enzyme recognizes and binds other parts of the lipid substrate, besides the glycerolphosphate head group. Furthermore, eLtaS activity was Mn(2+) ion dependent; Mg(2+) and Ca(2+) supported only weak enzyme activity. Addition of Zn(2+) or EDTA inhibited enzyme activity even in the presence of Mn(2+). The pH optimum of the enzyme was 6.5, characteristic for an enzyme that functions extracellularly. Lastly, we show that the in vitro assay can be used to study the enzyme activities of other members of the lipoteichoic acid synthase enzyme family.

ChemInform Abstract: Chemical Synthesis of Lipoteichoic Acid and Derivatives

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Identification of Lipoteichoic Acid as a Ligand for Draper in the Phagocytosis of Staphylococcus aureus by Drosophila Hemocytes

Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. We searched for the Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. We propose that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection.