Acids Of Gram-positive Bacteria Research Articles

Lactoferrin: a secret weapon in the war against pathogenic bacteria

The excessive use of antibiotics to treat bacterial infectious diseases in all living beings has caused a global epidemic of bacterial resistance to antibiotics, leading to the emergence of multidrug-resistant and pandrug-resistant strains. In 2019, the World Health Organization (WHO) reported that antimicrobial resistance causes at least 700,000 deaths per year worldwide. Therefore, in this global war against microorganisms, a therapeutic alternative is necessary to help us win this battle. A key in this race against the clock could be lactoferrin (Lf), a cationic glycoprotein of the mammalian innate immune system that is highly conserved among mammals. Lf is a multifunctional glycoprotein with immunomodulatory, anticarcinogenic, wound-healing, antioxidant, antimicrobial, and bone regeneration properties, in addition to improving the gut microbiota. Lf limits the growth of microorganisms through the sequestration of iron but can also interact directly with some components of the outer membrane of Gram-negative bacteria or bind to teichoic acids in Gram-positive bacteria, destabilizing the membrane and resulting in lysis. Moreover, cleavage of the Lf molecule could promote the production of lactoferricins (Lfcins) and lactoferrampin (Lfampin) from the N-terminal end, which are known to often have stronger antimicrobial effects than the native molecule, as well as analogous peptides, such as HLopt2, which have also shown enhanced antimicrobial activity. Bovine Lf (bLf) has been approved by the US Food and Drug Administration (FDA), and the European Food Safety Authority for its use as a dietary supplement in food products. Because of its effectiveness, accessibility, low cost, and nontoxicity, Lf could be a promising alternative for preventing or treating infections in animals and humans.

A cell-free multi-enzyme cascade for the synthesis of CDP-glycerol.

CDP-glycerol is a nucleotide-diphosphate-activated version of glycerol. In nature, it is required for the biosynthesis of teichoic acid in Gram-positive bacteria, which is an appealing target epitope for the development of new vaccines. Here, a cell-free multi-enzyme cascade was developed to synthetize nucleotide-activated glycerol from the inexpensive and readily available substrates cytidine and glycerol. The cascade comprises five recombinant enzymes expressed in E. coli that were purified by immobilized metal affinity chromatography. As part of the cascade, ATP is in-situ regenerated from polyphosphate to reduce synthesis costs. The enzymatic cascade was characterized at laboratory scale and the products analyzed by high performance anion exchange chromatography (HPAEC)-UV and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After the successful synthesis was confirmed, a design of experiments approach was used to screen for optimal operation conditions (temperature, pH value and MgCl2 concentration). Overall, a substrate conversion of 89% was achieved with respect to the substrate cytidine.

Altering Microbiomes with Hydroxyapatite Nanoparticles: A Metagenomic Analysis.

Hydroxyapatite (HAp), the most abundant biological material among mammals, has been recently demonstrated to possess moderate antibacterial properties. Metagenomics provides a series of tools for analyzing the simultaneous interaction of materials with larger communities of microbes, which may aid in optimizing the antibacterial activity of a material such as HAp. Here, a microbiome intrinsic to the sample of sandy soil collected from the base of an African Natal plum (Carissa macrocarpa) shrub surrounding the children’s sandbox at the Arrowhead Park in Irvine, California was challenged with HAp nanoparticles and analyzed with next-generation sequencing for hypervariable 16S ribosomal DNA base pair homologies. HAp nanoparticles overwhelmingly reduced the presence of Gram-negative phyla, classes, orders, families, genera and species, and consequently elevated the relative presence of their Gram-positive counterparts. Thermodynamic, electrostatic and chemical bonding arguments were combined in a model proposed to explain this selective affinity. The ability of amphiphilic surface protrusions of lipoteichoic acid in Gram-positive bacteria and mycolic acid in mycobacteria to increase the dispersibility of the bacterial cells and assist in their resistance to capture by the solid phase is highlighted. Within the Gram-negative group, the variability of the distal, O-antigen portion of the membrane lipopolysaccharide was shown to be excessive and the variability of its proximal, lipid A portion insufficient to explain the selectivity based on chemical sequence arguments. Instead, flagella-driven motility proves to be a factor favoring the evasion of binding to HAp. HAp displayed a preference toward binding to less pathogenic bacteria than those causative of disease in humans, while taxa having a positive agricultural effect were largely captured by HAp, indicating an evolutionary advantage this may have given it as a biological material. The capacity to selectively sequester Gram-negative microorganisms and correspondingly alter the composition of the microbiome may open up a new avenue in environmental and biomedical applications of HAp.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

Lipids as Biomarkers of Cancer and Bacterial Infections.

Lipids are ubiquitous molecules, known to play important roles in various cellular processes. Alterations to the lipidome can therefore be used as a read-out of the signs of disease, highlighting the importance to consider lipids as biomarkers in addition of nucleic acid and proteins. Lipids are among the primary structural and functional constituents of biological tissues, especially cell membranes. Along with membrane formation, lipids play also a crucial role in cell signalling, inflammation and energy storage. It was shown recently that lipid metabolism disorders play an important role in carcinogenesis and development. As well, the role of lipids in disease is particularly relevant for bacterial infections, during which several lipid bacterial virulence factors are recognized by the human innate immune response, such as lipopolysaccharide in Gram-negative bacteria, lipoteichoic acid in Gram-positive bacteria, and lipoglycans in mycobacteria. Compared to nucleic acids and proteins, a complete analysis of the lipidome, which is the comprehensive characterization of different lipid families, is usually very challenging due to the heterogeneity of lipid classes and their intrinsic physicoproperties caused by variations in the constituents of each class. Understanding the chemical diversity of lipids is therefore crucial to understanding their biological relevance and, as a consequence, their use as potential biomarkers for non-infectious and infectious diseases. This mini-review exposes the current knowledge and limitations of the use of lipids as biomarkers of the top global killers which are cancer and bacterial infections.

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-glucose-1-phosphate ( kcat = 1.1 s-1, Km = 1.05 mM, and kcat/ Km = 1.12 × 103 M-1 s-1). Additionally, it was also shown that in the presence of β-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-d-glucose-1-phosphate ( kcat = 21 s-1, Km = 18 μM, and kcat/ Km = 1.1 × 106 M-1 s-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-d-allose-1-phosphate, β-d-mannose-1-phosphate, and β-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a kcat = 2.1 s-1, Km = 69 μM, and kcat/ Km = 3.1 × 104 M-1 s-1.

The Central Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Septic Pathogen Lipid Transport and Clearance.

Microbial cell walls contain pathogenic lipids, including LPS in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and phospholipomannan in fungi. These pathogen lipids are major ligands for innate immune receptors and figure prominently in triggering the septic inflammatory response. Alternatively, pathogen lipids can be cleared and inactivated, thus limiting the inflammatory response. Accordingly, biological mechanisms for sequestering and clearing pathogen lipids from the circulation have evolved. Pathogen lipids released into the circulation are initially bound by transfer proteins, notably LPS binding protein and phospholipid transfer protein, and incorporated into high-density lipoprotein particles. Next, LPS binding protein, phospholipid transfer protein, and other transfer proteins transfer these lipids to ApoB-containing lipoproteins, including low-density (LDL) and very-low-density lipoproteins and chylomicrons. Pathogen lipids within these lipoproteins and their remnants are then cleared from the circulation by the liver. Hepatic clearance involves the LDL receptor (LDLR) and possibly other receptors. Once absorbed by the liver, these lipids are then excreted in the bile. Recent evidence suggests pathogen lipid clearance can be modulated. Importantly, reduced proprotein convertase subtilisin/kexin type 9 activity increases recycling of the LDLR and thereby increases LDLR on the surface of hepatocytes, which increases clearance by the liver of pathogen lipids transported in LDL. Increased pathogen lipid clearance, which can be achieved by inhibiting proprotein convertase subtilisin/kexin type 9, may decrease the systemic inflammatory response to sepsis and improve clinical outcomes.

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.

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.

DltA gene mutation in the teichoic acids alanylation system of Lactococcus garvieae results in diminished proliferation in its natural host

The dltABCD cluster is involved in the d-alanylation of teichoic acids in gram positive bacteria. In order to determine the role of this alanylation in the physiology and virulence of Lactococcus garvieae, a previously isolated dltAΔTn 917 signature tagged mutagenesis (STM) clone was analyzed. RT-PCR results revealed that dltABCD genes form an operon. No major differences could be established between the parental and mutant strains with respect to growth rate, autolytic properties, and susceptibility to acid conditions, lysozyme treatment, anionic detergents, or oxidant agents. However, the dltA mutant was more susceptible to nisin than the parental strain, with minimum inhibitory concentration (MIC) values of 8 and 16 μg/ml, respectively. Less proliferation of the mutant was observed in in vivo competence index experiments (CI = 0.08). Furthermore, the mutant strain had a 50% lethal dose (LD 50) 3-fold that of the parental strain. These results, together with the fact that the dltAΔTn 917 mutant was isolated as a STM clone, reveal that the dltA locus of Lactococcus garvieae is required for full growth and pathogenesis on rainbow trout.

Identification of the initial binding sites of α s2-casein f(183–207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α s2-casein f(183–207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α s2-casein f(183–207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α s2-casein peptide f(183–207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183–207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.

Toll-like Receptors 2 and 4 and Their Mutations in Patients with Otitis Media and Middle Ear Effusion

ObjectivesToll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion.MethodsMiddle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis.ResultsAmong the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20±60.94 ng/mL, 21.59±7.96 ng/mL and 11.55±16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile).ConclusionTLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.

YerQ is a soluble diacylglycerol kinase in Bacillus subtilis required for lipoteichoic acid synthesis

The biosynthesis of lipoteichoic acid in Gram-positive bacteria involves the sequential transfer of multiple glycerophosphate units from phosphatidylglycerol to the glycolipid anchor with the concomitant release of sn-1,2-diacylglycerol (DAG). DAG is reintroduced into the phospholipid biosynthetic pathway by DAG kinase. This reaction is carried out by an integral membrane protein encoded by the dgkA gene in E. coli; however, the related dgkA gene of Bacillus subtilis was not a DAG kinase. There are three B. subtilis genes, yerQ, bmrU and ytlR, that belong to the DAG kinase protein family established based on the similarities between the soluble mammalian DAG kinases (Pfam00781). Only yerQ complemented the DAG kinase deficiency in the E. coli dgkA mutant. YerQ was essential in B. subtilis and a conditional yerQ knockout strain was constructed that expressed yerQ under the control of the Pspac promoter. Removal of the inducer resulted in the cessation of growth, the accumulation of DAG, a reduction in the levels of phosphatidylglycerol and lysylphosphatidylglycerol, and the termination of lipoteichoic acid formation. YerQ was purified and possessed ATP:Mg2+-dependent DAG kinase activity in vitro. Thus, B. subtilis has a soluble DAG kinase that is more closely related to the mammalian DAG kinase catalytic cores than to the prototypical, membrane-bound E. coli DgkA.

A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli 'lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.

A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria.

Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis.

New structures of the O-specific polysaccharides of bacteria of the genus Proteus. 1. Phosphate-containing polysaccharides.

The O-specific polysaccharide chains (O-antigens) of the lipopolysaccharides of five Proteus strains, P. vulgaris O17, P. mirabilis O16 and O33, and P. penneri 31and 103, were found to contain phosphate groups that link the non sugar components, e.g., ethanolamine and ribitol. The polysaccharides of P. mirabilis O16 and P. penneri 103 include ribitol phosphate in the main chain and thus resemble ribitol teichoic acids of Gram-positive bacteria. The structures of the polysaccharides were elucidated using NMR spectroscopy, including two-dimensional 1H,1H correlation spectroscopy (COSY and TOCSY), nuclear Overhauser effect spectroscopy (NOESY or ROESY), and H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence spectroscopy (HMQC), along with chemical methods. The structures determined are unique among the bacterial polysaccharides and, together with the data obtained earlier, represent the chemical basic for classification of Proteus strains. Based on structural similarities of the O-specific polysaccharides and serological relationships between the O-antigens, we propose to extend Proteus serogroups O17 and O19 by including P. penneri strains 16 and 31,respectively.

The role of immunostimulatory CpG-DNA in septic shock.

Septic shock is the most severe manifestation of infection, and in the intensive care unit (ICU) appears increasingly commonly (reviewed in [20]). As in other forms of acute circulatory failure, septic shock is best described as an imbalance between oxygen demand and oxygen delivery [18]. Older studies suggested that gram-negative bacteria are more commonly associated with septic shock; however, more recent studies have shown that gram-positive bacteria are just as common. Septic shock develops when specific microbial components gain access to the circulation and are recognized by the immune system which generates exaggerated mediator and cytokine responses. Cell wall constituents, such as LPS in gram-negative bacteria, or peptidoglycan and teichoic acid in gram-positive bacteria, or proteins liberated during growth (exotoxins) are considered as the principal microbial components responsible. Exotoxins are a group of bacterial proteins that have been termed superantigens (Sag) because of their unique mechanism to interact with antigen-presenting cells (APCs) and T lymphocytes [14]. All Sag share the ability to bind to MHC class II molecules outside the peptide binding groove as intact proteins. Once bound to MHC class II molecules Sag bind to certain regions of the T cell receptor (TCR) β chain encoded by Vβ gene segments. Consequently a trimolecular TCR-Sag-MHC complex is formed which causes cross-linking and immobilization of the TCR and activation of the respective T cells [17]. Finally, pore-forming bacterial exotoxins may also contribute to the pathogenesis of sepsis syndrome and septic shock [1].

Synthesis and reactivity of the monosaccharide esters of amino acids as models of teichoic acid fragment.

The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates further evaluation of the basic assumptions about the molecular pathogenesis of septic shock. Since diverse physiological functions of gram-positive bacteria are controlled by the degree of esterification of teichoic acids with D-alanine, we examined the reactivity of monosaccharide esters in which anomerically free or protected D-glucose is linked through its C-6 hydroxy group to either phenylalanyl or tyrosyl residues as models for teichoic acid fragment. We show that the attached sugar moiety induces activation of the amino acid residue. Due to the enhanced reactivity of the NH2 group in the monosaccharide esters studied, the formation of products generated by intramolecular and intermolecular glycation reactions is accelerated resulting in heterogeneous mixture of compounds. These findings suggest that, if similar adducts are formed by glycation of D-alanine in teichoic acid of gram-positive bacteria, they should be examined as potential bioactive ligands or chemical message for infection.

Pathogenic mechanisms induced by microbial proteases in microbial infections.

Most bacterial and fungal proteases excreted into infected hosts exhibit a wide range of pathogenic potentials ranging from pain, edema or even shock to translocation of bacteria from the site of infection into systemic circulation, thus resulting in septicemia. The basic mechanism or principle common to all these phenomena is explained by kinin generation, either directly from high- and/or low-molecular weight kininogens or indirectly via activation of the bradykinin generating cascade: i.e. Hageman factor-->activated Hageman factor-->prekallikrein-->kallikrein-->high-molecular weight kininogen-->bradykinin. Some bacterial proteases are also involved in activation of other host protease zymogens such as plasminogen, procollagenase (matrix metallo proteases) and proenzymes of the clotting system. Furthermore, most bacterial proteases are not only resistant to plasma protease inhibitors of the hosts, most of which belong to a group of serine protease inhibitors called serpins (serine protease inhibitors), but they also quickly inactivate serpins. Some bacterial proteases may also activate bacterial toxins thus rendering toxigenic pathogenesis. They are also capable of degrading immunoglobulins and components of the complement system and facilitate propagation of micro organisms. All in all, microbial proteases are very critical in enhancing pathogenesis of severe diseases. It is also noteworthy that bacterial cell wall components themselves, i.e. endotoxin (or lipopolysaccharide) of gram negative bacteria and teichoic/lipoteichoic acid of gram positive bacteria, are also able to activate the bradykinin generating cascade-involving activation of Hageman factor as mentioned above.

A 28 kDa protein of normal mouse serum binds lipopolysaccharides of Gram-negative and lipoteichoic acids of Gram-positive bacteria

A 28 kDa protein from normal mouse serum known to bind to the inner core region of bacterial lipopolysaccharide (LPS) was found to bind also to bacterial poly(glycerophosphate) lipoteichoic acid (LTA). Twenty-nine preparations of LTA were isolated from 19 different bacterial species, purified, chemically analysed, and tested for their ability to bind the 28 kDa protein in a complement-dependent hemolysis and hemolysis inhibition assay. All but one were active in one or both systems and one half of the preparations were active in both. Reactivity patterns were not strictly correlated with the chemical structure of LTA considering the substitution of the poly(glycerophosphate) chain with alanine ester and glycosyl residues and the type of lipid anchor. The isolated lipid anchor alone was unable to bind the serum factor. Comparing the binding to LTA and LPS from Acinetobacter calcoaceticus indicated complete cross-reactivity of LTA and LPS in various serological approaches. Thus, LPS and LTA which are unique amphiphiles in Gram-negative and Gram-positive bacteria, respectively, share a similar function in terms of binding the 28 kDa mouse serum protein.