Binding of albumin promotes bacterial survival at the epithelial surface

Human serum albumin (HSA) is the dominating protein in human plasma. Many bacterial species, especially streptococci, express surface proteins that bind HSA with high specificity and affinity, but the biological consequences of these protein-protein interactions are poorly understood. Group G streptococci (GGS), carrying the HSA-binding protein G, colonize the skin and the mucosa of the upper respiratory tract, mostly without causing disease. In the case of bacterial invasion, pro-inflammatory cytokines are released that activate the epithelium to produce antibacterial peptides, in particular the chemokine MIG/CXCL9. In addition, the inflammation causes capillary leakage and extravasation of HSA and other plasma proteins, environmental changes at the epithelial surface to which the bacteria need to respond. In this study, we found that GGS adsorbed HSA from both saliva and plasma via binding to protein G and that HSA bound to protein G bound and inactivated the antibacterial MIG/CXCL9 peptide. Another surface protein of GGS, FOG, was found to mediate adherence of the bacteria to pharyngeal epithelial cells through interaction with glycosaminoglycans. This adherence was not affected by activation of the epithelium with a combination of IFN-γ and TNF-α, leading to the production of MIG/CXCL9. However, at the activated epithelial surface, adherent GGS were protected against killing by MIG/CXCL9 through protein G-dependent HSA coating. The findings identify a previously unknown bacterial survival strategy that helps to explain the evolution of HSA-binding proteins among bacterial species of the normal human microbiota.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes

Fibronectin-binding surface proteins are found in many bacterial species. Most strains of Streptococcus pyogenes, a major human pathogen, express the fibronectin-binding protein F1, which promotes bacterial adherence to and entry into human cells. In this study, the role of fibronectin in S. pyogenes virulence was investigated by introducing the protein F1 gene in an S. pyogenes strain lacking this gene. Furthermore, transgenic mice lacking plasma fibronectin were used to examine the relative contribution of plasma and cellular fibronectin to S. pyogenes virulence. Unexpectedly, protein F1-expressing bacteria were less virulent to normal mice, and virulence was partly restored when these bacteria were used to infect mice lacking plasma fibronectin. Dissemination to the spleen of infected mice was less efficient for fibronectin-binding bacteria. These bacteria also disseminated more efficiently in mice lacking plasma fibronectin, demonstrating that plasma fibronectin bound to the bacterial surface downregulates S. pyogenes virulence by limiting bacterial spread. From an evolutionary point of view, these results suggest that reducing virulence by binding fibronectin adds selective advantages to the bacterium.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Proteomic analysis and identification of Streptococcus pyogenes surface-associated proteins

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.     

Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids.

The primary ligands of human serum albumin (HSA), an abundant plasma protein, are non-esterified fatty acids. In vivo, the majority of fatty acids associated with the protein are unsaturated. We present here the first high-resolution crystal structures of HSA complexed with two important unsaturated fatty acids, the monounsaturated oleic acid (C18:1) and the polyunsaturated arachidonic acid (C20:4). Both compounds are observed to occupy the seven binding sites distributed across the protein that are also bound by medium and long-chain saturated fatty acids. Although C18:1 fatty acid binds each site on HSA in a conformation almost identical with that of the corresponding saturated compound (C18:0), the presence of multiple cis double bonds in C20:4 induces distinct binding configurations at some sites. The observed restriction on binding configurations plausibly accounts for differences in the pattern of binding affinities for the primary sites between polyunsaturated fatty acids and their saturated or monounsaturated counterparts.     

Bilirubin,model membranes and serum albumin interaction: The influence of fatty acids

Electronic circular dichroism (ECD), absorption and fluorescence spectroscopy were used to study the enantioselective interactions which involved bilirubin (BR), liposomes, human serum albumin of two different purities, pure (HSA) and non-purified of fatty acids (FA-HSA), and individual fatty acids.The application of the ECD technique to such a complex problem provided a new perspective on the BR binding to liposomes. Our results demonstrated that in the presence of pure HSA, BR preferred to bind to the protein over the liposomes. However, in the presence of FA-HSA, BR significantly bound to the liposomes composed either of DMPC or of sphingomyelin and bound only moderately to the primary and secondary binding sites of FA-HSA even at high BR concentrations. For the DMPC liposomes, even a change of BR conformation upon binding to the primary binding site was observed. The individual saturated fatty acids influenced the BR binding to HSA and liposomes in a similar way as fatty acids from FA-HSA. The unsaturated fatty acids interacted with BR alone and prevented it from interacting with either 99-HSA or the liposomes. In the presence of arachidonic acid, BR interacted enantioselectively with the liposomes and only moderately with 99-HSA.Hence, our results show a substantial impact of the liposomes on the BR binding to HSA. As a consequence of the existence of fatty acids in the blood plasma and in the natural structure of HSA, BR may possibly bind to the cell membranes even though it is normally bound to HSA.     

The use of formic acid in carrier gas : Rapid method for identification and determination of phytanic acid by gas chromatography—mass spectrometry—chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography—mass spectrometry; fatty acids (C₁ to C_16:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M − 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures

Human serum albumin (HSA) is one of the most abundant proteins in the circulatory system and plays a key role in the transport of fatty acids, metabolites, and drugs. For many drugs, binding to serum albumin is a critical determinant of their distribution and pharmacokinetics; however, there have as yet been no high resolution crystal structures published of drug-albumin complexes. Here we describe high resolution crystal structures of HSA with two of the most widely used general anesthetics, propofol and halothane. In addition, we describe a crystal structure of HSA complexed with both halothane and the fatty acid, myristate. We show that the intravenous anesthetic propofol binds at two discrete sites on HSA in preformed pockets that have been shown to accommodate fatty acids. Similarly we show that the inhalational agent halothane binds (at concentrations in the pharmacologically relevant range) at three sites that are also fatty acid binding loci. At much higher halothane concentrations, we have identified additional sites that are occupied. All of the higher affinity anesthetic binding sites are amphiphilic in nature, with both polar and apolar parts, and anesthetic binding causes only minor changes in local structure.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that is responsible for the transport of fatty acids. HSA also binds and perturbs the pharmacokinetics of a wide range of drug compounds. Binding studies have revealed significant interactions between fatty acid and drug-binding sites on albumin but high-resolution structural information on ligand binding to the protein has been lacking. We report here a crystallographic study of five HSA-fatty acid complexes formed using saturated medium-chain and long-chain fatty acids (C10:0, C12:0, C14:0, C16:0 and C18:0). A total of seven binding sites that are occupied by all medium-chain and long-chain fatty acids have been identified, although medium-chain fatty acids are found to bind at additional sites on the protein, yielding a total of 11 distinct binding locations. Comparison of the different complexes reveals key similarities and significant differences in the modes of binding, and serves to rationalise much of the biochemical data on fatty acid interactions with albumin. The two principal drug-binding sites, in sub-domains IIA and IIIA, are observed to be occupied by fatty acids and one of them (in IIIA) appears to coincide with a high-affinity long-chain fatty acid binding site.     

A method for the estimation of bacterial biomass and community structure in mangrove-associated sediments

Many recent reports have proposed that certain monocarboxylic fatty acids found in sediments originate in the in situ bacterial population. In this study we have divided the acids derived from bacteria into nine subgroups, each characteristic of a distinct compositional group of bacteria. It is proposed that the abundance of selected marker acids from each bacterial subgroup (chemotype) can be used to estimate the biomass of that chemotype. Conversion factors from acid abundance to bacterial biomass have been estimated using literature data. Since this procedure results in nine biomass parameters, bacterial communities can be compared in terms of both total biomass and chemotype distribution, that is, biomass and community structure. The ability of this procedure to resolve community structure variations is illustrated with the interpretation of the fatty acid profiles of a spatially distributed set of mangrove-associated sediments.     

Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

Virulent aggregates of Streptococcus pyogenes are generated by homophilic protein-protein interactions

Many strains of the important human pathogen Streptococcus pyogenes form aggregates when grown in vitro in liquid medium. The present studies demonstrate that this property is crucial for the adherence, the resistance to phagocytosis and the virulence of S. pyogenes. A conserved sequence of 19 amino acid residues (designated AHP) was identified in surface proteins of common S. pyogenes serotypes. This sequence was found to promote bacterial aggregation through homophilic protein-protein interactions between AHP-containing surface proteins of neighbouring bacteria. A synthetic AHP peptide inhibited S. pyogenes aggregation, reduced the survival of S. pyogenes in human blood and attenuated its virulence in mice. In contrast, mutant bacteria devoid of surface proteins containing AHP-related sequences did not aggregate or adhere to epithelial cells. These bacteria are also rapidly killed in human blood and show reduced virulence in mice, underlining the pathogenic significance of the AHP sequence and S. pyogenes aggregation.     

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D(2)O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D(2)O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.     

Regulation of fatty acid biosynthesis in Escherichia coli.

Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.