Change in the surface hydrophobicity of substrate cells during bdelloplast formation by Bdellovibrio bacteriovorus 109J.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J, the substrate cell surface becomes more hydrophobic. This was shown (i) by comparing the sensitivity to hydrophobic antibiotics of wild-type and lipopolysaccharide mutant strains of Salmonella typhimurium to that of the bdellovibrio growing on these strains and (ii) by measuring the binding efficiency of these strains, Escherichia coli, and their derived bdelloplasts to octyl Sepharose. The kinetics of increase in surface hydrophobicity was similar to the kinetics of the conversion of the substrate cell peptidoglycan to a lysozyme-resistant form (M. Thomashow and S. Rittenberg, J. Bacteriol. 135:1008-1014, 1978), and hydrophobicity reached a maximum at about 60 min in a synchronous culture. The change in hydrophobicity was inhibited by chloramphenicol, suggesting that bdellovibrio protein synthesis was required. Control experiments revealed that the free-swimming bdellovibrio had a more hydrophobic surface than the deep rough mutants of S. typhimurium.     

The role of surface structures of Streptococcus agalactiae in adhesion to epithelial cells

Induced mutants of S. agalactiae which differed in surface structures were used for the study. The aim of using them was to try to correlate the presence of defined structures or surface properties with the ability of group B streptococci to attach to epithelial cells. The presence of protein antigen R conditioned strong binding of S. agalactiae cells to hydrophobic gel. Strains bearing clumping factor (CF) showed high surface hydrophobicity and presented compact growth in serum soft agar. However, there was no correlation between high surface hydrophobicity and the ability to adhere. Fibrinogen binding decreased the attachment to epithelial cells of CF-positive strains. Preincubation of bacterial cells with lectin (ConA) did not influence the attachment of S. agalactiae strains with protein surface antigen but increased the adhesion of the strains with polysaccharide antigen or untypable.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

Comparative evaluation of adhesion, surface properties, and surface protein composition of Listeria monocytogenes strains after cultivation at constant pH of 5 and 7

AIMS: To analyse the cellular mechanisms that influence Listeria monocytogenes adhesion onto inert surfaces under acidic growth conditions. METHODS AND RESULTS: The adhesion capability of all the strains was significantly reduced after cultivation at constant pH 5 than at constant pH 7 and the cell surface was significantly less hydrophobic at pH 5 than at 7. At pH 5, the analyses of surface protein composition revealed that the flagellin was downregulated for all strains, which was confirmed by the absence of flagella and the P60 protein was upregulated for L. monocytogenes EGD-e, X-Li-mo 500 and 111. The use of L. monocytogenes EGD mutants revealed that flagellin could be involved in the adhesion process, but not P60 protein. It was also observed that the hydrophobic character was not linked to the presence or the absence of flagellin or P60 protein at the cell surface of L. monocytogenes. CONCLUSIONS: The decrease of L. monocytogenes adhesion at pH 5 could be attributed to the downregulation of the flagellin synthesis under the acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Conservation of food product at pH 5 will delay bacterial adhesion and biofilm formation during food processing on inert surfaces when the product is contaminated with L. monocytogenes.     

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.     

Compositional factors influencing cell surface hydrophobicity ofPasteurella multocida variants

The influence of macromolecules other than lipopolysaccharide on the hydrophobic properties ofPasteurella multocida was investigated by assessing cell surface hydrophobicity (CSH) after experimentally modifying surfaces of various strains. CSH of hydrophobic variants was enhanced by growth on blood-supplemented medium and mechanical shearing, whereas chloramphenicol, oxytetracycline, trypsin, and pronase E treatments decreased CSH. No such modifications were observed for hydrophilic strains. Microscopic observations revealed hydrophilic strains to be heavily encapsulated in contrast to hydrophobic strains. Repeated subculturing reduced encapsulation with a concomitant increase in CSH for one hydrophilic strain while exerting no changes in the other hydrophilic strain examined. Hyaluronidase removal of capsular material from a serotype A strain resulted in increased CSH; subsequent exposure to pronase E resulted in partial restoration of hydrophilicity. These data suggest the encapsulation of hydrophilicP. multocida strains masks a relatively hydrophobic surface that is conferred, at least in part, by the presence of one or more surface-exposed proteins common to both hydrophilic and hydrophobic variants.     

Dynamic cell surface hydrophobicity of Lactobacillus strains with and without surface layer proteins

Variations in surface hydrophobicity of six Lactobacillus strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.     

Cell surface hydrophobicity ofStaphylococcus aureus measured by the salt aggregation test (SAT)

Well-defined laboratory strains as well as 72 clinical strains ofStaphylococcus aureus isolated from bovine mastitis were investigated for surface hydrophobicity by the salt aggregation test (SAT).Staphylococcus aureus strain Cowan 1, rich in protein A and fibronectin-binding surface proteins, was found to show high surface hydrophobicity, whereas strain Wood 46, deficient in these surface proteins, showed low surface hydrophobicity. SAT showed a significant difference in surface hydrophobicity (P<0.001) between protein A-positive and A-negative strains measured by ²-test analysis. Comparison of SAT values with results obtained from hydrophobic interaction chromatography (HIC) showed a good correlation (P<0.025). A high-level protein-A-producing mutant (SA 113prA-3) showed increased surface hydrophobicity as compared with the parent strain (SA 113), whereas ten protein-A-negative mutants showed low surface hydrophobicity in SAT. Of the 72 clinical isolates tested by SAT, 47 (65%) showed autoaggregating properties, i.e., the strains aggregated even in isotonic buffers. Tween 80 (1% vol/vol) and ethylene glycol (50% vol/vol) prevented autoaggregation of some hydrophobic strains aggregating in phosphate-buffered saline. However, 2M of a chaotropic agent (NaSCN) was more efficient in preventing autoaggregation of the strains tested. Heating of cell suspensions to 80°C or 100°C as well as trypsin andStreptomyces griseus protease treatment generally caused a decrease in the cell surface hydrophobicity. This indicates that protein A, fibronectin-binding proteins, and probably other as yet unidentified proteins contribute to the high surface hydrophobicity of most strains isolated from bovine mastitis.     

A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.     

Adhesion to bile drain materials and physicochemical surface properties of Enterococcus faecalis strains grown in the presence of bile

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.     

Computational protein design with explicit consideration of surface hydrophobic patches

De novo protein design requires the identification of amino-acid sequences that favor the target-folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of protein-protein interactions. A less restrictive approach for surface design that is used by the modeling program Rosetta is to parameterize the energy function so that the number of hydrophobic amino acids designed on the protein surface is similar to what is observed in naturally occurring monomeric proteins. Previous studies with Rosetta have shown that this limits surface hydrophobics to the naturally occurring frequency (～28%), but that it does not prevent the formation of hydrophobic patches that are considerably larger than those observed in naturally occurring proteins. Here, we describe a new score term that explicitly detects and penalizes the formation of hydrophobic patches during computational protein design. With the new term, we are able to design protein surfaces that include hydrophobic amino acids at naturally occurring frequencies, but do not have large hydrophobic patches. By adjusting the strength of the new score term, the emphasis of surface redesigns can be switched between maintaining solubility and maximizing folding free energy.     

Cell surface hydrophobicity and macrophage association ofAeromonas salmonicida

Hydrophobic interaction chromatography and salt aggregation were used to compare the call surface hydrophobicity of strains of the fish pathogenAeromonas salmonicida which differed in their ability to produce the surface protein array known as A-layer. Presence of this superficial protein layer is crucial to the virulence of this organism and was found to coincide with a dramatic increase in cell surface hydrophobicity. Assays with in vitro cultured macrophages from either rainbow trout or mice revealed that this hydrophobic A-layer providedA. salmonicida cells with an enhanced ability to associate with phagocytic monocytes. This enhanced association was demonstrated in the absence of opsonizing antibody and may have important implications in the virulence ofA. salmonicida for fish.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

Crystal structure of Neisserial surface protein A (NspA), a conserved outer membrane protein with vaccine potential

The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. NspA is a homolog of the Opa proteins, which mediate adhesion to host cells. Here, we present the crystal structure of NspA, determined to 2.55-A resolution. NspA forms an eight-stranded antiparallel beta-barrel. The four loops at the extracellular side of the NspA molecule form a long cleft, which contains mainly hydrophobic residues and harbors a detergent molecule, suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids. In addition, the structure provides a starting point for structure-based vaccine design.     

Protein patterning on silicon-based surface using background hydrophobic thin film

A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37 degrees C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

The effects of seven growth media on cell surface hydrophobicity of a collection of Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis were compared in the salt-aggregation test. Thirty-three per cent of Staph. aureus strains showed extremely high cell surface hydrophobicity (auto-aggregated) and 28% were moderately hydrophobic while 26% were hydrophilic after growth on horse blood agar at 37°C for 18 h. There were great variations in the proportion and degree of the hydrophobicity depending on the medium used. Cultivations on/in capsule-inducing media caused a shift from a high to a low degree of hydrophobicity, although a microscopically detectable capsule or slime layer was seen in only one strain. This strain and encapsulated reference strains had a hydrophilic cell surface and migrated faster in free zone electrophoresis than cells of unencapsulated strains. Cells of strains grown on staphylococcus medium 110 agar migrated faster than those grown on horse blood agar regardless of their capsule production. Coagulase-negative staphylococci showed uniformly hydrophilic cell surface after cultivation on horse blood agar, but not when grown in tryptic soy broth or proteose peptone broth. It was concluded that most of the Staph. aureus strains from bovine mastitis under a variety of growth conditions in stationary phase culture constantly expressed hydrophobic cell surface.     

Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface

The influenza A virus M2 protein is expressed abundantly at the cell surface, and in addition to the hemagglutinin (HA) and neuraminidase (NA), is a third virus-specific membrane protein. M2 has an internal hydrophobic membrane anchorage domain and associates with the same cellular membrane fractions as HA and NA. Trypsin treatment of infected cells and immunoprecipitation with site-specific antisera indicate that a minimum of 18 NH2-terminal amino acids of M2 are exposed at the cell surface. Ten NH2-terminal residues are conserved in all strains of influenza A virus for which sequences are available. Antibodies can recognize M2 on the cell surface and therefore it may be an infected-cell surface antigen. We discuss properties of M2 that match it to the elusive major target molecule on influenza A virus-infected cells for cross-reactive cytotoxic T cells.     

Analysis of accessible surface of residues in proteins

We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity.