Surface-associated Proteins Research Articles

Atomic force microscopy: A powerful molecular toolkit in nanoproteomics

Analysing microbial cell surface proteins is a challenging task in current microbial proteomic research, which has major implications for drug design, vaccine development, and microbial monitoring. In this context, atomic force microscopy (AFM) has recently emerged has a powerful characterization platform, providing valuable insights into the surface proteome of microbial cells. The aim of this article is to show how advanced AFM techniques, that all have in common functionalization of the AFM tip with specific molecules, can be used to answer pertinent questions related to surface-associated proteins, such as what is their spatial arrangement on the cell surface, and what are the forces driving their interaction with the environment?

Protein expression by planktonic and biofilm cells of clinical isolations of Streptococcus mutans

To investigate the changes in the surface-associated protein expression of planktonic cells and biofilm cells of clinical isolations of Streptococcus mutans (Sm). The proteins were extracted by the method of Homer from the nonadhered planktonic and the adhered biofilm cells and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis followed by image analysis. Proteins were identified by matrix-assisted laser desorption time of flight mass spectrometry and computer-assisted protein sequence analysis. Image analysis revealed that 30% - 31% of the protein spots in biofilm was modulated. A total of 238 proteins changed 1.3-fold or greater in biofilm cells compared to planktonic cells in Sm18, with 5 only expressed in biofilm cells and 5 not expressed in biofilm cells. A total of 279 proteins changed 1.3-fold or greater in biofilm cells compared to planktonic cells in Sm593, with 12 only expressed in biofilm cells and 2 not expressed in biofilm cells. Two clinical isolations in biofilm cells had three identical protein expressions whose functions were associated with biosynthetic processes. The two clinical isolations in biofilm status have high expression of some special proteins, which are presumed to be key proteins essential for formation of biofilm. The difference of protein expression in biofilm cells of the two clinical isolations may represent their distinct characters.

Abstract B52: Annexin A6 promotes cellular attachment and invasiveness of breast cancer cells

Abstract Annexin A6 (ANXA6) is a member of a family of Ca2+-dependent phospholipid-binding proteins capable of associating with negatively charged membranes in its Ca2+-bound state. Although the functions of this protein are yet to be clearly defined, various physiological roles have been proposed, including a role in the regulation of cell growth and tumorigenesis. We previously showed that ANXA2, ANXA4 and ANXA6 are receptors of the serum factor fetuin-A on the surface of breast carcinoma cells. To determine which of these annexins is the bona fide cell surface receptor for fetuin-A, we have now examined the surface expression of ANXA2 and ANXA6 in the fetuin-A expressing hepatocellular carcinoma cell line HepG2 and in the invasive breast carcinoma cell line BT549. By glycerol gradient centrifugation of the post nuclear supernatants of HepG2 cells briefly cultivated at high extracellular Ca2+, we show that while ANXA2 predominantly associated with heavy membranes corresponding to the endogenous fetuin-A profile, the total membrane-associated ANXA6 on the contrary, diminished. Analysis of the EGTA-solubilized surface-associated proteins from HepG2 and BT549 cells revealed that ANXA6 is predominantly associated with the cell surface suggesting that it is externalized. Further analysis of the secreted proteins by differential velocity centrifugation and in particular the nanovesicle-associated (exosomal) secreted proteins confirmed that while secreted ANXA2 was barely detectable, ANXA6 was abundantly secreted via the exosomal pathway. From these data we hypothesized that the surface associated ANXA6 may be the major fetuin-A receptor on the surface of the invasive BT549 breast cancer cells and that it may be important in the growth, attachment and migration of these tumor cells. Using ANXA6 targeted shRNA we show that down regulation of ANXA6 inhibited the attachment of these cells to collagen Type I, Collagen Type IV and to the serum factor fetuin-A but attachment to and spreading on fibronectin was not affected. Based on the detection of vinculin and focal adhesion kinase, we demonstrate the complete absence of adhesion plaques in the ANXA6-depleted cells compared to the parental BT549 cells. This supports our observation that the ANXA6-depleted BT549 cells lost their ability to spread and hence are predominantly spindle-shaped; Moreover, the ANXA6-depleted BT549 cells proliferate faster than the parental cells; they grow in three-dimensional cultures as spheroid colonies resembling the ANXA6-negative MCF-10A acini rather than as stellate colonies produced by the parental cells. Finally, ANXA6 depletion in BT549 cells inhibited their migration and in vitro invasion of matrigel. Together our data demonstrate that ANXA6 expression in this metastatic breast cancer cells correlates with the invasive phenotype and that its association with the surface of these breast cancer cells modulates at least in part, attachment of these cells to the ECM and their overall morphology. Citation Information: Cancer Res 2009;69(23 Suppl):B52.

Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2

Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA − mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA − mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.

Identification and characterisation of Mycosphaerella graminicola secreted or surface-associated proteins with variable intragenic coding repeats

Pathogenic micro-organisms have been suggested to vary the number of intragenic repeats present within secreted or cell membrane/cell wall-associated proteins in order to manipulate host immune responses. We have identified a number of genes predicted to encode secreted proteins possessing internal tandem repeats in the genome sequence of Mycosphaerella graminicola (isolate IPO323), a wheat leaf-specific fungal pathogen and causal agent of Septoria tritici blotch disease. Twenty-three M. graminicola Tandem Repeat Proteins ( MgTRPs) were subject to further analysis. Many MgTRPs varied in the number of intragenic repeats between isolates and almost all were expressed. Peak gene expression was frequently observed towards the end of the symptomless phase of wheat leaf colonisation which typically lasts for 8–10 days after inoculation. In contrast, with one exception, increased expression of the majority of MgTRPs was not detected during interactions with resistant host genotypes. Repeat number differences detected in genomic DNA were retained in different transcript sizes produced during plant infection by different isolates. One in planta expressed MgTRP was found to reside within a ∼6 kb region that appears to be absent from a number of tested isolates and also from individual members of a modern field population. Sequence analysis of another in planta expressed MgTRP from six isolates highlighted the potential for structural changes which may occur as a consequence of varying internal repeat numbers and provided support for repeat variation occurring as a consequence of intragenic recombination. These data provide new insights into the genetic variation which exists within M. graminicola populations at the level of in planta expressed secreted/surface-associated proteins which are candidate effectors in the host–pathogen interaction.

Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-A resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.

Cell surface-bound IL-1α is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network

Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1alpha is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1alpha mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1alpha antibodies, and IL-1alpha depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1alpha neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1alpha depletion reduced the DNA binding activity of NF-kappaB and C/EBPbeta, which stimulate IL-6/IL-8 transcription. IL-1alpha was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1alpha blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1alpha-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1alpha regulates the biological effects of these cytokines. Thus, cell surface IL-1alpha is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

Development of a non-invasive murine infection model for acute otitis media

Otitis media (OM) is one of the most frequent diseases in childhood, and Streptococcus pneumoniae is among the main causative bacterial agents. Since current experimental models used to study the bacterial pathogenesis of OM have several limitations, such as the invasiveness of the experimental procedures, we developed a non-invasive murine OM model. In our model, adapted from a previously developed rat OM model, a pressure cabin is used in which a 40 kPa pressure increase is applied to translocate pneumococci from the nasopharyngeal cavity into both mouse middle ears. Wild-type pneumococci were found to persist in the middle ear cavity for 144 h after infection, with a maximum bacterial load at 96 h. Inflammation was confirmed at 96 and 144 h post-infection by IL-1beta and TNF-alpha cytokine analysis and histopathology. Subsequently, we investigated the contribution of two surface-associated pneumococcal proteins, the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA), to experimental OM in our model. Pneumococci lacking the slrA gene, but not those lacking the ppmA gene, were significantly reduced in virulence in the OM model. Importantly, pneumococci lacking both genes were significantly more attenuated than the DeltaslrA single mutant. This additive effect suggests that SlrA and PpmA exert complementary functions during experimental OM. In conclusion, we have developed a highly reproducible and non-invasive murine infection model for pneumococcal OM using a pressure cabin, which is very suitable to study pneumococcal pathogenesis and virulence in vivo.

Identification of secreted and surface proteins from Enterococcus faecalis

Secreted and surface proteins of bacteria are key molecules that interface the cell with the environment. Some of them, corresponding to virulence factors, have already been described for Enterococcus faecalis, the predominant species involved in enterococcal nosocomial infections. In a global proteomic approach, the identification of the most abundant secreted and surface-associated proteins of E. faecalis JH2-2 strain was carried out. These proteins were separated by gel electrophoresis or directly subjected to in vivo trypsinolysis and then analyzed by liquid chromatography - electrospray ion trap tandem mass spectrometry. Putative functions were assigned by homology to the translated genomic database of E. faecalis. A total of 44 proteins were identified, eight secreted proteins from the supernatant culture and 38 cell surface proteins from two-dimensional gel electrophoresis and in vivo trypsinolysis among which two are common to the two groups. Their sequences analysis revealed that 35 of the 44 proteins harbour characteristic features (signal peptide or transmembrane domains) consistent with an extracellular localization. This study may be considered as an important step to encourage proteomic-based investigations of E. faecalis cell surface associated proteins that could lead to the discovery of virulence factors and to the development of new therapeutic tools.

Proteomic profiling of Cronobacter turicensis 3032, a food‐borne opportunistic pathogen

Members of the genus Cronobacter are opportunistic pathogens for neonates and are often associated with contaminated milk powder formulas. At present little is known about the virulence mechanisms or the natural reservoir of these organisms. The proteome of Cronobacter turicensis 3032, which has recently caused two deaths, was mapped aiming at a better understanding of physiology and putative pathogenic traits of this clinical isolate. Our analyses of extracellular, surface-associated and whole-cell proteins by two complementary proteomics approaches, 1D-SDS-PAGE combined with LC-ESI-MS/MS and 2D-LC-MALDI-TOF/TOF MS, lead to the identification of 832 proteins corresponding to a remarkable 19% of the theoretically expressed protein complement of C. turicensis. The majority of the identified proteins are involved in central metabolic pathways, translation, protein folding and stability. Several putative virulence factors, whose expressions were confirmed by phenotypic assays, could be identified: a macrophage infectivity potentiator involved in C. turicensis persistence in host cells, a superoxide dismutase protecting the pathogen against reactive oxygen species and an enterobactin-receptor protein for the uptake of siderophore-bound iron. Most interestingly, a chitinase and a metalloprotease that might act against insects and fungi but no casein hydrolysing enzymes were found, suggesting that there is an environmental natural habitat of C. turicensis 3032.

Surfome Analysis as a Fast Track to Vaccine Discovery: IDENTIFICATION OF A NOVEL PROTECTIVE ANTIGEN FOR GROUP B STREPTOCOCCUS HYPERVIRULENT STRAIN COH1

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.

Proteomic analysis of cell surface-associated proteins from probioticLactobacillus plantarum

In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.

Lactobacillus fermentumBCS87 expresses mucus- and mucin-binding proteins on the cell surface

To identify and characterize adhesion-associated proteins in the potential probiotic Lactobacillus fermentum BCS87. Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1(-1) LiCl were analysed by Western blotting using HRP-labelled porcine mucus and mucin. Two adhesion-associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N-terminal and internal peptides of the 32 kDa protein (32-Mmubp) were sequenced, and the corresponding gene (32-mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32-mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47-99% identity to solute-binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC-conserved domain was identified and phylogenetic relationship analysis confirmed that 32-Mmubp belongs to the OpuAC family. Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface-associated proteins. 32-Mmubp is a component of ABC transporter system that also functions as an adhesin. Characterization of 32-Mmubp and 32-mmub will contribute to understanding the host-bacteria interactions of Lact. fermentum with the intestinal tract of pigs.

Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin

Several Bacillus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus cereus(CH) displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. cereus(CH) were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. cereus(CH) were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated, Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.

Protective capacities of cell surface-associated proteins of Streptococcus suis mutants deficient in divalent cation-uptake regulators

Many cell surface-associated, divalent cation-regulated proteins are immunogenic, and some of them confer protection against the bacterial species from which they are derived. In this work, two Streptococcus suis divalent cation uptake regulator genes controlling zinc/manganese and iron uptake (adcR and fur, respectively) were inactivated in order to study the protective capacities of their cell surface-associated proteins. The results obtained showed overexpression of a set of immunogenic proteins (including members of the pneumococcal histidine triad family previously reported to confer protection against streptococcal pathogens) in S. suis adcR mutant cell surface extracts. Likewise, genes encoding zinc transporters, putative virulence factors and a ribosomal protein paralogue related to zinc starvation appeared to be derepressed in this mutant strain. Moreover, protection assays in mice showed that although neither adcR- nor fur-regulated cell surface-associated proteins were sufficient to confer protection in mice, the combination of both adcR- and fur-regulated cell surface-associated proteins is able to confer significant protection (50 %, P=0.038) against a challenge to mice vaccinated with them.

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

Host-bacteria interactions are often mediated via surface-associated proteins. The identification of these proteins is an important goal of bacterial proteomics. To address how bile can influence the cell-envelope proteome of Bifidobacterium longum biotype longum NCIMB 8809, we analysed its membrane protein fraction using stable isotope labelling of amino acids in cell culture (SILAC). We were able to identify 141 proteins in the membrane fraction, including a large percentage of the theoretical transporters of this species. Moreover, the envelope-associated soluble fraction was analysed using different subfractionation techniques and differential in-gel fluorescence electrophoresis (DIGE). This approach identified 128 different proteins. Some of them were well-known cell wall proteins, but others were highly conserved cytoplasmic proteins probably displaying a 'moonlighting' function. We were able to identify 11 proteins in the membrane fraction and 6 proteins in the envelope-associated soluble fraction whose concentration varied in the presence of bile. Bile promoted changes in the levels of proteins with important biological functions, such as some ribosomal proteins and enolase. Also, oligopeptide-binding proteins were accumulated on the cell surface, which was reflected in a different tripeptide transport rate in the cells grown with bile. The data reported here will provide the first cell-envelope proteome map for B. longum, and may contribute to understanding the bile tolerance of these bacteria.

High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells

Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.

Surface analysis of pure and complex mucin coatings on a real-type substrate using individual and combined mBCA, ELLA, and ELISA

In the past, we introduced the idea of using mucin coatings to improve biomaterials performance. Here, we evaluate non-radioactive methods for the analysis of pure and human host protein-containing (complex) mucin coatings on a real-type substrate (Thermanox). A common protein quantification assay (mBCA) was combined with mass-calibrated, enzyme-amplified assays based on lectin (ELLA) and antibody (ELISA) recognition, to determine the total and specific amounts of surface-associated proteins. Model studies showed the mBCA assay to be of limited use at low mass loads, and steric effects to influence the ELLA at high surface layer densities. Non-specific responses due to substrate interaction were low for the ELLA and ELISAs. Cross-reactions were observed during ELLA analysis of analytes sharing high degree of O-glycosylation. Combined mBCA–ELLA–ELISA analysis suggested that mucin desorption was low upon protein addition and that low concentrations of ELISA-determined protein for the complex coatings could be explained in terms of low accessibility of proteins to the bulk environment. Specifically, a methodology is presented for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating. Finally, X-ray photoelectron spectroscopy and infrared reflectance spectroscopy combined with multivariate data analysis were proven useful in the evaluation of mucin-based coatings.

Virulence Gene Expression in Human Community‐AcquiredStaphylococcus aureusInfection

Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospital-associated infections; however, community-acquired MRSA (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript levels of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton-Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated protein A was linked only with invasive disease. Comparisons with laboratory-grown corresponding clinical isolates revealed that tissue-specific expression profiles reflect the activity of the staphylococcal accessory gene regulator during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.