Whole genome and RNA sequencing of oral commensal bacterium Streptococcus anginosus subsp. anginosus with vancomycin tolerance.

"Antibiotic tolerance" promotes the rapid subsequent evolution of "antibiotic resistance," however, it is often overlooked because it is difficult to distinguish between tolerant and susceptible organisms. A commensal bacterium S. anginosus subsp. anginosus strain KHUD_S1, isolated from dental biofilm was found to exhibit a high MBC/MIC ratio of 32 against vancomycin. We observed KHUD_S1 cells exposed to vancomycin did not grow but maintained viability. Transmission electron microscope showed KHUD_S1 cells possessed a dense, thick capsule and maintained the cell wall integrity upon vancomycin exposure. To infer the underlying mechanisms of the vancomycin tolerance in KHUD_S1, we performed whole genome sequencing and RNA sequencing. The KHUD_S1 genome carried three genes encoding branching enzymes that can affect peptidoglycan structure through interpeptide bridge formation. Global gene expression profiling revealed that the vancomycin-induced downregulation of carbohydrate and inorganic ion transport/metabolism as well as translation is less prominent in KHUD_S1 than in the vancomycin susceptible strain KHUD_S3. Based on the transcriptional levels of genes related to peptidoglycan synthesis, KHUD_S1 was determined to have a 3D peptidoglycan architecture distinct from KHUD_S3. It was found that, under vancomycin exposure, the peptidoglycan was remodeled through changes in the interpeptide bridge and transpeptidation reactions. Collectively, these features of S. anginosus KHUD_S1, including a dense capsule and differential gene expression in peptidoglycan synthesis, may contribute to vancomycin tolerance. Our results showing the occurrence of vancomycin tolerance amongst oral commensal bacteria highlight the need for considering future strategies for screening of antibiotic tolerance as an effort to reduce antibiotic resistance.

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.

The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.

Streptococcus koreensis sp. nov., Isolated from Human Subgingival Dental Plaque of Periodontitis Lesion.

A novel facultative anaerobic, Gram-stain-positive coccus, strain JS71T, was isolated from the human subgingival dental plaque of a periodontitis lesion. Phylogenetic analysis based on the 16S ribosomal RNA gene (16S rDNA) revealed that the strain belonged to the genus Streptococcus. The 16S rDNA sequence had high similarity with Streptococcus rubneri DSM 26920T (98.6%), Streptococcus parasanguinis ATCC 15912T (98.5%), and Streptococcus australis CCUG 45919T (98.3%). The genome of strain JS71T was 2,009,592 bp in length. The DNA G+C content of the strain was 42.1 mol%. Average nucleotide identity values between strain JS71T and S. rubneri DSM 26920T, S. parasanguinis ATCC 15912T, and S. australis CCUG 45919T were 88.9%, 80.8%, and 92.4%, respectively. Genome-to-genome distance values between strain JS71TS. rubneri DSM 26920T, S. parasanguinis ATCC 15912T, and S. australis CCUG 45919T were 36.5% (34-39%), 26.3% (23.9-28.7%), and 48.0% (45.4-50.6%), respectively. The major fatty acids of the strain were C16:0 (39.7%), C18:1 ω6c/C18:1 ω7c (15.5%), and C18:0 (10.4%). Based on these results, strain JS71T (= KCOM 2890T = JCM 33454T) should be a novel species of the genus Streptococcus, for which the name Streptococcus koreensis sp. nov. is proposed.

Regulation of Cell Division in Streptococci: Comparing with the Model Rods.

Streptococcus is a genus of oval-shaped bacteria that act as both commensals and pathogens. Streptococcal infections are relevant to high morbidity and huge socioeconomic costs, with drug resistant strains becoming an increasing threat. Cell division plays an essential role during streptococcal colonization and infection, rendering it an ideal target for antibiotics. Substantial progress has been made to uncover the molecular biology and cellular processes of cell division, favoring the target strategies. This review discusses recent advances in our understanding of streptococcal cell division and its regulatory mechanisms regarding the conserved proteins, by comparing with model rods. Peptidoglycan synthesis that involved in septum formation and the maintenance of the unique oval shape have been spatiotemporally controlled in concert with the pace of division. With newly available tools of genetic and cytological study, streptococci will become an additional model bacterial system for cytokinesis and novel therapeutic agents that target cell division.

Streptococcus gwangjuense sp. nov., Isolated from Human Pericoronitis.

A novel facultative anaerobic, Gram-stain-negative coccus, designated strain ChDC B345T, was isolated from human pericoronitis lesion and was characterized by polyphasic taxonomic analysis. The 16S ribosomal RNA gene (16S rDNA) sequence revealed that the strain belonged to the genus Streptococcus. The 16S rDNA sequence of strain ChDC B345T was most closely related to those of  Streptococcus mitis NCTC 12261T (99.5%) and Streptococcus pseudopneumoniae ATCC BAA-960T (99.5%). Complete genome of strain ChDC B345T was 1,972,471 bp in length and the G + C content was 40.2 mol%. Average nucleotide identity values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 92.17% and 93.63%, respectively. Genome-to-genome distance values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 47.8% (45.2-50.4%) and 53.0% (51.0-56.4%), respectively. Based on these results, strain ChDC B345T (= KCOM 1679T = JCM 33299T) should be classified as a novel species of genus Streptococcus, for which we propose the name Streptococcus gwangjuense sp. nov.

Metabolic-Activity-Based Assessment of Antimicrobial Effects by D2O-Labeled Single-Cell Raman Microspectroscopy.

To combat the spread of antibiotic resistance, methods that quantitatively assess the metabolism-inhibiting effects of drugs in a rapid and culture-independent manner are urgently needed. Here using four oral bacteria as models, we show that heavy water (D2O)-based single-cell Raman microspectroscopy (D2O-Raman) can probe bacterial response to different drugs using the Raman shift at the C-D (carbon-deuterium vibration) band in 2040 to 2300 cm-1 as a universal biomarker for metabolic activity at single-bacterial-cell resolution. The "minimum inhibitory concentration based on metabolic activity" (MIC-MA), defined as the minimal dose under which the median ΔC-D-ratio at 8 h of drug exposure is ≤0 and the standard deviation (SD) of the ΔC-D ratio among individual cells is ≤0.005, was proposed to evaluate the metabolism-inhibiting efficacy of drugs. In addition, heterogeneity index of MIC-MA (MIC-MA-HI), defined as SD of C-D ratio among individual cells, quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens. When exposed to 1× MIC of sodium fluoride (NaF), 1× MIC of chlorhexidine (CHX), or 60× MIC of ampicillin, the cariogenic oral pathogen Streptococcus mutans UA159 ceased propagation yet remained metabolically highly active. This underscores the advantage of MIC-MA over the growth-based MIC in being able to detect the "nongrowing but metabolically active" (NGMA) cells that underlie many latent or recurring infections. Moreover, antibiotic susceptible and resistant S. mutans strains can be readily discriminated at as early as 0.5 h. Thus, D2O-Raman can serve as a universal method for rapid and quantitative assessment of antimicrobial effects based on general metabolic activity at single-cell resolution.

Construction of a Bioassay System to Identify Extracellular Agents Targeting Bacterial Cell Envelope.

sigE in Streptomyces coelicolor encodes an extracytoplasmic function (ECF) sigma factor, σ (E), which is part of a signal transduction system that senses and responds to general cell wall stress in S. coelicolor. Expression of sigE is induced by a wide variety of agents that stress the cell wall under the control of two-component signal transduction system, CseB/CseC encoded in the same operon where sigE was identified from. Here we describe a method developing a bioassay system in S. coelicolor via a transcriptional fusion in which the promoter of sigE operon and a reporter gene (neo) conferring resistance to kanamycin were used. The effectiveness of the resulting bioassay system was determined by monitoring various agents that cause bacterial cell wall stress such as lysozyme or some antibiotics that target cell wall. In consequence, the result confirms that the bioassay system has a potential to be a simple but effective screening tool for identifying novel extracellular agents targeting bacterial cell wall.

Functional analysis of an unusual type IV pilus in the Gram-positive Streptococcus sanguinis.

Type IV pili (Tfp), which have been studied extensively in a few Gram-negative species, are the paradigm of a group of widespread and functionally versatile nano-machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram-positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram-negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface-associated motility. Tfp power 'train-like' directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano-machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram-negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano-machines.

Codon-optimized fluorescent mTFP and mCherry for microscopic visualization and genetic counterselection of streptococci and enterococci.

Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multi-species oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expressions could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications.

Inhibition of Oral Streptococci Growth Induced by the Complementary Action of Berberine Chloride and Antibacterial Compounds.

Synergistic interactions between natural bioactive compounds from medicinal plants and antibiotics may exhibit therapeutic benefits, acting against oral cariogenic and opportunistic pathogens. The aim of the presented work was to assess the antibacterial activity of berberine chloride (BECl) in light of the effect exerted by common antibiotics on selected reference strains of oral streptococci (OST), and to evaluate the magnitude of interactions. Three representative oral microorganisms were investigated: Streptococcus mutans ATCC 25175 (SM), S. sanguinis ATCC 10556 (SS), S. oralis ATCC 9811 (SO) and microdilution tests, along with disc diffusion assays were applied. Here, we report that growth (viability) of all oral streptococci was reduced by exposure to BECl and was dependent primarily on exposure/ incubation time. A minimum inhibitory concentrations (MIC) of BECl against OST ranged from 512 µg/mL (SS) to 1024 µg/mL (SM, SO). The most noticeable antibacterial effects were observed for S. sanguinis (MIC 512 µg/mL) and the most significant synergistic action was found for the combinations BECl-penicillin, BECl-clindamycin and BECl-erythromycin. The S. oralis reflects the highest MBC value as assessed by the AlamarBlue assay (2058 µg/mL). The synergy between berberine and common antibiotics demonstrates its potential use as a novel antibacterial tool for opportunistic infections and also provides a rational basis for the use of berberine as an oral hygiene measure.

Molecular studies of the structural ecology of natural occlusal caries.

Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. This study shows for the first time the spatial distribution of bacterial taxa in vivo at various stages of occlusal caries, applying a molecular methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. Eleven freshly extracted teeth were classified according to their occlusal caries status. The teeth were fixed, embedded, sectioned and decalcified before FISH was performed using oligonucleotide probes for selected abundant species/genera associated with occlusal caries including Streptococcus, Actinomyces, Veillonella, Fusobacterium, Lactobacillus and Bifidobacterium. The sites showed distinct differences in the bacterial composition between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supragingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by low fluorescence signal intensity and presence of material of non-bacterial origin. Bacterial invasion (often Lactobacillus and Bifidobacterium spp.) into the dentinal tubules was seen only at advanced stages of caries with manifest cavity formation. It is concluded that the molecular methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.

An extracellular bacterial pathogen modulates host metabolism to regulate its own sensing and proliferation.

Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum-sensing sil system as a reporter, we found that, during adherence to host cells, GAS delivers streptolysin toxins, creating endoplasmic reticulum stress. This, in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria, altering the expression of ∼17% of GAS genes of which about one-third are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated, whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase, a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a therapeutic strategy against GAS infections.

Phenotypic diversity of multicellular filamentation in oral Streptococci.

Filamentous multicellular bacteria are among the most ancient multicellular organisms. They inhabit a great variety of environments and are present in the human body, including the oral cavity. Beside the selective advantages related to the larger size achieved through filamentation, the development of multicellular bacteria can be also driven by simple ecological factors such as birth and death rates at the cellular level. In order to extend earlier results obtained in aquatic species, we investigate the filamentation process of four different strains of oral streptococci, namely S. mutans, S. salivarius, S. oralis and S. anginosus. The results indicate differences in the capacities of different streptococcus species to form filaments, manifested in terms of length and the time-scale of filament elongation. The filamentation pattern of these oral streptococci resembles that of aquatic bacteria, whereby filaments reach a peak length during exponential growth and become short when the population reaches a steady state. Hence, this study validates that multicellularity can be an emergent property of filamentous bacteria of different ecological niches, and that phenotypic differences in filamentation can occur within species of the same genus, in this case oral streptococci. Moreover, given the role that specific oral streptococci can play in the etiology of oral diseases, these results can possibly open new perspectives in the study of the virulence properties of these species.

Rapid interferon-gamma release from natural killer cells induced by a streptococcal commensal.

Interferon-gamma (IFN-γ) is a critical cytokine for the initiation of immune responses against a variety of infectious agents and malignancies. We found that a range of Gram-positive and Gram-negative bacteria stimulated the rapid release (<24 h) of IFN-γ from murine leukocytes. Using fluorescence activated cell sorting and cd1d(-/-) and rag1(-/-) mice, we determined that dendritic cells (DCs) and natural killer (NK) cells were primarily responsible for IFN-γ release by Streptococcus salivarius, a Gram-positive commensal, previously noted to possess potent interleukin-12 (IL-12)-inducing potential. IFN-γ release from NK cells required DC:NK membrane contact and IL-12/IL-18 expression, but was independent of lymphocyte function-associated antigen-1-mediated interactions. IFN-γ release in response to bacteria was maintained in mice deficient for Toll-like receptor (TLR)-2 and TLR-4, suggesting that bacteria activate antigen-presenting cells via multiple, redundant pathways. Together, our results suggest that Gram-positive bacteria may be useful in driving NK cell activation and T helper 1 polarization and have the potential for development as effective adjuvants.

Comparing biocompatibility of gingival fibroblasts and bacterial strains on a different modified titanium discs.

The modification of implant surface situated in the area of peri-implant sulcus has important role in bacterial and cell adhesion. Six different chemically and physically modified titanium discs were prepared: glazed (Tis-MALP), unglazed (Tis-O), unglazed and alkali-etched (Tis-OA), unglazed and coated with ZrN (Tis-OZ), unglazed, sand blasted, and acid etched (Tis-OPAE), and unglazed, sand blasted, acid, and alkali etched (Tis-OPAAE). Analysis of surface topography was determined using scanning electron microscopy and atomic force microscopy (AFM). Biocompatibility of gingival fibroblasts was characterized by the production of tumor necrosis factor alpha, collagen I, matrix metalloproteinase 2 (MMP-2) after 24 and 72 h and expression of α3 ß1 integrin and vinculin using enzyme-linked immunosorbent assay (ELISA) or modified ELISA after 6 and 24 h. Microorganism adhesion (five bacterial strains) and biofilm formation was also evaluated. The adhesion of bacteria and gingival fibroblasts was significantly higher on titanium disc Tis-OPAAE and biofilm formation on the same surface for Streptococcus mutans, Streptococcus gordonii, and Streptococcus intermedius. The gingival fibroblasts on Tis-OPAAE disc had also significantly lower production of MMP-2. The collagen production was significantly lower on all surfaces with roughness higher than 0.2 µm. This study confirmed that the titanium disc with the surface roughness 3.39 µm (Tis-OPAAE) supported the adhesion of bacterial strains as well as gingival fibroblasts.

Streptococcus danieliae sp. nov., a novel bacterium isolated from the caecum of a mouse.

We report the characterization of one novel bacterium, strain ERD01G(T), isolated from the cecum of a TNF(deltaARE) mouse. The strain was found to belong to the genus Streptococcus based on phylogenetic analysis of partial 16S rRNA gene sequences. The bacterial species with standing name in nomenclature that was most closely related to our isolate was Streptococcus alactolyticus (97 %). The two bacteria were characterized by a DNA-DNA hybridization similarity value of 35 %, demonstrating that they belong to different species. The new isolate was negative for acetoin production, esculin hydrolysis, urease, α-galactosidase and ß-glucosidase, was able to produce acid from starch and trehalose, grew as beta-hemolytic coccobacilli on blood agar, did not grow at >40 °C, did not survive heat treatment at 60 °C for 20 min and showed negative agglutination in Lancefield tests. On the basis of these characteristics, strain ERD01G(T) differed from the most closely related species S. alactolyticus, Streptococcus gordonii, Streptococcus intermedius and Streptococcus sanguinis. Thus, based on genotypic and phenotypic evidence, we propose that the isolate belongs to a novel bacterial taxon within the genus Streptococcus, for which the name Streptococcus danieliae is proposed. The type strain is ERD01G(T) (= DSM 22233(T) = CCUG 57647(T)).

Role of operon aaoSo-mutT in antioxidant defense in Streptococcus oligofermentans.

Previously, we have found that an insertional inactivation of aao(So), a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aao(So) and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aao(So) and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aao(So) insertional mutant, indicating that gene polarity derived from the inactivation of aao(So) attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H(2)O(2) (min · mg protein)(-1)]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aao(So) mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant's growth by 11%, indicating that the aao(So) mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aao(So). Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aao(So) mutant. This demonstrated that inactivation of aao(So) caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aao(So)-mutT operon in antioxidant defense in S. oligofermentans.

Bacterial colonization or infection in chronic sinusitis.

The aim of this study was the determination of bacteria present in maxillary and ethmoid cavities in patients with chronic sinusitis and to correlate these findings with bacteria simultaneously present in their nasopharynx. The purpose of this correlation was to establish the role of bacteria found in chronically inflamed sinuses and to evaluate if the bacteria present colonized or infected sinus mucosa. Nasopharyngeal and sinus swabs of 65 patients that underwent functional endoscopic sinus surgery were cultivated and at the same time the presence of leukocytes were determined in each swab. The most frequently found bacteria in nasopharynx were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Streptococcus viridans and Streptococcus pneumoniae. Maxillary or ethmoidal sinus swabs yielded bacterial growth in 47 (72.31%) patients. The most frequently found bacteria in sinuses were Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella spp. and Streptococci (pneumoniae, viridans and spp.). The insignificant number of leukocytes was present in each sinus and nasopharyngeal swab. Every published microbiology study of chronic sinusitis proved that sinus mucosa were colonized with bacteria and not infected, yet antibiotic therapy was discussed making no difference between infection and colonization. Chronic sinusitis should be considered a chronic inflammatory condition rather than bacterial infection, so routine antibiotic therapy should be avoided. Empiric antibiotic therapy should be prescribed only in cases when the acute exacerbation of chronic sinusitis occurs and the antibiotics prescribed should aim the usual bacteria causing acute sinusitis. In case of therapy failure, antibiotics should be changed having in mind that under certain circumstances any bacteria colonizing sinus mucosa can cause acute exacerbation of chronic sinusitis.

Surface-affinity profiling to identify host-pathogen interactions.

Proteolytic treatment of intact bacterial cells has proven to be a convenient approach for the identification of surface-exposed proteins. This class of proteins directly interacts with the outside world, for instance, during adherence to human epithelial cells. Here, we aimed to identify host receptor proteins by introducing a preincubation step in which bacterial cells were first allowed to capture human proteins from epithelial cell lysates. Using Streptococcus gallolyticus as a model bacterium, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteolytically released peptides yielded the identification of a selective number of human epithelial proteins that were retained by the bacterial surface. Of these potential receptors for bacterial interference, (cyto)keratin-8 (CK8) was verified as the most significant hit, and its surface localization was investigated by subcellular fractionation and confocal microscopy. Interestingly, bacterial enolase could be assigned as an interaction partner of CK8 by MS/MS analysis of cross-linked protein complexes and complementary immunoblotting experiments. As surface-exposed enolase has a proposed role in epithelial adherence of several Gram-positive pathogens, its interaction with CK8 seems to point toward a more general virulence mechanism. In conclusion, our study shows that surface-affinity profiling is a valuable tool to identify novel adhesin-receptor pairs, which advocates its application in other hybrid biological systems.

High-level fluorescence labeling of gram-positive pathogens.

Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.