Biomedical subjects

Bernd Kreikemeyer

Publications and source records attributed to Bernd Kreikemeyer.

14 recordsLinked to original sources

PubMed2006

Characterization of biofilm formation by clinically relevant serotypes of group A streptococci.

Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.

Bacterial Adhesion↗

PubMed2005

Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

Amino Acid Sequence↗

PubMed2005

Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococci, GAS) is a human pathogen which probably varies its multiplication rate and thus, growth phases in association with the type of infection caused in its host. To create a basis for future determinations of such associations, the genome-wide growth phase-related GAS transcriptome was assessed in the present study. Therefore, the published serotype M1 S. pyogenes genome sequence as well as the partially sequenced serotype M18 and M49 GAS genomes were used to produce DNA microarrays that carried 2256 oligonucleotide probes matching 3662 open reading frames (ORFs). With these microarrays, the transcriptome of the serotype M49 GAS strain 591 grown to the exponential, transition, and early stationary growth phases was assessed in seven independent experiments. The gained data were compared to real-time RT-PCR assays. Data analysis was refined by a novel approach, i.e. grouping of expressed genes to four classes according to relative transcript abundance and gene functions. At the different growth phases, 86.7%, 79.5% and 55.7% of the at least 1883 ORFs contained in the serotype M49 genome were expressed above the defined detection level. Contrary to the general trend, transcript amounts of genes in the functional groups of transport and membrane proteins as well as stress response factors peaked at the transition phase. The most prominent changes in the transcript abundances were predominantly observed for sugar compound transport and turnover-related ORFs. The majority of known virulence genes had their maximum expression during the transition phase, consistent with the proposed associated change in virulence behavior of the bacteria. With these results, it will now be feasible to assess the in situ growth phase of a given GAS strain during any type of infection by measuring the expression of selected marker genes.

DNA, Bacterial↗

PubMed2005

MsmR, a specific positive regulator of the Streptococcus pyogenes FCT pathogenicity region and cytolysin-mediated translocation system genes.

As a prerequisite for colonization or causing local infections, Streptococcus pyogenes (group A streptococci, GAS) need to specifically adhere to eukaryotic cell surfaces. Predominantly responsible adhesin genes are contained in a genotype-specific pattern within the FCT region of the GAS genome. In this study, MsmR, belonging to AraC/XylS type transcriptional regulators, was identified in the FCT region as a positive regulator of the major fibronectin-binding adhesin protein F2 in a serotype M49 strain. Compared with the wild-type strain, the msmR mutant showed reduced binding to immobilized fibronectin and decreased adherence to and internalization into human pharyngeal epithelial cells. These results suggested that altered levels of fibronectin-binding proteins in the mutant affect eukaryotic cell attachment and internalization. Complete transcriptome and reporter fusion assay data revealed that MsmR positively regulates FCT region genes including Nra and cytolysin-mediated translocation system genes. Consistent with the genetic data, the mutant showed attenuated streptolysin O activity and eukaryotic cell cytotoxity. Direct binding of recombinant MsmR to nga, nra/cpa and prtF2 promoter regions was confirmed by EMSA assays. As prior analysis demonstrated the Nra regulator negatively affects gene expression from the FCT region, MsmR and Nra appear to adversely control crucial virulence factor expression in GAS and thus contribute to a fine-tuned balance between local destructive process and metastatic spreading of the bacteria.

Bacterial Adhesion↗

PubMed2005

Global epithelial cell transcriptional responses reveal Streptococcus pyogenes Fas regulator activity association with bacterial aggressiveness.

The bacterial human pathogen Streptococcus pyogenes (group A streptococci, GAS) is able to adhere to, internalize into and cross-talk on multiple levels with its host cells. To gain insight into the Fas function in pathogenesis we used Affymetrix human genome DNA-arrays to measure temporal and global transcriptional responses of HEp-2 cells infected with M49 S. pyogenes wild-type bacteria and DeltafasX, an isogenic S. pyogenes two-component-signal-transduction system mutant. A modified stringent statistical analysis method identified a total of 86 HEp-2 cell genes as differentially transcribed upon infection over the investigated time course. Increased expression of genes encoding proteins involved in GAS host cell adherence and internalization (fibronectin, integrin-alpha5) was found as a common response. In contrast to earlier reports investigating other GAS serotype strains, Ras superfamily and RhoA pathways are exploited by M49 GAS, suggesting serotype specific interactions with the host cell cytoskeleton. Despite transcriptional induction, secreted IL-8 levels of deltafasX mutant infected cells were below those of non-infected cells, indicating an absence of Fas expression could be important for GAS tissue colonization and long-term intracellular persistence. Oppositely, activity of the S. pyogenes Fas-system apparently promotes high adherence and internalization rates, massive cytokine gene transcription and cytokine release, host cell apoptosis via a caspase-2 activation pathway, and cytotoxicity. Thus, the S. pyogenes Fas two-component signal transduction system could be involved in local tissue destruction and general bacterial aggressiveness towards host cells.

Apoptosis↗

PubMed2004

Streptococcus pyogenes fibronectin-binding protein F2: expression profile, binding characteristics, and impact on eukaryotic cell interactions.

Some Streptococcus pyogenes (group A streptococci, GAS) strains have previously been shown to express the fibronectin-binding protein F2 instead of the functionally related but structurally dissimilar protein F1/SfbI. In this study, recombinant N-terminal and C-terminal portions and the two fibronectin-binding domains of protein F2 were used to assess affinity parameters of the interaction with fibronectin and its N-terminal 70-, 30-, and 45-kDa fragments. The association and dissociation equilibrium constants for both binding domains were in the nanomolar range, although the repeat domain of protein F2 exceeded the affinity of the unique domain by up to one order magnitude. Both domains primarily interacted with the 30-kDa fibronectin fragment. Using a prtF2 gene isogenic mutant of a serotype M49 GAS strain that does not harbor the protein F1/SfbI gene, the attachment values of whole bacteria to immobilized fibronectin and to HEp-2 epithelial cells were found to be 6- and 2-fold decreased, respectively. Reduction of prtF2 mutant internalization rates for eukaryotic cells exceeded the reduction of attachment rates, indicating an independent contribution of protein F2 to both processes. The prtF2 transcription and protein F2 expression profiles documented maximum expression at the transition to the stationary phase especially under aerobic growth condition. The protein F2 function as the major fibronectin-binding adhesin in a subset of GAS strains, its expression pattern, and highly specific interaction with fibronectin would be consistent with a status as an indispensable virulence factor for both earlier and later pathogenetic stages of GAS superficial infections.

Adhesins, Bacterial↗

PubMed2004

Cell density--dependent regulation: basic principles and effects on the virulence of Gram-positive cocci.

PRINCIPLES: Quorum sensing (QS) regulation appears to be a consequence of interbacterial communication by which bacteria of one or even different species learn about their current population density and react in a defined way to that information. QS-regulation is a three step process: in the first step specific signaling molecules are produced and secreted to the exterior space. In the second step, the molecules accumulate e.g. with growing population density. In the last step, a supra-threshold concentration of the molecules is extra- or intra-cellularly sensed by the bacteria and leads to a cascade of regulatory activities. While Gram-negative bacteria can employ five or more different chemical classes of signaling molecules, Gram-positive cocci predominantly use special oligopeptides for specific signaling. DESCRIPTIONS: Examples of QS-regulatory effects on virulence factor expression in Staphylococcus aureus, Streptococcus mutans, and Enterococcus faecalis are given. In these bacteria, QS-regulation appears to be crucial for displaying tissue invasiveness and/or biofilm formation. APPLICATIONS: The high specificity of the initial signal sensing and the importance for expressing special virulence traits make this type of gene expression control a promising target for the development of novel therapeutics. The options for such therapies are critically discussed based on practical experiences with interference in S. aureus QS-regulation.

Animals↗

PubMed2004

The intracellular status of Streptococcus pyogenes: role of extracellular matrix-binding proteins and their regulation.

Streptococcus pyogenes (group A streptococci, GAS) is an important and exclusively human pathogen. Adherence to and internalization into host cells significantly contributes to the pathogenesis of GAS infections. The adherence mechanism is a two-step process in which host extracellular matrix (ECM) proteins act as prime targets. GAS may express more than a dozen different microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that attach to fibronectin or collagen. One of them, protein F1/SfbI binds fibronectin and mediates adherence of GAS to host cells. Bound fibronectin acts as a bridging molecule towards host cell integrins, which in turn initialize the uptake process that leads to GAS internalization. In their safe intracellular niche GAS can persist protected from antibiotics and host defense, a scenario currently discussed in the context of treatment failure, asymptomatic GAS carriers and recurrent GAS infections. Patients with such low grade infections represent the main GAS reservoir from which the bacteria are spread in the general population. Due to their important function, expression of GAS MSCRAMMs is under control of several "stand alone" transcriptional regulators and two-component signal transduction systems. Several regulator genes are organized together with MSCRAMM genes on one of two potential pathogenicity islands, act together in a growth phase-dependent regulatory network and are expressed in a strain-specific manner. A detailed understanding of these mechanisms is crucial, since interference with MSCRAMM function alone or in conjunction with specific manipulations of regulators is an attractive goal for novel anti-infective strategies.

Adhesins, Bacterial↗

PubMed2004

Function of the fibronectin-binding serum opacity factor of Streptococcus pyogenes in adherence to epithelial cells.

The serum opacity factor (SOF) of Streptococcus pyogenes is a serotyping tool and pathogenesis factor. Using SOF-coated latex beads in cell adherence assays and antiserum directed against SOF in S. pyogenes-HEp-2 cell adherence inhibition experiments, we demonstrate SOF involvement in the fibronectin-mediated adherence of S. pyogenes to epithelial cells. SOF exclusively targets the 30-kDa N-terminal region of fibronectin. The interaction revealed association and dissociation constants 1 order of magnitude lower than those of other S. pyogenes fibronectin-binding proteins.

Binding Sites↗

PubMed2003

Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions.

Streptococcus pyogenes (group A streptococcus, GAS) is a very important human pathogen with remarkable adaptation capabilities. Survival within the harsh host surroundings requires sensing potential on the bacterial side, which leads in particular to coordinately regulated virulence factor expression. GAS 'stand-alone' response regulators (RRs) and two-component signal transduction systems (TCSs) link the signals from the host environment with adaptive responses of the bacterial cell. Numerous putative regulatory systems emerged from GAS genome sequences. Only three RRs [Mga, RofA-like protein (RALP) and Rgg/RopB] and three TCSs (CsrRS/CovRS, FasBCAX and Ihk/Irr) have been studied in some detail with respect to their growth-phase-dependent activity and their influence on GAS-host cell interaction. In particular, the Mga-, RALP- and Rgg/RopB-regulated pathways display interconnected activities that appear to influence GAS colonization, persistence and spreading mechanisms, in a growth-phase-related fashion. Here, we have summarized our current knowledge about these RRs and TCSs to highlight the questions that should be addressed in future research on GAS pathogenicity.

Bacterial Proteins↗

PubMed2003

Epidemiology and virulence gene expression of intracellular group A streptococci in tonsils of recurrently infected adults.

Intracellularly persistent group A streptococci (GAS, Streptococcus pyogenes) have been associated with recurrent tonsillopharyngitis and antibiotic treatment failure. As a supplementation of the published in vitro data, conventional bacteriology and molecular epidemiology was performed on material from 29 adult patients of a German army hospital with anamnestic signs of recurrent tonsillopharyngitis. Pre-surgery tonsil swabs and the surgically removed tonsils were examined with respect to growth of aerobic bacteria in absence and presence of antibiotics with exclusively extracellular activity. Under such antibiotic selection, Staphylococcus aureus and GAS were cultured from specimens of 13 and 3 patients, respectively. In every material GAS-positive by culture methods, the intracellular location of the penicillin-susceptible GAS isolates was confirmed by immunohistologic examination of tonsillar sections using a GAS-specific IgG antibody. The three intracellular GAS isolates were typed by emm gene sequencing and could be associated to types M6 and M49 (two isolates). The bacteria were serially passaged on sheep blood agar, and semiquantitative mRNA analysis from virulence genes was performed using bacteria of the 4th and 25th passage after isolation. An M-type-specific pattern of virulence gene expression and different gene expression levels in relation to the passage number were observed.

Adenoids↗

PubMed2002

The role of the map protein in Staphylococcus aureus matrix protein and eukaryotic cell adherence.

The Staphylococcus aureus Map protein was proposed to act as a multifunctional adhesin. Using a map- mutant, a complemented strain and recombinant Map, we demonstrated that Map was not a conventional adhesin and was not involved in binding soluble extracellular matrix (ECM) proteins and in staphylococcal adherence to immobilized ECM proteins. However, Map provided a substrate for efficient and species-specific adherence of staphylococcal cells. This interaction was dose-dependent and was inhibited by specific anti-Map antibodies. According to ligand blots, two staphylococcal surface proteins of 82 and 50 kDa appeared to function as Map receptors. Adherence of the mutant to epithelial cells was reduced by 80% as compared to wild-type and complemented strains. However, adherence was not followed by a similar high rate of internalization. In conclusion, Map can function as an endogenous adhesion substrate in the attachment to plastic surfaces and eukaryotic cells via interaction with staphylococcal surface adhesins.

Bacterial Adhesion↗

PubMed2002

Group A streptococcal RofA-type global regulators exhibit a strain-specific genomic presence and regulation pattern.

RofA-like protein (RALP) type regulators have been shown to exist in different forms in group A streptococci (GAS) and to regulate the expression of important bacterial adhesins. This study shows that the vast majority of strains from different GAS M serotypes carried a rofA virulence regulator gene in their genome and that this gene could be detected in combination with other RALP genes and RALP-dependent adhesin genes in a strain-specific manner. The gene encoding the Nra regulator was predominantly found in opacity factor (OF)-negative serotypes. When analysing a rofA mutant in a serotype M2 strain, the strain specificity was also found in the positive and negative regulatory functions of RALP genes as well as in the type and number of virulence genes and functions controlled by the RALP genes. Of 17 virulence-associated genes tested, only one, the putative streptolysin S gene, was observed to be derepressed in RALP mutants of three different GAS serotype strains. This strain-specific variability of RALP regulon sizes is associated with different patterns of host cell attachment and internalization. In addition, RofA2 was shown to control expression of the ribosomal protein gene rpsL. As a consequence, it was demonstrated for the first time in streptococci that aminoglycoside resistance mediated by rpsL expression is apparently controlled by a virulence gene regulator.

Bacterial Adhesion↗

PubMed2002

Involvement of Lsp, a member of the LraI-lipoprotein family in Streptococcus pyogenes, in eukaryotic cell adhesion and internalization.

Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn(2+) and Cu(2+) ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.

Bacterial Proteins↗