Biofilm formation is a major pathogenetic factor of Staphylococcus epidermidis. In S. epidermidis the alternative sigma factor sigma B was identified to regulate biofilm formation in S. epidermidis ...1457. In S. aureus sigma B dependent regulation plays a minor role, whereas sarA (Staphylococcus accessory regulator) is an essential regulator. Therefore, we investigated the impact of sigma B on sarA transcription and biofilm formation in three independent S. epidermidis isolates. Mutants with dysfunctional sigma B displayed a strongly reduced biofilm formation, whereas in mutants with constitutive sigma B activity biofilm formation was increased. Transcriptional analysis revealed that icaA transcription was down-regulated in all sigma B negative mutants while icaR transcription was up-regulated. However, transcriptional differences varied between individual strains, indicating that additional sigma B-dependent regulators are involved in biofilm expression. Interestingly, despite the presence of a sigma B promoter beside two sigma A promoters no differences, or only minor ones, were observed in sarA transcription, indicating that sigma B-dependent sarA transcript has no influence on the phenotypic changes. The data observed in independent clinical S. epidermidis isolates suggests that, in contrast to S. aureus, regulation of biofilm formation by sigma B is a general feature in S. epidermidis. Additionally, we were able to demonstrate that the sarA- dependent regulation is not involved in this regulatory pathway.
Due to its ability to attach to polymeric surfaces Staphylococcus epidermidis is a common pathogen in chronic, medical device-associated infections. Attached S. epidermidis displays reduced ...susceptibility against a variety of antimicrobial substances, and little correlation between standard susceptibility test results and clinical outcome of antibiotic treatment is observed. In this study we established a new, versatile, and easy method of antimicrobial susceptibility testing for attached Staphylococcus epidermidis, suitable for both biofilm-negative and biofilm-positive attached bacteria using readily available equipment. For three biofilm-positive wild-type strains and their biofilm-negative mutants minimal attachment killing concentrations (MAK) of penicillin, oxacillin, vancomycin, and gentamicin were determined. Depending on strain and investigated antibiotics, a heterogeneous MAK (MAK(hetero)) could be differentiated from a homogeneous resistance (MAK(homo)), favoring a model of few persisters within attached cells under antibiotic treatment. For the biofilm-negative mutants, a lower MAK(homo) was detected than for the corresponding wild types for some of the tested antibiotics, which probably resulted from higher bacterial inocula of wild-type strains, whereas the MAK(hetero) were comparable for mutants and wild types for most of the tested antibiotics and strains. These data indicate that biofilm formation is not a necessary prerequisite for persistence of attached S. epidermidis cells under antibiotic treatment, which could explain therapeutic failure in foreign body-associated infections due to biofilm-negative S. epidermidis isolates. The highly individual resistance phenotypes of the investigated strains with different antibiotics suggests that MAK determination could help to predict the therapeutic outcome of foreign body-associated infections with both biofilm-positive and biofilm-negative S. epidermidis.
In the embryonic lens, cells of the anterior epithelium proliferate, migrate through the equatorial zone, and elongate to form primary lens fibers at the posterior pole. During this stage of ...development, cholinesterase (ChE) activity has been described as in other embryonic tissues implicated in morphogenesis. The purpose of the study was to demonstrate in addition to ChE the presence of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) and to test whether the muscarinic cholinergic system is involved in the regulation of cellular movements.
In the chick embryo lens (Hamburger-Hamilton HH stage 21), the expression of mAChR and ChAT were demonstrated by immunohistochemistry. Isolated whole embryonic lenses were loaded with fura-2/AM, and changes of cytosolic calcium were measured after muscarinic stimulation. Size changes were assessed by morphometry with the use of time-lapse videos.
mAChR was present in the equatorial zone of the lens and in the elongating primary lens fibers. Anti-ChAT immunoreactivity was restricted to the primary lens fibers. Addition of carbachol induced an intracellular Ca2+ peak followed by a plateau phase of extracellular Ca2+ influx. The plateau phase was reversed by addition of atropine. Concomitant with the calcium release, a contraction of the lens and an increase in opacity was observed.
The localization of ChAT in the differentiating fibers of the lens indicates that acetylcholine is synthesized during differentiation and modulates morphogenesis and elongation of mAChR-positive progenitor cells in the equatorial zone. The carbachol-induced contraction indicates that the embryonic muscarinic system may be involved in the regulation of cellular movements.
... expression of STAT3 and CDH2/N-cadherin in human ESCs was nearly identical to that in human fibroblasts (Fig. 1i). ... the expression of these genes cannot be used to distinguish pluripotent ...cells from fibroblasts. According to the Microarray Facility Tübingen, one of the critical points of the challenge by Ko et al.1 is that they use our data set for comparison with their own expression profiles from different cell types.The use of microarrays that are generated several days ormonths apart introduces systematic batch effects or non-biological differences, which make it meaningless to compare samples from different batches directly.
IcaADBC-encoded proteins mediate synthesis of the intercellular polysaccharide adhesin (PIA), which is essentially involved in Staphylococcus epidermidis biofilm formation. Seventy S. epidermidis ...isolates were investigated for their ability to form biofilm and synthesize PIA in different growth media including trypticase soy broth obtained from Becton Dickinson (TSBBBL), or Oxoid (TSB(OXOID)), and TSB(OXOID) supplemented with 0.5% N-acetylglucosamine, and for the presence of icaADBC. Dependent on the medium used (TSB(BBL) or TSB(OXOID)), the isolates exhibited a differential expression of PIA and biofilm formation, with 51 (72.85%) and 34 (48.57%) being biofilm positive, respectively. Using these growth media four different expression phenotypes were differentiated: similar quantities of biofilm formation in both TSBBBL and TSB(OXOID) (11 isolates, type A), significantly reduced biofilm expression in TSB(OXOID) compared to TSB(BBL) (23 isolates, type B), biofilm negative in TSB(OXOID) but biofilm producing in TSB(BBL) (17 isolates, type C) and biofilm negative in both media (19 isolates, type D). For all strains a biofilm-positive phenotype in a specific medium was closely linked to expression of PIA in that medium. All but one strain of expression type A-C and 7/19 expression type D strains were icaADBC positive. On the basis of restriction fragment length polymorphisms, the isolates were classified into two main icaADBC genotypes. There was no association between the observed biofilm-expression types and a defined icaADBC genotype. In the biofilm-negative S. epidermidis 5179, isolated from a ventriculo-atrial shunt infection, the insertion of IS257 interrupted the transcription of icaADBC, resulting in a PIA- and biofilm-negative phenotype. In all other icaADBC-positive, biofilm-negative isolates no major alterations of the icaADBC gene locus were identified. Obviously, expression of icaADBC, PIA synthesis and biofilm formation are integrated into a complex regulatory network involving other determinants independent of icaADBC genotype. Inactivation of icaADBC by IS elements is apparently a rare cause of a biofilm-negative phenotype in clinical S. epidermidis isolates.
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the ...regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at alpha -, beta -, gamma -, and epsilon -sites to generate soluble ectodomains, soluble A beta -like-, and intracellular fragments termed mEpEX, mEp- beta , and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the alpha - and beta -sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble A beta -like fragments mEp- beta and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the gamma -secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent gamma -cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, gamma -secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.
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