Biomedical subjects

Friedrich Götz

Publications and source records attributed to Friedrich Götz.

16 recordsLinked to original sources

PubMed2005

Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus.

Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild-type strain was O-acetylated at C6-OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan-specific O-acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.

Acetylation↗

PubMed2005

Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation.

A lipoprotein diacylglyceryl transferase (lgt) deletion mutant of Staphylococcus aureus SA113 was constructed. The lipoprotein and prelipoprotein expression, the growth behavior, and the ability of the mutant to elicit an immune response in various host cells were studied. In the wild type, the majority of [14C]palmitate-labeled lipoproteins were located in the membrane fraction, although some lipoproteins were also present on the cell surface and in the culture supernatant. The lgt mutant completely lacked palmitate-labeled lipoproteins and released high amounts of some unmodified prelipoproteins, e.g., the oligopeptide-binding protein OppA, the peptidyl-prolyl cis-trans isomerase PrsA, and the staphylococcal iron transporter SitC, into the culture supernatant. The growth of the lgt mutant was hardly affected in rich medium but was retarded under nutrient limitation. The lgt mutant and its crude lysate induced much fewer proinflammatory cytokines and chemokines in human monocytic (MonoMac6), epithelial (pulmonary A549), and endothelial (human umbilical vein endothelial) cells than the wild type. However, in whole blood samples, the culture supernatant of the lgt mutant was equal or even superior to the wild-type supernatant in tumor necrosis factor alpha induction. Lipoprotein fractionation experiments provided evidence that a small proportion of the mature lipoproteins are released by the S. aureus wild type despite the lipid anchor and are trapped in part by the cell wall, thereby exposing the immune-activating lipid structure on the cell surface. Bacterial lipoproteins appear to be essential for a complete immune stimulation by gram-positive bacteria.

Genome, Bacterial↗

PubMed2005

Solitary spinal artery aneurysms as a rare source of spinal subarachnoid hemorrhage: potential etiology and treatment strategy.

Solitary aneurysms of spinal arteries lacking associated vascular malformations are rare. We report three patients with spinal subarachnoid hemorrhage (SAH) due to rupture of such aneurysms, which regressed spontaneously, as confirmed on conventional angiography. One patient had spinal SAH with presumed spontaneous dissection of a segmental artery. In the other two, SAH resulted from ruptured fusiform aneurysms of the artery of Adamkiewicz immediately proximal to the anterior spinal artery. Solitary aneurysms of the spinal arteries appear to be etiopathologic entities completely different from intracranial aneurysms. Spontaneous occlusion seems to be common, justifying a wait-and-see strategy rather than urgent treatment.

Aged↗

PubMed2004

Bacterial evasion of innate host defenses--the Staphylococcus aureus lesson.

Bacterial pathogens such as Staphylococcus aureus use highly efficient mechanisms to evade recognition and elimination by the innate immune system. S. aureus produces sophisticated anti-inflammatory molecules and it employs several mechanisms protecting the bacteria against host cationic antimicrobial molecules such as defensin-like peptides and bacteriolytic enzymes such as lysozyme. Cell wall teichoic acids and lipoteichoic acids, complex Gram-positive surface polymers, and modified membrane lipids such as lysylphosphatidylglycerol are crucial in defensin resistance and other important aspects of staphylococcal virulence such as nasal colonization and biofilm formation on biomaterials. Certain S. aureus genes conferring escape from innate host defenses are conserved in many human pathogens suggesting that the underlying mechanisms are of general significance in bacterial virulence.

Biofilms↗

PubMed2004

Staphylococci in colonization and disease: prospective targets for drugs and vaccines.

Pathogenic staphylococci are now regarded in the scientific community as antibiotic resistant 'superbugs' because they have an amazing capacity to acquire resistance traits. Surprisingly, antibiotic development has decelerated. Promising targets for drug development are enzymes involved in the biosynthesis of cell envelope structures such as peptidoglycan, teichoic acids, membrane lipids, or cell wall associated adhesins. Compounds that inactivate or neutralize the most aggressive toxins such as the superantigens and the pore forming toxins have also been considered. In the past decade, global regulatory systems have been studied that contribute to virulence and might be candidates for target development. Targets that are particularly promising include all enzymatic reactions that are unique to bacteria and that are involved in central metabolism, such as methionine-tRNA(fMet) formyltransferase or the peptide deformylase, which have been successfully used for designing new inhibitors. There are also several known antibiotics that have roused new interest especially if they are active against multi-resistant staphylococci. Various cell wall components are promising candidates for active and passive immunization strategies such as capsule, slime, teichoic acids or cell wall bound adhesins. Several new targets for drugs or vaccines will arise from the functional analysis of the staphylococcal genomes that contain many hitherto unknown targets.

Anti-Bacterial Agents↗

PubMed2004

The ability of biofilm formation does not influence virulence of Staphylococcus aureus and host response in a mouse tissue cage infection model.

The virulence of Staphylococcus aureus Sa113 (SA113) and an isogenic ica deletion mutant (ica-), deficient in the production of polysaccharide intercellular adhesin (PIA), which is crucial for biofilm formation, was compared in a mouse tissue cage infection model. The minimal infective doses for the induction of persistent tissue infections in C57BL/6 mice were 10(3) CFU for both SA113 and the ica- mutant. Bacterial growth, initial adherence to surfaces within the implants and the course of inflammation including growth-dependent host TNF and MIP-2 release, influx of phagocytes and an accumulation of dead leukocytes were similar as well. Since SA113 expressed PIA in vivo, we could demonstrate that PIA and the lack of biofilm formation did not influence the capacity of S. aureus to induce persistent infections and did not modulate host responses in the mouse tissue cage model.

Animals↗

PubMed2004

Staphylococcal NreB: an O(2)-sensing histidine protein kinase with an O(2)-labile iron-sulphur cluster of the FNR type.

The nreABC (nitrogen regulation) operon encodes a new staphylococcal two-component regulatory system that controls dissimilatory nitrate/nitrite reduction in response to oxygen. Unlike other two-component sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB-cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desulphurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron-sulphur cluster formation) developed. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no iron-sulphur cluster was incorporated; both findings corroborated the importance of the cysteine residues. Comparison of the kinase activity of (i). the reconstituted (ii). the unreconstituted, and (iii). the unreconstituted and deferrated NreB-His indicated that NreB kinase activity depended on iron availability and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4-8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1-2 iron-sulphur [4Fe-4S](2+) clusters of the FNR-type. Unlike FNR, NreB does not act directly as transcriptional activator, but transfers the phosphoryl group to the response regulator NreC.

Adenosine Triphosphate↗

PubMed2003

Lack of biofilm contribution to bacterial colonisation in an experimental model of foreign body infection by Staphylococcus aureus and Staphylococcus epidermidis.

The contribution of in vivo biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis was studied in an experimental model of foreign body infections. Increasing inocula (from 10(2) to 10(7) organisms) of ica-positive strains of S. aureus and S. epidermidis and their ica-negative isogenic mutants (the ica locus codes for a major polysaccharide component of biofilm) were injected into subcutaneously implanted tissue cages in guinea pigs. Surprisingly, bacterial counts and time-course of tissue cage infection by ica-positive strains of S. aureus or S. epidermidis were equivalent to those of their respective ica-negative mutants, in the locally infected fluids and on tissue-cage-inserted plastic coverslips.

Animals↗

PubMed2003

Control of antimicrobial peptide synthesis by the agr quorum sensing system in Staphylococcus epidermidis: activity of the lantibiotic epidermin is regulated at the level of precursor peptide processing.

The accessory gene regulator (agr) quorum sensing system in staphylococci is responsible for the regulation of surface proteins and exoproteins, including many virulence factors in the pathogenic species Staphylococcus aureus and S. epidermidis. Strain S. epidermidis Tü3298 produces the lantibiotic epidermin. An isogenic agr deletion mutant of this strain showed a strong reduction of epidermin production. Detailed analysis of the impact of agr on epidermin biosynthesis revealed that agr does not interfere with the transcription of epidermin biosynthetic genes, but controls the extracellular processing of the N-terminal leader peptide by the EpiP protease.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01.

Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 microg of extracellular alkaline, Ca(2+)-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5alpha and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5alpha culture, 280 microg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level.

Amidohydrolases↗

PubMed2003

Identification of a 5-nucleotide sequence that controls expression of the ica locus in Staphylococcus aureus and characterization of the DNA-binding properties of IcaR.

Biofilm formation is an important aspect of the pathogenesis of staphylococcal infections. A beta-1,6-linked N-acetyl glucosamine polysaccharide is critical to biofilm elaboration and is synthesized by proteins encoded by the intercellular adhesion (ica) locus. These studies were undertaken to characterize the mechanism by which transcription of the ica locus in S. aureus is regulated using isogenic S. aureus MN8 and MN8 mucoid (MN8m) strains, the latter of which constitutively overproduces biofilm. Transformation of the ica locus from MN8m to the ica knock-out mutants of two strains, MN8 and NCTC 10833, conferred a strong biofilm-producing phenotype. Sequence analysis revealed a 5-nucleotide deletion within the promoter region of the ica locus in MN8m compared with the sequence in the wild-type locus. Deletion or substitution of these 5 nucleotides within the wild-type ica locus augmented transcription of the ica locus and induced the strong biofilm-producing phenotype. Gel shift analysis demonstrated that a protein(s) within cell-free lysates from strain MN8 bind(s) specifically to oligonucleotides representative of the wild-type ica promoter sequence and that this binding is greatly diminished by the deletion or substitution of the 5 nucleotides. DNase I footprint analysis revealed that purified IcaR, thought to be a regulator of ica transcription, also binds to the ica promoter sequence just upstream of the ica start codon, but its affinity for the ica promoter is unaffected by deletion of the 5-nucleotide motif. These findings identify a 5-nucleotide motif within the ica promoter region that has a functional role in transcriptional regulation of the ica locus that is independent of IcaR, and also show that IcaR binds to the promoter region of the S. aureus ica locus.

Bacterial Adhesion↗

PubMed2003

Amyotrophic lateral sclerosis (ALS) and mirror movements in a patient with polymicrogyria.

We report on a case of polymicrogyria with benign childhood epilepsy and amyotrophic lateral sclerosis (ALS). While performing dexterity tasks with either hand, strong unsustained mirror movements of the unintended hand were observed. The patient was seen over a period of three years and, as often seen in ALS, there was a moderate progress of the motor neuron disease affecting the upper and lower motor neuron in an asymmetrical manner. In addition, more rapidly progressive bulbar symptoms could be observed by the clinical and neurophysiological examination. MRI revealed polymicrogyria of the right frontal lobe with irregular bumpy inner and outer surface and abnormally thick and smooth cortex, dysplastic insular cortex and asymmetrically widened Sylvial fissure. No signs of atrophy, especially of the motorcortex and corticospinal tract were observed. The corpus callosum was completely developed and of normal size. We hypothesize an enrolment of the dysplastic right frontal lobe pathophysiology of the observed mirror movements.

Amyotrophic Lateral Sclerosis↗

PubMed2002

Staphylococcus aureus strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice.

Staphylococcus aureus is resistant to alpha-defensins, antimicrobial peptides that play an important role in oxygen-independent killing of human neutrophils. The dlt operon mediates d-alanine incorporation into teichoic acids in the staphylococcal cell envelope and is a determinant of defensin resistance. By using S. aureus wild-type (WT) and Dlt- bacteria, the relative contributions of oxygen-dependent and -independent antimicrobial phagocyte components were analyzed. The Dlt- strain was efficiently killed by human neutrophils even in the absence of a functional respiratory burst, whereas the killing of the WT organism was strongly diminished when the respiratory burst was inhibited. Human monocytes, which do not produce defensins, inactivated the WT and Dlt- bacteria with similar efficiencies. In addition, mice injected with the Dlt- strain had significantly lower rates of sepsis and septic arthritis and fewer bacteria in the kidneys, compared with mice infected with the WT strain.

Alanine↗

PubMed2002

Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01.

Ceramidase (CDase) hydrolyses the N-acyl linkage of the sphingolipid ceramide. We synthesized the non-fluorescent ceramide analogue (4E,2S,3R)-2-N-(10-pyrenedecanoyl)-1,3,17-trihydroxy-17-(3,5-dinitrobenzoyl)-4-heptadecene (10) that becomes fluorescent upon hydrolysis of its N-acyl bond. This novel substrate was used to study several kinetic aspects of the recombinant CDase from the pathogenic bacterium Pseudomonas aeruginosa PA01. Maximum CDase activity was observed above 1.5 microM substrate, with an apparent K(m) of 0.5+/-0.1 microM and a turnover of 5.5 min(-1). CDase activity depends on divalent cations without a strong specificity. CDase is inhibited by sphingosine and by several sphingosine analogues. The lack of inhibition by several mammalian CDase inhibitors such as D-erythro-MAPP, L-erythro-MAPP or N-oleoylethanolamine points to a novel active site and/or substrate binding region. The CDase assay described here offers the opportunity to develop and screen for specific bacterial CDase inhibitors of pharmaceutical interest.

Amidohydrolases↗

PubMed2002

Staphylococcus and biofilms.

The genetic and molecular basis of biofilm formation in staphylococci is multifaceted. The ability to form a biofilm affords at least two properties: the adherence of cells to a surface and accumulation to form multilayered cell clusters. A trademark is the production of the slime substance PIA, a polysaccharide composed of beta-1,6-linked N-acetylglucosamines with partly deacetylated residues, in which the cells are embedded and protected against the host's immune defence and antibiotic treatment. Mutations in the corresponding biosynthesis genes (ica operon) lead to a pleiotropic phenotype; the cells are biofilm and haemagglutination negative, less virulent and less adhesive on hydrophilic surfaces. ica expression is modulated by various environmental conditions, appears to be controlled by SigB and can be turned on and off by insertion sequence (IS) elements. A number of biofilm-negative mutants have been isolated in which polysaccharide intercellular adhesin (PIA) production appears to be unaffected. Two of the characterized mutants are affected in the major autolysin (atlE) and in D-alanine esterification of teichoic acids (dltA). Proteins have been identified that are also involved in biofilm formation, such as the accumulation-associated protein (AAP), the clumping factor A (ClfA), the staphylococcal surface protein (SSP1) and the biofilm-associated protein (Bap). Concepts for the prevention of obstinate polymer-associated infections include the search for new anti-infectives active in biofilms and new biocompatible materials that complicate biofilm formation and the development of vaccines.

Bacterial Proteins↗

PubMed1998

Physical and genetic map of the genome of Staphylococcus carnosus TM300.

A genome map of Staphylococcus carnosus TM300, an important micro-organism in the food industry and long used as a starter culture, was constructed by pulsed-field gel electrophoresis of DNA fragments obtained after digestion with NotI, SfiI and ApaI. The size of the chromosome was estimated to be 2590 kb. The fragments were assembled into a physical map using a combination of complementary methods including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. Fifteen genes or gene clusters were positioned on the physical map by Southern hybridization analysis. The map provides a basis for further analysis of the S. carnosus chromosome.

Chromosome Mapping↗