Publications by authors named "Annett Petrich"

12 Publications

  • Page 1 of 1

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Biomolecules 2020 05 22;10(5). Epub 2020 May 22.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Street 24-25, 14476 Potsdam, Germany.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.
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http://dx.doi.org/10.3390/biom10050802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277205PMC
May 2020

Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus.

J Virol 2020 07 1;94(14). Epub 2020 Jul 1.

Department of Molecular Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany

Viruses from the family are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with case fatality rates of up to 50%. Here, we comprehensively investigated entry of the Old World hantavirus Puumala virus (PUUV) into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are greatly reduced. We demonstrate that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, decreases PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV particles and show colocalization of stained virions and markers of macropinosomes. Finally, we report a significant increase in the fluid-phase uptake of cells infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis. The family comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.
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http://dx.doi.org/10.1128/JVI.00184-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343216PMC
July 2020

Aerococcus urinae - A potent biofilm builder in endocarditis.

PLoS One 2020 23;15(4):e0231827. Epub 2020 Apr 23.

Biofilmzentrum, Dept. of Microbiology, Infectious Disease and Immunology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231827PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180067PMC
July 2020

Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis.

Anaerobe 2017 Oct 12;47:25-32. Epub 2017 Mar 12.

Biofilmcenter, Deutsches Herzzentrum Berlin, Berlin, Germany; Former German Consultant Laboratory for Treponema Identification, Charité - Universitätsmedizin Berlin, Berlin, Germany. Electronic address:

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).
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http://dx.doi.org/10.1016/j.anaerobe.2017.03.012DOI Listing
October 2017

Fluorescence in situ hybridization for the identification of Treponema pallidum in tissue sections.

Int J Med Microbiol 2015 Oct 28;305(7):709-18. Epub 2015 Aug 28.

Biofilmzentrum, Deutsches Herzzentrum Berlin, Consultant Laboratory for Tropheryma whipplei, Berlin, Germany. Electronic address:

Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models.
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http://dx.doi.org/10.1016/j.ijmm.2015.08.022DOI Listing
October 2015

Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years' experience in a National Reference Laboratory.

J Clin Microbiol 2013 Nov 21;51(11):3858-61. Epub 2013 Aug 21.

Institut für Mikrobiologie und Hygiene, Charité-Universitätsmedizin Berlin, Berlin, Germany.

The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens.
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http://dx.doi.org/10.1128/JCM.01703-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889782PMC
November 2013

Co-localized or randomly distributed? Pair cross correlation of in vivo grown subgingival biofilm bacteria quantified by digital image analysis.

PLoS One 2012 24;7(5):e37583. Epub 2012 May 24.

Institut für Mikrobiologie und Hygiene, Charité-Universitätsmedizin Berlin, Berlin, Germany.

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037583PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360060PMC
September 2012

Molecular epidemiology and spatial distribution of Selenomonas spp. in subgingival biofilms.

Eur J Oral Sci 2010 Oct;118(5):466-74

Institut für Mikrobiologie und Hygiene, Charité- Universitätsmedizin Berlin, Berlin, Germany.

The aetiology of periodontal disease has been a field of intensive research in the past decades. Along with a variety of other putative pathogens, different members of the genus Selenomonas have repeatedly been associated with both generalized aggressive periodontitis and chronic periodontitis. For the present study, a specific oligonucleotide probe targeting the majority of all oral Selenomonas spp. was designed. Their prevalence was determined, using dot-blot hybridization, in a total of 742 subgingival samples collected from patients with generalized aggressive (n=62) and chronic periodontitis (n=82), and from periodontitis-resistant subjects (n=19). In addition, fluorescence in situ hybridization (FISH) and electron microscopy were performed to analyze the spatial arrangement of Selenomonas in subgingival biofilms collected from patients with generalized aggressive periodontitis. In the samples from patients, Selenomonas spp. showed a lower prevalence in both diseased groups compared with other putative pathogens, and a relatively high prevalence in the periodontitis-resistant group. Consequently, Selenomonas spp. do not seem to be suitable diagnostic marker organisms for periodontal disease. By contrast, FISH and electron microscopic analysis of periodontal carriers revealed that Selenomonas spp. appeared in large numbers in all parts of the collected biofilms and seemed, if present in a site from patients, to make a relevant contribution to their structural organization.
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http://dx.doi.org/10.1111/j.1600-0722.2010.00765.xDOI Listing
October 2010

Filifactor alocis--involvement in periodontal biofilms.

BMC Microbiol 2010 Mar 1;10:66. Epub 2010 Mar 1.

Institut für Mikrobiologie und Hygiene, Charité - Universitätsmedizin Berlin, Dorotheenstrasse 96, 10117 Berlin, Germany.

Background: Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.

Results: A species-specific oligonucleotide probe, FIAL, was designed and evaluated. A total of 490 subgingival plaque samples were submitted to PCR and subsequent dot blot hybridization to compare the prevalence of F. alocis in patients suffering from generalized aggressive periodontitis (GAP), chronic periodontitis (CP), and control subjects resistant to periodontitis. Moreover, a specially designed carrier system was used to collect in vivo grown subgingival biofilms from GAP patients. Subsequent topographic analysis was performed using fluorescence in situ hybridization.While the majority of patients suffering from GAP or CP harboured F. alocis, it was rarely detected in the control group. In the examined carrier-borne biofilms the organism predominantly colonized apical parts of the pocket in close proximity to the soft tissues and was involved in numerous structures that constitute characteristic architectural features of subgingival periodontal biofilms.

Conclusions: F. alocis is likely to make a relevant contribution to the pathogenetic structure of biofilms accounting for periodontal inflammation and can be considered an excellent marker organism for periodontal disease.
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http://dx.doi.org/10.1186/1471-2180-10-66DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846919PMC
March 2010

Genetic control of the innate immune response to Borrelia hermsii influences the course of relapsing fever in inbred strains of mice.

Infect Immun 2010 Feb 7;78(2):586-94. Epub 2009 Dec 7.

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

Host susceptibility to infection is controlled in large measure by the genetic makeup of the host. Spirochetes of the genus Borrelia include nearly 40 species of vector-borne spirochetes that are capable of infecting a wide range of mammalian hosts, causing Lyme disease and relapsing fever. Relapsing fever is associated with high-level bacteremia, as well as hematologic manifestations, such as thrombocytopenia (i.e., low platelet numbers) and anemia. To facilitate studies of genetic control of susceptibility to Borrelia hermsii infection, we performed a systematic analysis of the course of infection using immunocompetent and immunocompromised inbred strains of mice. Our analysis revealed that sensitivity to B. hermsii infections is genetically controlled. In addition, whereas the role of adaptive immunity to relapsing fever-causing spirochetes is well documented, we found that innate immunity contributes significantly to the reduction of bacterial burden. Similar to human infection, the progression of the disease in mice was associated with thrombocytopenia and anemia. Histological and fluorescence in situ hybridization (FISH) analysis of infected tissues indicated that red blood cells (RBCs) were removed by tissue-resident macrophages, a process that could lead to anemia. Spirochetes in the spleen and liver were often visualized associated with RBCs, lending support to the hypothesis that direct interaction of B. hermsii spirochetes with RBCs leads to clearance of bacteria from the bloodstream by tissue phagocytes.
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http://dx.doi.org/10.1128/IAI.01216-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812184PMC
February 2010

Rapid and accurate diagnosis of human intestinal spirochetosis by fluorescence in situ hybridization.

J Clin Microbiol 2009 May 11;47(5):1393-401. Epub 2009 Mar 11.

Institut für Mikrobiologie und Hygiene, Charité-Universitätsmedizin Berlin, Charité Campus Mitte, Berlin, Germany.

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.
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http://dx.doi.org/10.1128/JCM.02469-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681872PMC
May 2009

Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows.

Vet Microbiol 2008 Apr 7;128(1-2):118-25. Epub 2007 Oct 7.

Institut für Mikrobiologie und Hygiene, Charité - Universitätsmedizin, Dorotheenstrasse 96, Berlin, Germany.

Digital dermatitis (DD) of cattle leads to lameness and a decrease of milk production and is responsible for major economic losses worldwide. Although a bacterial aetiology is generally accepted, it still is unclear which microorganisms cause and/or maintain the disease. Recently, a previously undiscovered bacterial species, Guggenheimella bovis, has been isolated from the front of two DD lesions in Swiss cattle and suggested as a potential pathogen. The aims of the present study were to determine the prevalence of G. bovis in 58 German cows suffering from DD via dot blot hybridization, and to analyse the spatial distribution of G. bovis within the affected tissue by fluorescence in situ hybridization (FISH). A species-specific probe, GUBO1, was designed and evaluated. In none of the 58 samples Guggenheimella could be detected, while cultured G. bovis was reliably identified by GUBO1. Further FISH experiments were carried out on two additional biopsies of Swiss cattle tested positive for G. bovis by quantitative PCR and permitted visualization of the newly discovered bacteria in situ. In these biopsies G. bovis proved to be tissue invasive forming characteristic spherical microcolonies not only within the bacterial biofilm but also in seemingly unaffected parts of the tissue not yet reached by the advancing bacterial front. Although the presence of G. bovis does not constitute an essential premise for DD, it seems likely that the bacterial species involved in DD vary, and that in some cases G. bovis is crucial for the development of DD lesions.
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http://dx.doi.org/10.1016/j.vetmic.2007.09.024DOI Listing
April 2008