Papers by Elisabeth Grohmann
Frontiers in molecular biosciences, Feb 6, 2024
Conjugation is a major mechanism that facilitates the exchange of antibiotic resistance genes amo... more Conjugation is a major mechanism that facilitates the exchange of antibiotic resistance genes among bacteria. The broad-host-range Inc18 plasmid pIP501 harbors 15 genes that encode for a type IV secretion system (T4SS). It is a membrane-spanning multiprotein complex formed between conjugating donor and recipient cells. The penultimate gene of the pIP501 operon encodes for the cytosolic monomeric protein TraN. This acts as a transcriptional regulator by binding upstream of the operon promotor, partially overlapping with the origin of transfer. Additionally, TraN regulates traN and traO expression by binding upstream of the P traNO promoter. This study investigates the impact of nine TraN amino acids involved in binding to pIP501 DNA through site-directed mutagenesis by exchanging one to three residues by alanine. For three traN variants, complementation of the pIP501ΔtraN knockout resulted in an increase of the transfer rate by more than 1.5 orders of magnitude compared to complementation of the mutant with native traN. Microscale thermophoresis (MST) was used to assess the binding affinities of three TraN double-substituted variants and one triple-substituted variant to its cognate pIP501 double-stranded DNA. The MST data strongly correlated with the transfer rates obtained by biparental mating assays in Enterococcus faecalis. The TraN variants TraN_ R23A-N24A-Q28A, TraN_H82A-R86A, and TraN_G100A-K101A not only exhibited significantly lower DNA binding affinities but also, upon complementation of the pIP501ΔtraN knockout, resulted in the highest pIP501 transfer rates. This confirms the important role of the TraN residues R23, N24, Q28, H82, R86, G100, and K101 in downregulating pIP501 transfer. Although TraN is not part of the mating pair formation complex, TraE, TraF, TraH, TraJ, TraK, and TraM were coeluted with TraN in a pull-down. Moreover, TraN homologs are present not only in Inc18 plasmids but also in RepA_N and Rep_3
FEMS Microbiology Ecology, Dec 22, 2016
Long-term monitoring of in situ aided phytostabilisation at a metal-contaminated mine soil reveal... more Long-term monitoring of in situ aided phytostabilisation at a metal-contaminated mine soil revealed variable effects in terms of microbial activity, biomass and diversity.
ASM Press eBooks, May 26, 2015
Journal of Dental Research, Jun 23, 2021
Genome-wide association studies identified various loci associated with periodontal diseases, but... more Genome-wide association studies identified various loci associated with periodontal diseases, but assigning causal alleles remains difficult. Likewise, the generation of biological meaning underlying a statistical association has been challenging. Here, we characterized the genetic association at the gene ST8SIA1 that increases the risk for severe periodontitis in smokers. We used CRISPR/dCas9 activation and RNA-sequencing to identify genetic interaction partners of ST8SIA1 and to determine its function in the cell. We used reporter gene assays to identify regulatory elements at the associated single-nucleotide polymorphisms (SNPs) and to determine effect directions and allele-specific changes of enhancer activity. Antibody electrophoretic mobility shift assays proved allele-specific transcription factor binding at the putative causal SNPs. We found the reported periodontitis risk gene ABCA1 as the top upregulated gene following ST8SIA1 activation. Gene set enrichment analysis showed highest effects on integrin cell surface interactions (area under the curve [AUC] = 0.85; q = 4.9 × 10−6) and cell cycle regulation (AUC = 0.89; q = 1.6 × 10−5). We identified 2 associated repressor elements in the introns of ST8SIA1 that bind the transcriptional repressor BACH1. The putative causative variant rs2012722 decreased BACH1 binding by 40%. We also pinpointed ST8SIA1 as the target gene of the association. ST8SIA1 inhibits cell adhesion with extracellular matrix proteins, integrins, and cell cycle, as well as enhances apoptosis. Likewise, tobacco smoke reportedly results in an inhibition of cell adhesion and a decrease in integrin-positive cells and cell growth. We conclude that impaired ST8SIA1 repression, independently caused by reduced BACH1 binding at the effect T allele, as well as by tobacco smoke, contributes to higher ST8SIA1 levels, and in smokers who carry the effect T allele, both factors would be additive with damaging effects on the gingival barrier integrity. The activity of ST8SIA1 is also linked with the periodontitis risk gene ABCA1.
Journal of Biological Chemistry, 2013
Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistan... more Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistance genes among bacteria. Results: Surface exposure of transfer protein TraM from the Gram-positive (Gϩ) plasmid pIP501 was confirmed, and its crystal structure was solved. Conclusion: Structural relations to type IV secretion (T4S) proteins provide a novel classification scheme. Significance: The novel classification will help elucidate structure-function relationships in Gϩ T4S systems. Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gramnegative (G؊) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G؉), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G؉ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G؊) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G؉). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G؉ and G؊ bacteria, we propose a new classification scheme of VirB8-like proteins.
Microbiology, Dec 1, 2011
Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the... more Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Enterococci, especially Enterococcus faecium, are well-known pathogens of hospitalized patients and are frequently linked with resistance against multiple antibiotics, which compromises effective therapy. Rabbit immune serum raised against heat-killed E. faecium E155, a HiRECC clone, was used in an opsonophagocytic assay, an inhibition assay and a mouse bacteraemia model to identify targets of opsonic and protective antibodies. Serum against whole heat-killed bacteria was opsonic and recognized a protein of about 72 kDa that was abundantly secreted. This protein, identified as SagA by LC-ES-MS/MS, was expressed in Escherichia coli and purified. Rabbit serum raised against the purified protein showed opsonic killing activity that was inhibited by almost 100 % using 100 mg purified protein ml "1. In a mouse bacteraemia model, a statistically significant reduction of the colony counts in blood was shown with immune rabbit serum compared with preimmune serum using the homologous and a heterologous vancomycin-resistant enterococci (VRE) strain. These results indicate that SagA could be used as a promising vaccine target to treat and/or prevent VRE bacteraemia.
Materials Science and Engineering: C, Feb 1, 2021
Multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause n... more Multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause nosocomial infections that can have deleterious effects on human health. Thus, it is imperative to find solutions to treat these detrimental infections as well as to control their spread. We tested the effect of two different antimicrobial materials, functionalised graphene oxide (GOX), and AGXX® coated on cellulose fibres, on the growth and transcriptome of the clinical MRSA strain S. aureus 04-02981. In addition, we investigated the effect of a third material as a combination of GOX and AGXX® fibres on S. aureus 04-02981. Standard plate count assay revealed that the combination of fibres, GOX-AGXX® inhibited the growth of S. aureus 04-02981 by 99.98%. To assess the effect of these antimicrobials on the transcriptome of our strain, cultures of S. aureus 04-02981 were incubated with GOX, AGXX®, or GOX-AGXX® fibres for different time periods and then subjected to RNA-sequencing. Uncoated cellulose fibres were used as a negative control. The antimicrobial fibres had a huge impact on the transcriptome of S. aureus 04-02981 affecting the expression of 2650 genes. Primarily genes related to biofilm formation and virulence (such as agr, sarA, and those of the two-component system SaeRS), and genes crucial for survival in biofilms (like arginine metabolism arc genes) were repressed. In contrast, the expression of siderophore biosynthesis genes (sbn) was induced, a probable response to stress imposed by the antimicrobials and the conditions of iron-deficiency. Genes associated with potassium transport, intracellular survival and pathogenesis (kdp) were also differentially expressed. Our data suggest that the combination of GOX and AGXX® acts as an efficient antimicrobial against S. aureus 04-02981. Thus, these materials are potential candidates for applications in antimicrobial surface coatings.
Antibiotics, Feb 4, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Microorganisms
Thermophilic composting is a suitable treatment for the recycling of organic wastes for agricultu... more Thermophilic composting is a suitable treatment for the recycling of organic wastes for agriculture. However, using human excreta as feedstock for composting raises concerns about antibiotic resistances. We analyzed samples from the start and end of a thermophilic composting trial of human excreta, together with green cuttings and straw, with and without biochar. Beta-lactamase genes blaCTX-M, blaIMP, and blaTEM conferring resistance to broad-spectrum beta-lactam antibiotics, as well as horizontal gene transfer marker genes, intI1 and korB, were quantified using qPCR. We found low concentrations of the beta-lactamase genes in all samples, with non-significant mean decreases in blaCTX-M and blaTEM copy numbers and a mean increase in blaIMP copy numbers. The decrease in both intI1 and korB genes from start to end of composting indicated that thermophilic composting can decrease the horizontal spread of resistance genes. Thus, thermophilic composting can be a suitable treatment for the...
Frontiers in Microbiology
In times of climate change, practicing a form of sustainable, climate-resilient and productive ag... more In times of climate change, practicing a form of sustainable, climate-resilient and productive agriculture is of primordial importance. Compost could be one form of sustainable fertilizer, which is increasing humus, water holding capacity, and nutrient contents of soils. It could thereby strengthen agriculture toward the adverse effects of climate change, especially when additionally combined with biochar. To get access to sufficient amounts of suitable materials for composting, resources, which are currently treated as waste, such as human excreta, could be a promising option. However, the safety of the produced compost regarding human pathogens, pharmaceuticals (like antibiotics) and related resistance genes must be considered. In this context, we have investigated the effect of 140- and 154-days of thermophilic composting on the hygienization of human excreta and saw dust from dry toilets together with straw and green cuttings with and without addition of biochar. Compost samples...
New Biotechnology, 2018
Respiratory bacterial and viral infections cause frequently mild to severe diseases worldwide, it... more Respiratory bacterial and viral infections cause frequently mild to severe diseases worldwide, it is a serious public health problem. To develop new anti-bacterial and viral drugs, new relevant and efficient tools are needed. We report herein a platform of 3D epithelia made of human primary airway epithelial cells, MucilAir TM , for anti-bacterial and viral drug screening. As proof-of-concept, typical disease-causing bacteria and viruses were used to infect MucilAir TM tissues. The effect of bacterial and viral infections can be easily and accurately monitored with several endpoints: Trans Epithelial Electrical Resistance (TEER); cytotoxicity (LDH), cilia activity, mucin and IL-8 release. Pseudomonas aeruginosa (PA) infection induces a loss of TEER, 20% cytotoxicity and an increase of Il-8 (+100 ng/ml). On the contrary, SP strongly increases the mucin production. Antibiotics like Meronem efficient inhibits the bacterial growth and abrogates the bacterial effect on endpoints. Clinically relevant Rhinovirus (A16, C15), Enterovirus (EV68) and Influenza A virus (H1N1, H3N2) strains were added to fully differentiated MucilAir TM-Pool. Released viral genome copy number, overall mucin secretion, cilia beating frequency, MCC and tissue integrity were assessed daily during 4 days. MucilAir TM supports high rate of replication for all tested viruses, including difficult-toculture HRV-C15. Anti-viral drug, Rupintrivir, efficiently inhibited the replication of HRV-A16 and HRV-C15 in a dose and time dependent manner and restored MCC impaired by EV68. Oseltamivir reduced the replication of H1N1 and H3N2 and restored the impaired barrier function. These results demonstrate that MucilAir TM is a robust, reliable and relevant tool for anti-bacterial and antiviral drug development.
Journal of Antimicrobial Chemotherapy, 2015
Objectives: Tigecycline represents one of the last-line therapeutics to combat multidrug-resistan... more Objectives: Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp. Methods: Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality. Results: Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus. Conclusions: Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.
Springer eBooks, 2013
The use of general descriptive names, registered names, trademarks, service marks, etc. in this p... more The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a speci fi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Springer eBooks, 2014
Heavy metals are important environmental pollutants which belong to the group of non-biodegradabl... more Heavy metals are important environmental pollutants which belong to the group of non-biodegradable and persistent compounds deposited in plant tissue (e.g. vegetables) which are then consumed by animals and humans. Increased pollution of natural environment with heavy metals, particularly in areas with anthropogenic pressure, also contributes to disorders in the natural balance of microbial populations. Molecular analysis carried out during the past decades revealed that density and diversity of microorganisms significantly correlated with increased contamination of the environment with heavy metals. As a result, a selective promotion of metal-tolerant genera of microorganisms was observed. In general, microorganisms are organisms with relatively high tolerance of unfavourable conditions, and these properties evolved over millions of years. In this chapter, a variety of mechanisms responsible for adaptation of microorganisms to high heavy metal concentrations, e.g. metal sorption, uptake and accumulation, extracellular precipitation and enzymatic oxidation or reduction, will be reported. Moreover, molecular mechanisms responsible for their metal tolerance will be described. The efficiency of accumulation of heavy metals in the microbial cells will be discussed and presented in photos from a reflection electron microscope (REM). The capacities of microorganisms for metal accumulation can be exploited to remove, concentrate and recover metals from polluted sites. This provides the basis for biotechnological solutions for the remediation of contaminated environments. Bioremediation has been regarded as an environment-friendly, inexpensive and efficient means of environmental restoration. Since microorganisms constitute a key factor of this technology, knowledge of the nature and molecular mechanisms of their tolerance of increased heavy metal concentrations is essential.
Microbiological Research, Mar 1, 2018
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
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Papers by Elisabeth Grohmann