Heart rhythm in vitro: measuring stem cell-derived pacemaker cells on microelectrode arrays.

Background: Cardiac arrhythmias have markedly increased in recent decades, highlighting the urgent need for appropriate test systems to evaluate the efficacy and safety of new pharmaceuticals and the potential side effects of established drugs. Methods: The Microelectrode Array (MEA) system may be a suitable option, as it provides both real-time and non-invasive monitoring of cellular networks of spontaneously active cells. However, there is currently no commercially available cell source to apply this technology in the context of the cardiac conduction system (CCS). In response to this problem, our group has previously developed a protocol for the generation of pure functional cardiac pacemaker cells from mouse embryonic stem cells (ESCs). In addition, we compared the hanging drop method, which was previously utilized, with spherical plate-derived embryoid bodies (EBs) and the pacemaker cells that are differentiated from these. Results: We described the application of these pacemaker cells on the MEA platform, which required a number of crucial optimization steps in terms of coating, dissociation, and cell density. As a result, we were able to generate a monolayer of pure pacemaker cells on an MEA surface that is viable and electromechanically active for weeks. Furthermore, we introduced spherical plates as a convenient and scalable method to be applied for the production of induced sinoatrial bodies. Conclusion: We provide a tool to transfer modeling and analysis of cardiac rhythm diseases to the cell culture dish. Our system allows answering CCS-related queries within a cellular network, both under baseline conditions and post-drug exposure in a reliable and affordable manner. Ultimately, our approach may provide valuable guidance not only for cardiac pacemaker cells but also for the generation of an MEA test platform using other sensitive non-proliferating cell types.

Effect of TraN key residues involved in DNA binding on pIP501 transfer rates in Enterococcus faecalis.

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 PtraNO 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 pIP501∆traN 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 pIP501∆traN 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 family plasmids, which are frequently found in enterococci, streptococci, and staphylococci. This points to a widespread role of this repressor in conjugative plasmid transfer among Firmicutes.

Horizontal Gene Transfer of Antibiotic Resistance Genes in Biofilms.

Most bacteria attach to biotic or abiotic surfaces and are embedded in a complex matrix which is known as biofilm. Biofilm formation is especially worrisome in clinical settings as it hinders the treatment of infections with antibiotics due to the facilitated acquisition of antibiotic resistance genes (ARGs). Environmental settings are now considered as pivotal for driving biofilm formation, biofilm-mediated antibiotic resistance development and dissemination. Several studies have demonstrated that environmental biofilms can be hotspots for the dissemination of ARGs. These genes can be encoded on mobile genetic elements (MGEs) such as conjugative and mobilizable plasmids or integrative and conjugative elements (ICEs). ARGs can be rapidly transferred through horizontal gene transfer (HGT) which has been shown to occur more frequently in biofilms than in planktonic cultures. Biofilm models are promising tools to mimic natural biofilms to study the dissemination of ARGs via HGT. This review summarizes the state-of-the-art of biofilm studies and the techniques that visualize the three main HGT mechanisms in biofilms: transformation, transduction, and conjugation.

Small Things Matter: The 11.6-kDa TraB Protein is Crucial for Antibiotic Resistance Transfer Among Enterococci.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely.

Conjugation Operons in Gram-Positive Bacteria with and without Antitermination Systems.

Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators inside the conjugation operon to be bypassed, but also the differential expression of a subset of genes. Here, we show that some conjugation operons on plasmids belonging to the Inc18 family of Gram-positive broad host-range plasmids do not possess an antitermination system, suggesting that the absence of an antitermination system may have advantages. The possible (dis)advantages of conjugation operons possessing (or not) an antitermination system are discussed.

The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.