Highly antibiotic resistant, microbial communities, referred to as biofilms, cause various life-threatening infections in humans. At least two-thirds of all clinical infections are biofilm associated, and antibiotic therapy regularly fails to cure patients. Anti-biofilm peptides represent a promising approach to treat these infections by targeting biofilm-specific characteristics such as highly conserved regulatory mechanisms. They are being considered for clinical application and we discuss here key factors in discovery, design, and application, particularly the implementation of host-mimicking conditions, that are required to enable the successful advancement of potent anti-biofilm peptides from the bench to the clinic.

Our findings further underline that HML-2 expression is commonly highly tissue-specific. In prostate cancer, a limited number of loci become activated, especially H22q and ERVK-5. As expressed and non-expressed proviruses do not differ significantly in TFBS, tissue- and tumor-specific expression may be governed primarily by chromatin context. Overexpression of HML-2 H22q is more likely consequence than cause of prostate cancer progression.

The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.

Host defence peptides (HDPs) can overcome biofilm-specific resistance and immune evasion mechanisms that render antibiotics ineffective. Infection models mimicking the resilience of biofilms are crucial for the development of these therapeutics.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

SummaryMembers of the bacterial genus Pseudomonas form mutualistic, commensal and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not hostspecific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles.

A large group of biopharmaceuticals is produced in cell lines. The yield of such products can be increased by genetic engineering of the corresponding cell lines. The prediction of promising genetic modifications by mathematical modeling is a valuable tool to facilitate experimental screening. Besides information on the intracellular kinetics and genetic modifications the mathematical model has to account for ubiquitous cell-to-cell variability. In this contribution, we establish a novel model-based methodology for influenza vaccine production in cell lines with overexpressed genes. The manipulation of the expression level of genes coding for host cell factors relevant for virus replication is achieved by lentiviral transduction. Since lentiviral transduction causes increased cell-to-cell variability due to different copy numbers and integration sites of the gene constructs we use a population balance modeling approach to account for this heterogeneity in terms of intracellular viral components and distributed kinetic parameters. The latter are estimated from experimental data of intracellular viral RNA levels and virus titers of infection experiments using cells overexpressing a single host cell gene. For experiments with cells overexpressing multiple host cell genes, only final virus titers were measured and thus, no direct estimation of the parameter distributions was possible. Instead, we evaluate four different computational strategies to infer these from single gene parameter sets. Finally, the best computational strategy is used to predict the most promising candidates for future modifications that show the highest potential for an increased virus yield in a combinatorial study. As expected, there is a trend to higher yields the more modifications are included.

Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.