Martin Loessner

Last Name

Loessner

First Name

Martin

ORCID

0000-0002-8162-2631

Organisational unit

03651 - Loessner, Martin / Loessner, Martin

Show all metadata

Search Results

Publications 1 - 10 of 24

Chimeric Peptidoglycan Hydrolases Kill Staphylococcal Mastitis Isolates in Raw Milk and within Bovine Mammary Gland Epithelial Cells
Keller, Anja P.; Ly, Shera; Daetwyler, Steven; et al. (2022)
Viruses
Staphylococcus aureus is a major causative agent of bovine mastitis, a disease considered one of the most economically devastating in the dairy sector. Considering the increasing prevalence of antibiotic-resistant strains, novel therapeutic approaches efficiently targeting extra- and intracellular bacteria and featuring high activity in the presence of raw milk components are needed. Here, we have screened a library of eighty peptidoglycan hydrolases (PGHs) for high activity against S. aureus in raw bovine milk, twelve of which were selected for further characterization and comparison in time-kill assays. The bacteriocins lysostaphin and ALE-1, and the chimeric PGH M23LST(L)_SH3b2638 reduced bacterial numbers in raw milk to the detection limit within 10 min. Three CHAP-based PGHs (CHAPGH15_SH3bAle1, CHAPK_SH3bLST_H, CHAPH5_LST_H) showed gradually improving activity with increasing dilution of the raw milk. Furthermore, we demonstrated synergistic activity of CHAPGH15_SH3bAle1 and LST when used in combination. Finally, modification of four PGHs (LST, M23LST(L)_SH3b2638, CHAPK_SH3bLST, CHAPGH15_SH3bAle1) with the cell-penetrating peptide TAT significantly enhanced the eradication of intracellular S. aureus in bovine mammary alveolar cells compared to the unmodified parentals in a concentration-dependent manner.
Computational Pipeline for Targeted Integration and Variable Payload Expression in Bacteriophage Engineering
Fernbach, Jonas; Hegedis, Emese; Loessner, Martin; et al. (2025)
ACS Synthetic Biology
Bacteriophages offer a promising alternative to conventional antimicrobials, especially when such treatments fail. While natural phages are viable for therapy, advances in synthetic biology allow precise genome modifications to enhance their therapeutic potential. One approach involves inserting antimicrobial genetic payloads into the phage genome. These are typically placed behind late-expressed genes, such as the major capsid gene (cps). However, phages engineered with toxic payloads often fail to produce viable progeny due to premature host shutdown. To broaden the scope of viable genetic insertion sites, we developed a method to identify intergenic loci with favorable expression profiles using the machine learning-based promoter prediction tool, PhagePromoter. Guided by these predictions, we designed a computationally assisted engineering pipeline for targeted genomic payload integration. We validated this approach by engineering bioluminescent reporter genes into the genome of the strictly lytic Staphylococcus phage K at various predicted loci. Using homologous recombination, we generated three recombinant phages, each carrying the reporter at a distinct genomic location. These engineered phages exhibited expression levels consistent with computational predictions and demonstrated temporal expression patterns corresponding to early, middle, or late gene clusters. Our study highlights the power of combining computational tools with classical genome analysis to streamline phage engineering. This method supports rational design and enables high-throughput, automated phage modification, advancing the development of personalized phage therapy.
Listeria phages
Klumpp, Jochen; Loessner, Martin (2013)
Bacteriophage
Listeria is an important foodborne pathogen and the causative agent of Listeriosis, a potentially fatal infection. Several hundred Listeria bacteriophages have been described over the past decades, but only few have actually been characterized in some detail, and genome sequences are available for less than twenty of them. We here present an overview of what is currently known about Listeria phage genomics, their role in host evolution and pathogenicity, and their various applications in biotechnology and diagnostics.
The Listeria Cell Wall and Associated Carbohydrate Polymers
Loessner, Martin J.; Eugster, Marcel R.; Loessner, Martin (2014)
Methods in Molecular Biology ~ Listeria Monocytogenes
Understanding molecular interactions of bacteria with their environment requires the purification and characterization of cell wall components. Here, we describe detailed experimental methods for the extraction, purification, and analysis of wall teichoic acids (WTA), which assume important roles as major constituents of Gram-positive cell walls, such as mediating interaction with cell wall-associated proteins, eukaryotic host cells, and bacteriophages. Specifically, we present a procedure for compositional WTA characterization to study large diversity of carbohydrate substitution on Listeria monocytogenes WTA. This protocol may also be used and adapted to analyze WTA from other bacteria.
Spontaneous Resistance of Erwinia amylovora Against Bacteriophage Y2 Affects Infectivity of Multiple Phages
Knecht, Leandra E.; Born, Yannick; Pelludat, Cosima; et al. (2022)
Frontiers in Microbiology
Broad application of antibiotics gave rise to increasing numbers of antibiotic resistant bacteria. Therefore, effective alternatives are currently investigated. Bacteriophages, natural predators of bacteria, could work as such an alternative. Although phages can be highly effective at eliminating specific bacteria, phage resistance can be observed after application. The nature of this resistance, however, can differ depending on the phage. Exposing Erwinia amylovora CFBP 1430, the causative agent of fire blight, to the different phages Bue1, L1, S2, S6, or M7 led to transient resistance. The bacteria reversed to a phage sensitive state after the phage was eliminated. When wild type bacteria were incubated with Y2, permanently resistant colonies (1430(Y2R)) formed spontaneously. In addition, 1430(Y2R) revealed cross-resistance against other phages (Bue1) or lowered the efficiency of plating (L1, S2, and S6). Pull down experiments revealed that Y2 is no longer able to bind to the mutant suggesting mutation or masking of the Y2 receptor. Other phages tested were still able to bind to 1430(Y2R). Bue1 was observed to still adsorb to the mutant, but no host lysis was found. These findings indicated that, in addition to the alterations of the Y2 receptor, the 1430(Y2R) mutant might block phage attack at different stage of infection. Whole genome sequencing of 1430(Y2R) revealed a deletion in the gene with the locus tag EAMY_2231. The gene, which encodes a putative galactosyltransferase, was truncated due to the resulting frameshift. The mutant 1430(Y2R) was monitored for potential defects or fitness loss. Weaker growth was observed in LB medium compared to the wild type but not in minimal medium. Strain 1430(Y2R) was still highly virulent in blossoms even though amylovoran production was observed to be reduced. Additionally, LPS structures were analyzed and were clearly shown to be altered in the mutant. Complementation of the truncated EAMY_2231 in trans restored the wild type phenotype. The truncation of EAMY_2231 can therefore be associated with manifold modifications in 1430(Y2R), which can affect different phages simultaneously.
Draft genome sequences of two wide-host-range phages of Listeria monocytogenes from food processing environments in the United States
Brown, Phillip; Kilcher, Samuel; Kim, Jae-Won; et al. (2024)
Microbiology Resource Announcements
Listeria monocytogenes is notorious for persistence in food facilities. Phages can significantly impact the ecology of Listeria, but there is a dearth of genome sequence data for Listeria phages from food processing ecosystems. We report the genome sequences of two Listeria phages from turkey processing facilities in the USA.
Random encounters and amoeba locomotion drive the predation of Listeria monocytogenes by Acanthamoeba castellanii
de Schaetzen, Frédéric; Fan, Mingzhen; Alcolombri, Uria; et al. (2022)
Proceedings of the National Academy of Sciences of the United States of America
Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.
A perfect fit: Bacteriophage receptor-binding proteins for diagnostic and therapeutic applications
Klumpp, Jochen; Dunne, Matthew; Loessner, Martin (2023)
Current Opinion in Microbiology
Bacteriophages are the most abundant biological entity on earth, acting as the predators and evolutionary drivers of bacteria. Owing to their inherent ability to specifically infect and kill bacteria, phages and their encoded endolysins and receptor-binding proteins (RBPs) have enormous potential for development into precision antimicrobials for treatment of bacterial infections and microbial disbalances; or as biocontrol agents to tackle bacterial contaminations during various biotechnological processes. The extraordinary binding specificity of phages and RBPs can be exploited in various areas of bacterial diagnostics and monitoring, from food production to health care. We review and describe the distinctive features of phage RBPs, explain why they are attractive candidates for use as therapeutics and in diagnostics, discuss recent applications using RBPs, and finally provide our perspective on how synthetic technology and artificial intelligence-driven approaches will revolutionize how we use these tools in the future.
Use of Bacteriophage Cell Wall-Binding Proteins for Rapid Diagnostics of Listeria
Schmelcher, Mathias; Loessner, Martin (2014)
Methods in Molecular Biology ~ Listeria Monocytogenes
Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.
Enzyme-responsive nanoparticles: enhancing the ability of endolysins to eradicate Staphylococcus aureus biofilm
Blanco Massani, Mariana; To, Dennis; Meile, Susanne; et al. (2024)
Journal of Materials Chemistry B
Stimuli-responsive nanomaterials show promise in eradicating Staphylococcus aureus biofilm from implants. Peptidoglycan hydrolases (PGHs) are cationic antimicrobials that can be bioengineered to improve the targeting of persisters and drug-resistant bacteria. However, these molecules can be degraded before reaching the target and/or present limited efficacy against biofilm. Therefore, there is an urgent need to improve their potency. Herein, PGH-polyphosphate nanoparticles (PGH-PP NPs) are formed by ionotropic gelation between cationic PGHs and anionic polyphosphate, with the aim of protecting PHGs and delivering them at the target site triggered by alkaline phosphatase (AP) from S. aureus biofilm. Optimized conditions for obtaining M23-PP NPs and GH15-PP NPs are presented. Size, zeta potential, and transmission electron microscopy imaging confirm the nanoscale size. The system demonstrates outstanding performance, as evidenced by a dramatic reduction in PGHs' minimum inhibitory concentration and minimum bactericidal concentration, together with protection against proteolytic effects, storage stability, and cytotoxicity towards the Caco-2 and HeLa cell lines. Time-kill experiments show the great potential of these negatively charged delivery systems in overcoming the staphylococcal biofilm barrier. Efficacy under conditions inhibiting AP proves the enzyme-triggered delivery of PGHs. The enzyme-responsive PGH-PP NPs significantly enhance the effectiveness of PGHs against bacteria residing in biofilm, offering a promising strategy for eradicating S. aureus biofilm.

Publications 1 - 10 of 24

Martin Loessner

Last Name

First Name

ORCID

Organisational unit

Filters

Search Results