Biology subjects

Aspholm, M. E.

Publications and source records attributed to Aspholm, M. E..

6 recordsLinked to original sources

bioRxiv2025

Endospore Appendages Enhance Bacillus cereus Spore Adhesion to Industrial Surfaces, Modulated by Physicochemical Factors

Spores of species belonging to the Bacillus cereus group (B. cereus spp.) are common contaminants in food processing environments due to their ability to adhere to surfaces and resist cleaning procedures. These spores are equipped with pilus-like endospore appendages (ENAs), which are believed to promote surface adhesion. We have investigated the role of ENAs in spore adhesion to abiotic surfaces using wild-type (WT) B. cereus spp. strains and isogenic mutants lacking ENAs or an intact exosporium. WT spores expressing both short and long ENAs (S+L+) adhered significantly more to stainless steel (SS) and polypropylene (PP) compared to bald spores (S-L-) and spores of an exosporium-deficient mutant ({Delta}exsY), while adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The {Delta}exsY mutant showed the lowest adhesion across all tested surfaces, a pattern also observed for vegetative cells. The individual roles of S-ENA and L-ENA were also assessed, where the strongest adhesion occurred when both fibers were present on PP. A trend also emerged on PP; WT remained adhered for an hour, while bald spores tended to detach within that time. For saline conditions and pH, bald spores adhered strongly to SS, while for non-ionic surfactant and concentrated protein solutions, WT spores exhibited the strongest adhesion. These results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrial relevant surfaces, providing mechanistic insight into spore persistence. This understanding supports the design of surface treatments to prevent contamination, spoilage, and foodborne illness. ImportanceBacteria belonging to the Bacillus cereus group represent a persistent challenge in food production due to their highly resilient endospores (spores), which resist cleaning and disinfection as well as food processing. Understanding the adhesion properties of these spores is essential for designing effective surface treatments that reduce chemical use, enhance food safety and quality, and minimize environmental impact. This study underscores the important role of endospore appendages (ENAs) in spore adhesion to common materials in food processing and laboratory environments. Wild-type spores expressing both S-ENA and L-ENA adhered significantly more than mutants lacking ENAs or the exosporium, highlighting ENAs as potential targets for disrupting spore adhesion. Time-dependent adhesion tests on polypropylene revealed strong, sustained attachment by wild-type spores, contrasting with weaker, transient adhesion by ENA-depleted mutants. These findings offer valuable insights into B. cereus spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.

microbiology↗

bioRxiv2025

Cryo-EM analysis of the Bacillus thuringiensis extrasporal matrix identifies F-ENA as a widespread family of endospore appendages across the Firmicutes phylum

For over 100 years, Bacillus thuringiensis (Bt) has been used as an agricultural biopesticide to control pests caused by insect species in the orders of Lepidoptera, Diptera and Coleoptera. Under nutrient starvation, Bt cells differentiate into spores and associated toxin crystals that can adopt biofilm-like aggregates. We reveal that such Bt spore/toxin biofilms are embedded in a fibrous extrasporal matrix (ESM), and using cryoID, we resolved the structure and molecular identity of an uncharacterized type of pili, referred to here as Fibrillar ENdospore Appendages or F-ENA. F-ENA are monomolecular protein polymers tethered to the exosporium of Bt and are decorated with a flexible tip fibrillum. Phylogenetic analysis reveals that F-ENA is widespread not only in the class Bacilli, but also in the class Clostridia, and the cryoEM structures of F-ENA filaments from Bacillus, Anaerovorax and Paenibaccilus reveal subunits with a generic head-neck domain structure, where the {beta}-barrel neck of variable length latch onto a preceding head domain through short N-terminal hook peptides. In Bacillus, two collagen-like proteins (CLP) respectively tether F-ENA to the exosporium (F-Anchor), or constitute the tip fibrillum at the distal terminus of F-ENA (F-BclA). Sedimentation assays point towards F-ENA involvement in spore-spore clustering, likely mediated via F-BclA contacts and F-ENA bundling through the antiparallel interlocking of the head-neck units.

molecular biology↗

bioRxiv2024

The role of endospore appendages in spore-spore contacts in pathogenic bacilli

Species within the spore-forming Bacillus cereus sensu lato group are recognized for their role in food spoilage and food poisoning. B. cereus spores are decorated with numerous pilus-like appendages, called S-ENAs and L-ENAs. These appendages are believed to play crucial roles in self-aggregation, adhesion, and biofilm formation. By using both bulk and single-cell approaches, we investigate the role of S-and L-ENAs as well as the impact of different environmental factors in spore-to-spore contacts and in the interaction between spores and vegetative cells. Our findings reveal that ENAs, and particularly their tip fibrilla, play an essential role in spore self-aggregation but not in the adhesion of spores to vegetative cells. The absence of L-BclA, which builds the L-ENA tip fibrillum, reduced both S-and L-ENA mediated spore aggregation, emphasizing the interconnected roles of S-and L-ENAs. Increased salt concentrations in the liquid environment significantly reduced spore aggregation, implying a charge dependency of spore-spore interactions. By elucidating these complex interactions, our study provides valuable insights into spore dynamics. This knowledge can guide future studies on spore behavior in environmental settings and aids in developing strategies to manage bacterial aggregation for beneficial purposes, like controlling biofilms in food production equipment.

microbiology↗

bioRxiv2023

A novel class of ultra-stable endospore appendages decorated with collagen-like tip fibrillae

Bacterial endospores are remarkable examples of biological resilience, representing a dormant and heavily fortified differentiation form capable of withstanding physical and chemical stressors detrimental to vegetative cells. In pathogenic firmicutes, spores also form an infectious particle and can take up a central role in the environmental persistence and dissemination of disease. A poorly understood aspect of spore-mediated infection is the fibrous structures or endospore appendages (ENAs) that have been seen to decorate the spores of pathogenic Bacilli and Clostridia. New methodological approaches are opening an unprecedented window on these long enigmatic structures. Using cryoID, Alphafold modelling and genetic approaches we identify a novel class of ultra-robust ENAs formed by Bacillus paranthracis. We demonstrate that L-ENA are encoded by a three-gene cluster (ena3) that contains all components for the self-assembly of ladder-like protein nanofibers of stacked heptameric rings, their anchoring to the exosporium, and their termination in a trimeric ruffle made of a collagen-like BclA paralogue. Phylogenomic analyses shows the ena3 gene cluster as a mobile element with a polyphyletic distribution across pathogenic Bacilli.

microbiology↗

bioRxiv2021

High diversity in the regulatory region of Stx-converting bacteriophage genomes

Shiga toxin (Stx) is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor. CI maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and the three already described Erus, ten novel Erus (named Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 seem to be widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain. IMPORTANCEEHEC is an emerging health challenge worldwide and outbreaks caused by this pathogen tend to be more frequent and severe. Increased knowledge on how characteristics of the replication region influence the virulence of E. coli may be used for more precise identification of high-risk EHEC strains.

microbiology↗

bioRxiv2021

WGS analysis of Listeria monocytogenes from rural, urban, and farm environments in Norway: Genetic diversity, persistence, and relation to clinical and food isolates

Listeria monocytogenes is a ubiquitous environmental bacterium associated with a wide variety of natural and man-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation of L. monocytogenes from various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole genome sequencing (WGS) was performed for the collected isolates and for L. monocytogenes from Norwegian dairy farms and slugs, in total 218 isolates. The data was compared with available datasets from clinical and food associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0-8 wgMLST allelic differences were collected over time in the same location, demonstrating persistence of L. monocytogenes in natural, urban and farm environments. Furthermore, several clusters with 6-20 wgMLST allelic differences containing isolates collected across different locations, times and habitats were identified, including nine clusters harbouring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, CC1) and food (CC9, CC121, CC7, CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases. ImportanceListeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment. For effective management, both public health authorities and food producers need reliable tools for source tracking, surveillance, and risk assessment. For this, whole genome sequencing (WGS) is regarded as the present and future gold standard. In the current study, we use WGS to show that L. monocytogenes can persist for months and years in natural, urban and dairy farm environments. Notably, clusters of almost identical isolates, with genetic distances within the thresholds often suggested for defining an outbreak cluster, can be collected from geographically and temporally unrelated sources. The work highlights the need for a greater knowledge of the genetic relationships between clinical isolates and isolates of L. monocytogenes from a wide range of environments, including natural, urban, agricultural, livestock, food production, and food processing environments, in order to correctly interpret and use results from WGS analyses.

microbiology↗