Using a ligate intestinal loop mouse model to investigate Clostridioides difficile adherence to the intestinal mucosa in aged mice

Interaction of Clostridioides difficile spores with the intestinal mucosa contributes to the persistence and recurrence of the infection. Advanced age is one of the main risk factors for C. difficile infection and recurrence of the disease. However, interaction of C. difficile spores with the intestinal mucosa during aging has not been evaluated. In the present work, using intestinal ligated loop technique in a mouse model, we analyzed C. difficile spore adherence and internalization to the ileum and colonic mucosa during aging. Additionally, we provide visual documentation of the critical steps of the procedure. Consequently, our data suggest that spore internalization in the ileum and colonic mucosa is higher in elderly mice rather than adults or young mice. Also, our data suggest that spore adherence to the ileum and colonic mucosa decreases with aging.

Pathogenicity of Cordyceps javanica (Hypocreales: Cordycipitaceae) to Diaphorina citri (Hemiptera: Liviidae) Adults, with Ultrastructural Observations on the Fungal Infection Process

Entomopathogenic fungi are proposed biological control agents against the Asian citrus psyllid (Diaphorina citri). We quantified the pathogenicity of Cordyceps javanica strain Apopka 97 strain (Cja Apopka 97) (blastospores and conidia) against D. citri using the spray technique. We also used light and scanning electron microscopy to observe the Cja Apopka 97, infection process against D. citri adults at different stages pre- and post-mortem. Our findings demonstrated that psyllid mortality in the blastospore treatment ranged from 8 to 25% within 24–48 h of exposure, compared to 0% in the conidial and control treatments. However, psyllid mortality rate had reached 100% by 7 days after exposure at a concentration of 107 spores/mL, under both fungal treatments compared to the controls (0%). SEM and light microscopy revealed several stages in in the Cja Apopka 97 infection process of D. citri, including spore adherence and germ tube formation within 24 h post-inoculation, penetration pegs and mycelia growth on wings after 72 h, rupturing of cuticle after 96 h and mycelial mass colonizing host body after 144 h. Our study findings provide basic information on the interaction of entomopathogenic fungi with D. citri which will assist in the understanding of the infection process and the potential roles of entomopathogenic fungi in its management.

E-cadherin redistribution mediated by Clostridioides difficile toxins increases spore-association to adherens junctions

Nearly ~20% of patients with C. difficile infection (CDI) manifest recurrence of CDI (R-CDI). During CDI, C. difficile forms spores essential for R-CDI. Interactions of C. difficile spores with intestinal epithelial cells (IECs) contribute to R-CDI. However, this interaction remains poorly understood. Here, we provide evidence that C. difficile spores interact with E-cadherin, contributing to spore-adherence and internalization into IECs. C. difficile toxins TcdA/TcdB lead to adherens junctions opening and increase spore-adherence to IECs. Confocal micrographs demonstrate that C. difficile spores associate with accessible E-cadherin; spore-E-cadherin association increases upon TcdA/TcdB intoxication. The presence of anti-E-cadherin antibodies decreased spore adherence and entry into IECs. By ELISA, immunofluorescence, and immunogold labelling, we observed that E-cadherin binds to C. difficile spores, specifically to the hair-like projections of the spore. Overall, these results expand our knowledge of how C. difficile spores bind to IECs and how toxin-mediated damage affects spore interactions with IECs.

A ligated intestinal loop mouse model protocol to study the interactions of Clostridioides difficile spores with the intestinal mucosa during aging.

The interaction of the Clostridioides difficile spores with the intestinal mucosa contribute to the persistence and recurrence of the infection. Advanced age is one of the main risk factors to manifest C. difficile infection and recurrence. However, the interaction of C. difficile spores with the intestinal mucosa during aging has not been evaluated. In the present work, we provide a detailed protocol with all the critical information to perform an intestinal ligated loop. Using this technique in a mouse model, we evaluated C. difficile spore adherence and internalization to the ileum and colonic mucosa during aging. Consequently, our data suggest that spore internalization in the ileum and colonic mucosa is higher in elderly than in adults or young mice. Also, our data suggest that spore-adherence to the ileum and colonic mucosa decreases with aging.

Expression and Localization of an Hsp70 Protein in the MicrosporidianEncephalitozoon cuniculi

Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; “C1”) was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and matureE. cuniculispores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the ‘C1’ Hsp70 will be used to delineate spore surface protein expression.

Recovery of Spores from Thermophilic Dairy Bacilli and Effects of Their Surface Characteristics on Attachment to Different Surfaces

ABSTRACT Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of ∼107 spores ml−1; spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from −10 to −20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 × 104 spores cm−2), with fewer spores attaching to glass (3 × 103 spores cm−2). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (∼1 × 103 spores cm−2), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.

Role of Sulfated Glycans in Adherence of the Microsporidian Encephalitozoon intestinalis to Host Cells In Vitro

ABSTRACT Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.