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Background & AimClostridioides difficileinfection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced byC. difficileare the major determinants of disease. However, the physiological cause of diarrhea associated with CDI is not well understood. We investigated the effects of CDI on paracellular permeability and apical ion transporters.MethodsWe studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure regional differences in paracellular permeability and ion transporter function in intestinal mucosa. Intestinal tissues were collected from mice and analyzed by immunofluorescence microscopy and RNA-sequencing.ResultsCDI increased intestinal permeability through the size-selective leak pathwayin vivo, but permeability was not increased at the sites of pathological damage. Chloride secretion was reduced in the cecum during infection by decreased CaCC function. Infected mice had decreased SGLT1 (also called SLC5A1) activity in the cecum and colon along with diminished apical abundance and an increase in luminal glucose. SGLT1 and DRA (also called SLC26A3) expression was ablated by either TcdA or TcdB, but NHE3 (also called SLC9A3) was decreased in a TcdB-dependent manner. Finally, expression of these three ion transporters was drastically reduced at the transcriptional level.ConclusionsCDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination may cause a dysfunction in water and solute absorption in the lower gastrointestinal tract, leading to osmotic diarrhea. These findings may open novel pathways for attenuating CDI-associated diarrhea.GRAPHICAL ABSTRACT

The gram-positive actinobacterium Clavibacter michiganensis is the causal agent of bacterial canker of tomato, an economically impactful disease with a worldwide distribution. This seedborne pathogen systemically colonizes tomato xylem leading to unilateral leaflet wilt, marginal leaf necrosis, stem and petiole cankers, and plant death. Additionally, splash dispersal of the bacterium onto fruit exteriors causes bird’s-eye lesions, which are characterized as necrotic centers surrounded by white halos. The pathogen can colonize developing seeds systemically through xylem and through penetration of fruit tissues from the exterior. There are currently no commercially available resistant cultivars, and bactericidal sprays have limited efficacy for managing the disease once the pathogen is in the vascular system. In this review, we summarize research on epidemiology, host colonization, the bacterial genetics underlying virulence, and management of bacterial canker. Finally, we highlight important areas of research into this pathosystem that have the potential to generate new strategies for prevention and mitigation of bacterial canker.

Clavibacter michiganensis is the Gram-positive causal agent of bacterial canker of tomato, an economically devastating disease with a worldwide distribution. C. michiganensis colonizes the xylem, leading to unilateral wilt, stem canker, and plant death. C. michiganensis can also infect developing tomato fruit through splash dispersal, forming exterior bird’s eye lesions. There are no documented sources of qualitative resistance in Solanum spp.; however, quantitative trait loci conferring tolerance in Solanum arcanum and Solanum habrochaites have been identified. Mechanisms of tolerance and C. michiganensis colonization patterns in wild tomato species remain poorly understood. This study describes differences in symptom development and colonization patterns of the wild type (WT) and a hypervirulent bacterial expansin knockout (ΔCmEXLX2) in wild and cultivated tomato genotypes. Overall, WT and ΔCmEXLX2 cause less severe symptoms in wild tomato species and are impeded in spread and colonization of the vascular system. Laser scanning confocal microscopy and scanning electron microscopy were used to observe preferential colonization of protoxylem vessels and reduced intravascular spread in wild tomatoes. Differences in C. michiganensis in vitro growth and aggregation were determined in xylem sap, which may suggest that responses to pathogen colonization are occurring, leading to reduced colonization density in wild tomato species. Finally, wild tomato fruit was determined to be susceptible to C. michiganensis through in vivo inoculations and assessing lesion numbers and size. Fruit symptom severity was in some cases unrelated to severity of symptoms during vascular infection, suggesting different mechanisms for colonization of different tissues.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474–4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Clostridioides difficile is linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains of C. difficile produce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota et al. (S. A. Hirota et al., Infect Immun 80:4474–4484, 2012) first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 (VPI 10463 reference strain [ATCC 43255]) and 630 C. difficile strains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reported in vivo and ex vivo. Furthermore, TcdB mutants lacking either endosomal pore formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared to slides from a human C. difficile infection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant‐like expansin genes in numerous xylem‐colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non‐chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem‐inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β‐proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm ΔCmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the ΔCmEXLX2 mutant, even though the strains grew as well as the wild‐type in vitro. Similarly, when inoculated onto tomato fruit, ΔCmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs ΔRsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, ΔRsEXLX attached to tomato seedling roots better than the wild‐type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non‐chimeric bacterial expansins and highlight their importance in plant–bacterial interactions. Published version