Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance

Abstract

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.

The Levels of Brachyspira hyodysenteriae Binding to Porcine Colonic Mucins Differ between Individuals, and Binding Is Increased to Mucins from Infected Pigs with De Novo MUC5AC Synthesis

“…Fluorescence microscopy of the pig colon tissue revealed that the mucus layer in the healthy pig colon, including in the colon of the inoculated pigs that did not develop SD, was organized in striations parallel to the mucosa and consisted mainly of the MUC2 mucin (Fig. 1A to C), similarly to the mucus structure reported for the mouse colon (2,27). In contrast, during infection with B. hyodysenteriae, a massive increase in MUC2 levels and de novo production of MUC5AC was observed in the four inoculated pigs with severe clinical signs of dysentery ( Fig.…”

“…The antibodies used have previously been shown to detect their specific targets in humans and mice (27,28), but no MUC2 or MUC5AC antibodies have been verified for use in pigs. To be certain that the stain represented MUC5AC and MUC2, we confirmed that the antibodies we used for the immunofluorescence indeed bound to the isolated mucins in specific manners that differed between the antibodies ( Fig.…”

“…During B. hyodysenteriae infection, we found a loss of the striated organization and a substantial increase in MUC2 and de novo secretion of MUC5AC mucins. We recently reported dynamic changes in the mucus barrier during Citrobacter rodentium infection in mice, with structural loss and decrease of the inner mucus layer at the onset and middle time points of infection (27). However, during the clearance phase, the mucus layer thickness increased but had an organization similar to that seen in uninfected mice, and no Muc5ac was detected (27).…”

“…We recently reported dynamic changes in the mucus barrier during Citrobacter rodentium infection in mice, with structural loss and decrease of the inner mucus layer at the onset and middle time points of infection (27). However, during the clearance phase, the mucus layer thickness increased but had an organization similar to that seen in uninfected mice, and no Muc5ac was detected (27). The changes observed in B. hyodysenteriae-infected pigs are thus completely different from any of the mucus changes identified during C. rodentium infection and clearance.…”

Dynamic polymer systems with self-regulated secretion for the control of surface properties and material healing

“…Storage droplets or vesicles continuously adjust their size, shape and contents through ongoing exchange with the surroundings, creating intrinsically responsive control mechanisms that tie secretion to a wide range of chemical and physical stimuli and feedback signals. [10][11][12][13] At the same time, collective changes in the stores are reported to the organism, alerting it that it needs to drink or eat to replenish the limited supply. Many synthetic approaches have been developed to enable triggered release from microcapsules, hydrogels, nanoparticles, vesicles, micelles, mesoporous carriers and other containers.…”