Introduction the epithelial mucus layer as its C

Introduction One of the most diverse and abundant microbial niches is found in the human body. TheGram-negative anaerobe Akkemansia muciniphila, which belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum, is found in the alimentary canal of more than90% of the evaluated cases. A. muciniphila is well adapted to the human gut environment anduses glycosolated proteins of the epithelial mucus layer as its C and N source. Earlier studiesshow that there is a relation between A. muciniphila not being abundant in the intestinal trackand gut health.

For example, when the density of A. muciniphila is low this is related todiabetes type 1, Crohn’s disease (CD) and in Ulcerative colitis (UC). Furthermore, therecovery of the mucus layer in the intestines and decreasing endotoxemia are accociated withA. muciniphila. State of the art There is not much known about the interactions between A. muciniphila and the host, nor howit handles the different environmental circumstances.

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Previous studies do suggest that A.muciniphila has a positive consequence on gut health so further investigation is needed forfuture implementations. The interaction of the bacteria and its hosts starts with colonizing inwhich they can adhere by binding to the mucus layer of the intestines epithelium or via thecells underneath, the enterocytes. To which surface A. muciniphila adheres hasn’t beenstudied yet even as the ability of coping with an oxygen rich environment. This study willanswer which mechanisms A.

muciniphila uses to adhere to the mucus layer or the epitheliumcells of the gastrointestinal tract. Recent findings Although the human colon consists out of an anaerobic microbe community, it turned out thatA. muciniphila is capable of surviving in both oxic as well as anoxic conditions. As A.

muciniphila is aerotolerant, contrasting incubation conditions were compared in an adhesionexperiment. The binding efficiency with epithelial cells HT29 and Caco-2 do not differbetween aerobic and anaerobic atmosphere. Thus, A. muciniphila does not have to be treatedas a true anaerobe, but is able to cope with oxygen. Also, it turned out that the only significant binding of A. muciniphila, compared to BSA,occurred with laminin.

The bindingprocess of A. muciniphila with other extracellular matrix(ECM) proteins, was not significant. As the adhesion between A. muciniphila and theintestinal mucus is less than 1%, it can be stated that there is no adhesion at all. As otherbacteria, for example L. rhamnosus and B. bifidum, show strong connection to human colonicmucus, it was unexpected that A.

muciniphila did not. An explanation for this is that thesespecies do not utilize and degrade the mucus, like A. muciniphila does. Although the adhesion of A. muciniphila and L. rhamnosus on colonic mucus was not compareble, A.

muciniphilaadhered to both enterocrytes equally well as L. rhamnosus. This might indicate that theenterocytes are true binding sites for A. muciniphila. A. muciniphila and B. fragilis were both cocultivated for 24 hours and indicated an expansionin transepithelial electrical resistance (TER).

Compared to the Caco-2 cultures, withoutbacteria the TER of Caco-2 cocultures of Escherichia coli declined significantly. This showsthat at this timepoint E. coli cells increased and that there is no cell suspension for the OD600values of A. muciniphila and B. fragilis, which indicates a stagnation of growth. The positiveimpact of cell monolayer integrity for the first 24 hours was the most succesfull with B.fragilis, followed by A. muciniphila.

After 48 hours the transepithelial electrical resistance ofCaco-2 cocultures of A. muciniphila became equal to the cocultures of B. fragilis.

During the48 hour incubation the cell density of B. fragilis and A. muciniphila did not diversify, neitherseems that the bacteria are severly affected. Under the same circumstances, E. coli affectedTER development negatively and the coculture increased during the second 24 hours, whichwill most likely result in a further decline of the transepithelial electrical resistance in E. colicocultures.

Earlier studies have associated obesity and diabetes to decreased gut health and inflammation,which result in lipopolysaccharide (LPS) induced endotoxemia. When LPS is released,enterocytes start producing the chemokine interleukin-8 (IL-8) which leads to inflammation.Needless inflammation can cause disorder in the intestinal epithelium and can disturb thehomeastasis of the colonal mucus. Compared to the IL-8 production by E. coli, A. muciniphilaproduced less IL-8 in HT-29 cells. Thus, there will be no strong inflammation when A.muciniphila is present in the gastrointestinal tract.

Since there almost was no inflammatoryresponse in the presence of A. muciniphila, it was checked wether it does or does not produceLPS and whether it is different compared to E. coli. The results show that A. muciniphila doesproduce LPS, however it does not activate HT-29 cells to produce a lot of interleukin-8.Therefor it is likely that the produced LPS by A.

muciniphila is different compared to that ofE. coli. Discussion The results of this study show that A. muciniphila does not bind to the intestinal mucus butprefers to bind to the epithelial cells Caco-2 and HT-29 and the ECM laminin.

It remainsunknown how this organism is able to live in this continually adjusting habitat and should bestudies to answere this question. A possible justification could be that A. muciniphila releasesa certain enzyme which decreases the colonic mucus, making it hard for the bacteria toeffectively bind to the mucus. As A. muciniphila was able to connect to the extracellular matrix laminin it might suggest thatpathogens are competing with A. muciniphila for bindingsites at locations where the epithelialcell layer of the colon is damaged.

Furthermore, it is likely that A. muciniphila is able to strengthen the barrier of the intestinaltrack. Future research could study the helpful role of A. muciniphila in connection with itshost in for example obesity and diabetes 



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