Introduction the epithelial mucus layer as its C

Introduction

One of the most diverse and abundant microbial niches is found in the human body. The
Gram-negative anaerobe Akkemansia muciniphila, which belongs to the Planctomycetes-
Verrucomicrobia-Chlamydiae superphylum, is found in the alimentary canal of more than
90% of the evaluated cases. A. muciniphila is well adapted to the human gut environment and
uses glycosolated proteins of the epithelial mucus layer as its C and N source. Earlier studies
show that there is a relation between A. muciniphila not being abundant in the intestinal track
and gut health. For example, when the density of A. muciniphila is low this is related to
diabetes type 1, Crohn’s disease (CD) and in Ulcerative colitis (UC). Furthermore, the
recovery of the mucus layer in the intestines and decreasing endotoxemia are accociated with
A. muciniphila.

State of the art

There is not much known about the interactions between A. muciniphila and the host, nor how
it handles the different environmental circumstances. Previous studies do suggest that A.
muciniphila has a positive consequence on gut health so further investigation is needed for
future implementations. The interaction of the bacteria and its hosts starts with colonizing in
which they can adhere by binding to the mucus layer of the intestines epithelium or via the
cells underneath, the enterocytes. To which surface A. muciniphila adheres hasn’t been
studied yet even as the ability of coping with an oxygen rich environment. This study will
answer which mechanisms A. muciniphila uses to adhere to the mucus layer or the epithelium
cells of the gastrointestinal tract.

Recent findings

Although the human colon consists out of an anaerobic microbe community, it turned out that
A. 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 adhesion
experiment. The binding efficiency with epithelial cells HT29 and Caco-2 do not differ
between aerobic and anaerobic atmosphere. Thus, A. muciniphila does not have to be treated
as 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 the
intestinal mucus is less than 1%, it can be stated that there is no adhesion at all. As other
bacteria, for example L. rhamnosus and B. bifidum, show strong connection to human colonic
mucus, it was unexpected that A. muciniphila did not. An explanation for this is that these
species 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. muciniphila
adhered to both enterocrytes equally well as L. rhamnosus. This might indicate that the
enterocytes are true binding sites for A. muciniphila.

A. muciniphila and B. fragilis were both cocultivated for 24 hours and indicated an expansion
in transepithelial electrical resistance (TER). Compared to the Caco-2 cultures, without
bacteria the TER of Caco-2 cocultures of Escherichia coli declined significantly. This shows
that at this timepoint E. coli cells increased and that there is no cell suspension for the OD600
values of A. muciniphila and B. fragilis, which indicates a stagnation of growth. The positive
impact 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 of
Caco-2 cocultures of A. muciniphila became equal to the cocultures of B. fragilis. During the
48 hour incubation the cell density of B. fragilis and A. muciniphila did not diversify, neither
seems that the bacteria are severly affected. Under the same circumstances, E. coli affected
TER development negatively and the coculture increased during the second 24 hours, which
will most likely result in a further decline of the transepithelial electrical resistance in E. coli
cocultures.

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 the
homeastasis of the colonal mucus. Compared to the IL-8 production by E. coli, A. muciniphila
produced 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 inflammatory
response in the presence of A. muciniphila, it was checked wether it does or does not produce
LPS and whether it is different compared to E. coli. The results show that A. muciniphila does
produce 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 of
E. coli.

Discussion

The results of this study show that A. muciniphila does not bind to the intestinal mucus but
prefers to bind to the epithelial cells Caco-2 and HT-29 and the ECM laminin. It remains
unknown how this organism is able to live in this continually adjusting habitat and should be
studies to answere this question. A possible justification could be that A. muciniphila releases
a certain enzyme which decreases the colonic mucus, making it hard for the bacteria to
effectively bind to the mucus.

As A. muciniphila was able to connect to the extracellular matrix laminin it might suggest that
pathogens are competing with A. muciniphila for bindingsites at locations where the epithelial
cell layer of the colon is damaged.
Furthermore, it is likely that A. muciniphila is able to strengthen the barrier of the intestinal
track. Future research could study the helpful role of A. muciniphila in connection with its
host in for example obesity and diabetes 

Author: