Introduction and Crohn’s disease. Furthermore, A. muciniphila

Introduction

We live in a microbial world. Everywhere we go, we are surrounded by microbes. Even the human body
itself is home to a large amount of these microscopic organisms. The area with the greatest diversity and
abundance of microbes in the human body, is the intestine. It houses more than a thousand different
species. One of these species that is commonly present is the Gram-negative anaerobe bacteria
Akkermansia muciniphila. This species is especially adapted to the gut environment and specializes in the
utilization of intestinal mucus as a source of carbon and nitrogen. Unsurprisingly, it has been detected in
high numbers in the mucus of the human colon, an area completely covered by a thick mucus layer when
healthy.

State of the art

Previous studies have associated the presence of A. muciniphila with intestinal health and improved
metabolic status. For example, a low cell count of A. muciniphila has been correlated to type 1 diabetes,
obesity and Crohn’s disease. Furthermore, A. muciniphila plays a role in restoring the mucus layer thickness
in the intestines and reducing endotoxemia.

Even though evidence of the positive effects of A. muciniphila grows, still little is known about the basic
ways it interacts with the host and how it copes with different environmental circumstances. In order for a
bacteria to colonize and interact with the host, it has to bind to the intestine epithelium. This can be
achieved by either binding to the protective mucus layer or to the cells underneath this layer, the
enterocytes. Which surface A. muciniphila adheres to has not been studied thus far. Also, the coping
capacities of this species in aerobic environments are unknown. Gaining knowledge on the mechanisms
that A. muciniphila uses can be of large value for possible future implementations of this promising gut
bacteria.

Recent findings

In the human colon all bacteria are anaerobe. However, not all species cope with oxygen the same way.
The survival of 80% of A. muciniphila cells exposed to atmospheric oxygen in a plate count, indicate that
this bacterium is an aerotolerant anaerobic species. Also, in an aerobic atmosphere the levels of binding
efficiency with the human colonic epithelial cells, Caco-2 and HT-29, did not differ from an anoxic
atmosphere. Thus, when working with this bacteria it does not have to be treated as a highly oxygen
sensitive anaerobe.

Also, the following possible binding sites in the human colon for A. muciniphila were studied: human
enterocytes (Caco-2 and HT-29 cell lines), colonic mucus and extracellular matrix (ECM) proteins. First, the
binding levels of A. muciniphila with colonic mucus were less than 1%, thus negligible. This was unexpected,
because of the close interaction between the mucus and the bacterium. Other intestinal bacteria such as L.
rhamnosus and B. bifidum do show strong binding to human colonic mucus. However, these species do not
degrade and utilize the mucus, which A. muciniphila does. Thus, this result may reflect the mucinolytic
nature of A. muciniphila. Secondly, A. muciniphila did show strong binding to the human enterocyte lines,
Caco-2 and HT-29. This may indicate that the enterocytes are the true docking sites for A. muciniphila cells.
In the colon these enterocytes are normally covered in a thick mucus layer. However, in the small intestine

this layer is more permeable to bacteria. Thus, there A. muciniphila may bind directly to the enterocytes.
The binding levels of A. muciniphila to enterocytes were not much affected in vitro by variation in
developmental state of the enterocytes. This indicates that surface molecules on the host cell surface, used
by A. muciniphila for adhesion, are expressed irrespective of the cell’s developmental state. Thus, A.
muciniphila may also be able to bind to enterocytes of different states in vivo. Third, binding of A.
muciniphila to ECM proteins was at background levels.

Furthermore, the results show that the trans-epithelial electrical resistance (TER) of a Caco-2 monolayer
significantly increased after 24h and 48h of cocultivation with A. muciniphila. Whereas, cocultivation with
E. coli significantly decreased the TER. This indicates that the presence of A. muciniphila positively affected
the integrity of the Caco-2 monolayer. It is also speculated that A. muciniphila may competitively exclude
pathobionts in areas where the epithelium is recently damaged. When the epithelium is damaged, ECM
proteins will be exposed. These proteins can serve as binding sites for pathobionts. However, because A.
muciniphila is able to bind to undifferentiated Caco-2 cells it may outcompete other bacteria in the early
stages of epithelial recovery. The role of A. muciniphila in the strengthening of the epithelial barrier could
explain previous observations linked to gut health and systemic health.

Finally, previous research has linked diabetes and obesity to decreased gut epithelium integrity and low-
grade inflammation, which can lead to LPS-induced endotoxemia. The release of LPS induces the production
of Interleukin-8 by enterocytes. This production leads to inflammation, which is a defence against
pathogens. Unnecessary inflammation in a healthy intestinal epithelium can lead to a disturbance of the
mucosal homeostasis. The results in this study show that A. muciniphila only provokes a minor IL-8
production in HT-29 cells, compared to E. coli. Thus, the presence of A. muciniphila does not lead to a strong
inflammation of the epithelium. Because of the low inflammatory response it was also studied whether A.
muciniphila produces LPS and if it is different from that of E. coli. Genome analysis and immunoelectron
microscopic analysis shows that A. muciniphila can and most likely does produce LPS. However, it does not
induce a strong IL-8 release by HT-29 cells. Thus, the LPS produced by A. muciniphila probably differs
structurally from that of E. coli.

Discussion and future developments

The results showed that A. muciniphila does strongly bind to enterocytes, but not to colon mucus. However,
this study cannot conclude with certainty that this is due to the binding capacities of A. muciniphila or due
to the aerobic experimental conditions used in the mucus binding assay. It is also still unknown how A.
muciniphila manages to colonize the thick mucus layer of the colon. Therefore, future research is needed.

Also, future research on the properties of LPS produced by A. muciniphila is needed. This can give more
insight into the precise immuno-signalling properties of the bacterium and the host receptors involved.

Furthermore, the in vitro results suggest a fortifying effect of A. muciniphila on the epithelial barrier. This
can be used to hypothesise in future in vivo research about the beneficial role of host interaction with A.
muciniphila in for example diabetes, obesity and the reduction of high-fat-diet-induced LPS endotoxemia in
obese mice.