Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Speaker:  Clara Belzer

Department: Laboratory of Microbiology, Wageningen University, The Netherlands

Subject: Verrucomicrobia and the intestinal microbiome; the case of the Akkermansia muciniphila

Location: BioTechnology department TU Delft

Date: 29-06-2017

Author: Mirte Golverdingen

To broaden my view of the studies done in the Applied Science building I visited a lunch lecture in the BioTechnology department. The microbiologist Clara Belzer from Wageningen University was the speaker and she spoke of the newly discovered Akkermansia muciniphila bacteria. Belzer and her group are interested in anaerobic micro-organisms that can degrade ligases and mucus in the gut. Verrucomicrobia is one of the phyla that lives in the gut, the bacteria Akkermansia muciniphila is one of the Verrucomicrobia and isolated by the group for a quest of new bacteria.

They discovered that the bacteria are very dominant along the mucus gut and an important player in human microbiota composition. The bacteria are a potential maker for healthy intestine and has an extraordinary mucus degrading capacity. The importance of the bacteria is shown in the fact that after a month after birth the bacteria can already be detected in the gut system. This is certainly not the case for all gut bacteria.

The human microbiota changes during life stages and perturbations, this is partly caused by the place of the bacteria on the body. However, over a life time the microbiota can also change due to physiological changes of the body by diet changes. Therefore, the change in microbiota can contribute to or starting diseases. Moreover, some microbiotia do not depend on food, they depend on mucus and mother milk.

Bacteria influence the host cell by a difference in glycal level. This has a microbial benefit; mucosal glycan foraging can solve the problem of the competition for glycans in the gut. For the host, the mucosal microbes can provide resistance against the colonization of pathogens. It directly inhibits the pathogens by production of antimicrobial compounds. Moreover, the depletion of nutrients modulates the immune response of the host.

In the gut, there are 5 major microbiological fila, some are mucus degraders and some are mucus binders. Mucus can be used as the source of growth because mucus glycans can be created by the bacteria. However, there are not only glucose molecules in these mucus glycans, there are also other more ‘awkward’ sugars which are hard to break down. These sugars need to be broken down by enzymes. The Akkermansia muciniphila is adapted to digest mucin, it contains a high number of genes that encodes for enzymes which can degrade mucin. None of all Verrucomicrobia have the same number of enzymes that are able to digest mucin as the A. muciniphila.  So, the A. muciniphila is really adapted to the work of mucin digestion.

So, to find out if A. muciniphila is also present in other animals, Belzer et al. went to the zoo to find the bacteria in mucus of mammals and non-mammals (see figure 1). The A. muciniphila found in these animals differ as the animals have different digestive physiologies, diets and mucus structure. Still, the bacteria were not very different, which is very remarkable. The only difference between the animals A. muciniphila was found in the antibiotic resistance and CRISPR Cas system, as the animals differ in defending to bacteria and viruses in their environment.

So, why are the bacteria so similar? This could be due to a spontaneous infection very recently, so the bacteria were not able to evolve since. The bacteria could also have a very stable genome that does not evolve. Or the bacteria are not specific on the type of mucus.  One genome, however, was different from the rest. The python strain of the A. muciniphila looks really different. Moreover, only a small number of all animals are tested on the A. muciniphila. Therefore, more different genomes could be discovered.

Belzer saw a similarity between organisms and the A. muciniphila system: As soon as the host was on a diet or starving, the A. muciniphila levels are going up. This is similar for all organisms and systems. This could indicate that A. muciniphila could be an obese related bacterium. Belzer moreover, showed that the A. muciniphila works as a growth support of beneficial microbes in the gut. Moreover, when the A. muciniphila levels rises in the gut, organisms were metabolic healthier.

It was very interesting to visit the BioTechonolgy department for this seminar. Some metabolic details were hard to follow for me; however, I was still able to follow her talk. I was surprised by the complex microbiota in the gut. The similarity of the A. muciniphila is very interesting and research for this bacteria in other animals can contribute to the understanding of this bacteria.

Honours11

Figure 1: Akkermansia muciniphila is universally distributed in intestinal tracts all over the animal kingdom. (a) Phylogenetic tree indicating the position of A. muciniphila among selected full-length 16S rRNA clones from mammalian gut samples. Red colored samples derive from human sources. Thermotoga thermarum is used as an outgroup. The tree was generated using the neighbor joining method. Full details and high-resolution information are provided in Supplementary Figure S1. (b) Schematic representation of the tree in (a) with the five different clades their position and similarity to A. muciniphila. (c) Taxonomic tree of mammals generated using iTol webtool from tree of life project using all available sequences from NCBI (Letunic and Bork). Animal silhouettes indicate single species as a representative of that order. When an animal species from the mammalian orders was positive for Akkermansia-like sequences the animal logo belonging to that order is colored red, when it was negative the animal logo is colored gray. No Akkermansia sequences have been reported yet in any of the animals belonging to the mammalian orders depicted in black.

Adapted from: Belzer, C., & De Vos, W. M. (2012). Microbes inside—from diversity to function: the case of Akkermansia. The ISME journal, 6(8), 1449-1458.

CRISPR-based interference in prokaryotes from exploration to exploitation

Speaker:         Prof. dr. John van der Oost

Department:    Bionanoscience

Subject:          CRISPR-based interference in prokaryotes

Location:         Delft

Date:               12-11-2015

 Author: Carolien Bastiaanssen

Prof. John van der Oost from the Laboratory of Microbiology of Wageningen University gave a BN seminar about CRISPR. This is a defence system which about 85% of archaea and 40% of bacteria have. The topics that were covered in this seminar were the discovery and mechanism of CRISPR, the different classes and their characteristics and the applications of the system.

Bacteria and archaea have developed various ways of dealing with viral threats: inhibition of adsorption, inhibition of DNA injection, degradation of DNA and abortive infection. In the 1980s a Japanese research group discovered a peculiar structure in the DNA of bacteria. At that time they did not know its function, but it turned out to be yet another defence mechanism of archaea and bacteria. The name of this mechanism, CRISPR, is an acronym that stands for Clustered Regularly Interspaced Short Palindromic Repeats. Between these repeats there are sequences, called spacers, which are homologous to viral DNA. Situated next to the CRISPR loci are the CRISPR associated (Cas) genes. The whole pathway is often referred to as CRISPR-Cas and it provides a form of adaptive and heritable immunity.

The process can be divided into three stages. The first stage is the acquisition of spacers. If a virus injects DNA into a bacteria, this DNA is recognized as foreign and it will be cut into pieces. Part of it will be incorporated in the bacteria’s own genome as a spacer. In the next stage the Cas genes are transcribed and pre-crRNA (CRISPR RNA) is produced. This is processed into mature crRNA. The final stage is the actual target interference where the Cas proteins and the crRNA locate foreign DNA and disable it. A very important aspect is that the system has to discriminate between non-self and self DNA. Because the foreign sequences are incorporated into the DNA of the bacterium itself, only looking at the sequence is not enough. CRISPR therefore relies on a protospacer adjacent motif (PAM). This short sequence is present in the viral DNA but not in the spacer. A second control point is the seed sequence, if there are any mismatches in this sequence the crRNA will not bind.

Overview of the CRISPR cas system

The figure above gives an overview of the CRISPR-Cas system. Source: Van der Oost, J. et all, Unravellling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology (2014)

There is a huge diversity of CRISPR-Cas systems, primarily in the Cas genes and proteins. There are three main types and each of them has several subtypes. Type I and Type III are very similar and are also referred to as Class I. Type II and a recently discovered Type V are referred to as Class 2. In Class I the different Cas genes produce a multisubunit complex, named CRISPR-associated complex for antiviral defence (Cascade). Together with Cas6, Cascade is responsible for pre-crRNA processing. When a target is near, Cascade undergoes a conformational change, probably to recruit Cas3. Together they target the DNA. The spacer acquisition is done by Cas1 and Cas2. In Class 2 there is no Cascade but only a single protein, for Type II this is Cas9. The unique features of Type II are that there is tracrRNA, which stands for trans-activating CRISPR RNA and that the nuclease used to cut the DNA is RNaseIII. The most recently discovered Type V (or Cpf1) also has a single subunit like Cas9. However it has no tracrRNA and here the PAM is at the 5’ side instead of at the 3’ side. It uses a non-Cas RNase and the double-strand DNA break that it makes is staggered unlike the cut in the other types. All types target DNA except for Type III which targets RNA.

Apart from protecting bacteria from viral attacks CRISPR can be used for various other purposes. Prof. Van der Oost and his co-workers engineered spacers from the lambda phage and introduced them into E. Coli. In this way they made E. coli immune for the lambda phage. Another application of CRISPR could be an interesting alternative for antibiotics. Using CRISPR to engineer good viruses to target harmful bacteria. And last but not least CRISPR has great possibilities as a genome editing tool.