Monday, February 18, 2019

Dialister and Coprococcus species in the Gut Microbiome has a role in Mental Health

If I had asked you to identify what are the most important organs of the human body, you are very likely to miss out on "microbiome". Though in a traditional textbook sense we never think of Microbiome as a part of the human body there is growing evidence and debate that perhaps microbiome contributes to the human gene pool. Some scientists have begun calling it a "virtual endocrine organ", thanks to many of the chemicals that they contribute. The concept that human is a holobiont and has implications on physiology is overwhelmingly taking over.

Fig 1: The concept of humans being a holobiont. Source

The point of this post is not to discuss the concept of humans being a holobiont, but to talk about a recent paper on the importance of gut microbiome in mental health. If you haven't read my previous posts on this topic, I suggest you read my earlier posts here and here.

Fig 2: Associations between QoL scores or depression and bacterial genera.
Source
Papers that have been published on the relationship between the microbiome and mental health have studied a few small numbers of cases which brings into question as to how valid the studies are in reality. So, a group of Dutch microbiologists from VIB-KU Leuven Center for Microbiology decided to look bioinformatically into the microbiome of 1,054 individuals enrolled in the Flemish Gut Flora Project (FGFP) and correlated their findings with the quality of life (QoL). They discovered that people presenting with depression had depleted levels of two bacteria- Coprococcus species and Dialister species irrespective of antidepressants. They also validated their results against a second independent cohort of 1,063 individuals from the Dutch LifeLinesDEEP (LLD) cohort and in a cohort of clinically depressed patients at the University Hospitals Leuven, Belgium. Fig 2 from the paper, shows a summary of microbial associations. With some additional bioinformatic analysis, they specifically looked for the presence of Bacteroides enterotype 2, which was based on their previous publication (Link) and found they indeed had a significant association with depression.

Jeroen Raes summarises the findings in his comment, "The relationship between gut microbial metabolism and mental health is a controversial topic in microbiome research. The notion that microbial metabolites can interact with our brain – and thus behaviour and feelings – is intriguing, but gut microbiome-brain communication has mostly been explored in animal models, with human research lagging behind. In our population-level study we identified several groups of bacteria that co-varied with human depression and QoL across populations. This finding adds more evidence pointing to the potentially dysbiotic nature of the Bacteroides 2 enterotype we identified earlier. Apparently, microbial communities that can be linked to intestinal inflammation and reduced wellbeing share a set of common features."

A small digression. Dialister species is known with reference to humans for some time as a potential pathogen, though very rare. They are small, anaerobic or microaerophilic gram-negative coccobacilli found to be involved with oral infections such as periodontitis, acute necrotizing ulcerative gingivitis, and endodontic infections especially D. pneumosintes and D. invisus. Other known human pathogens include D micraerophilus and D propionicifaciens. Coprococcus species are gram positive, anaerobic cocci knwon for its butyrate-producing capacity is a member of Clostridia group known to be a part of the human gut microbiome and found in faecal microbiome. Currently, three species are known- Coprococcus catus, Coprococcus comes and Coprococcus eutactus. 

Finding an association is one thing but how does it work? To understand possible mechanisms, the researchers developed a framework and based on literature curated and annotated 56 Gut–Brain modules (GBM). In a simplified sense, GBM is created by taking the genome of all the known microbiome and look for coding sequences which represent the ability to produce chemicals that can involve with neural function. For example, some of the Fusobacterium genomes that were analysed carried the potential to synthesize histamine. By comparing the GBM between microbiomes of people with a high or low QoL the researchers found 3 GBM's of interest based on data from the FGFP data. These include

1. Synthesis of 3,4-dihydroxyphenylacetic acid (DOPAC; positive correlation),
2. Isovaleric acid synthesis (via α -keto-acid decarboxylase pathway; positive correlation)
3. Histamine synthesis (Negative correlation).

However, when the findings were validated against the LLD metagenomic database, they found that only DOPAC synthesis could be replicated. Though Coprococcus did not have a genetic system to directly synthesise DOPAC, there were genes for converting 3,4-dihydroxy phenylacetaldehyde to DOPAC, in the genomes of Coprococcus comes and Coprococcus catus.

As the first author Mireia Valles-Colomer simplifies it, “Many neuroactive compounds are produced in the human gut. We wanted to see which gut microbes could participate in producing, degrading, or modifying these molecules. Our toolbox not only allows to identify the different bacteria that could play a role in mental health conditions but also the mechanisms potentially involved in this interaction with the host. For example, we found that the ability of microorganisms to produce DOPAC, a metabolite of the human neurotransmitter dopamine, was associated with better mental quality of life."

There is a word of caution associated with these studies. The current paper is completely a computational-based study and they need to be validated. However, they do give a lead into the understanding that microbes are significantly connected with mental health and probably their gene pool contributes to human neuro-metabolism through the principle of holobiosis.

References:

Clarke G, Stilling RM, Kennedy PJ, Stanton C, Cryan JF, Dinan TG. Gut Microbiota: The Neglected Endocrine Organ. Mol Endocrinol. 2014 Aug;28(8):1221-38 Link


Maarten van de Guchte, Joël Doré. Humans as holobionts: implications for prevention and therapy. Microbiome 2018; 6:81. Link

Mireia Valles-Colomer, Gwen Falony, Youssef Darzi, Ettje F. Tigchelaar, Jun Wang, Raul Y. Tito, Carmen Schiweck, Alexander Kurilshikov, Marie Joossens, Cisca Wijmenga, Stephan Claes, Lukas Van Oudenhove, Alexandra Zhernakova, Sara Vieira-Silva & Jeroen Raes. The neuroactive potential of the human gut microbiota in quality of life and depression. Nature Microbiology (2019). https://doi.org/10.1038/s41564-018-0337-x Link

Friday, February 01, 2019

Blogger's Desk#12- Financial spending on R&D: India

Greetings

Though this is a blog space to discuss Microbiology, I have also used this platform to sometimes talk about science itself to a larger audience of interest. In April 2015, I wrote a post on the plight of Indian research scholars (Link). With time, nothing seems to have really improved in the country.  The brightest of research scholars in the country are demonstrating on the streets to gain the attention of the government and seeking to solve the long-standing issues (Link). In this post, I am trying to provide a snapshot of the current financial status of research(ers) in India.

Fig 1:  Investment on R&D by country. Source
Fig 2: %GDP expenditure on R&D
The prime minister of India aims to place India at the top 3 on a global scale. Evidently, though these are words of political interest, there is nothing much that has been done at the scale of reality for the long term. In a study published in 2013 by Meo et al, it has been convincingly shown that there is a direct relationship between the spending on R&D and the quality of publications. In this light, India stands really down. Fig 2, shows a % GDP spending of India (in the year 2015) in comparison to some a few countries, as per the data available from the world bank. Despite this situation, the Indian spend on R&D has been the same at 0.6% GDP from the year 1998 to 2017. This financial crunch in research funding is totally reflected in our scientific publications. India stands at the 9th position on a global scale in producing citable documents but ranks 184/239 when it comes to the average citations per document. A large chunk of the citations is contributed by self-citations. Clearly, the government is not funding enough research labs to do their research work.

Fig 3: Stipend amounts received by PhD Scholars.
There has been a recent demand from researchers like me, who are pursuing our PhD under government fellowship (Through agencies such as CSIR/UGC/DST/ICMR). The developments so far have not been in favour of PhD scientists as a whole. It is largely the opinion of lots of scientists that the brain drain is due to this unfavourable financial situation. In fact, India contributes as a top source for emigration globally. Figure 3 shows the trend of stipend received by PhD students for performing research, who have qualified through national level competitive examinations. It appears from the graph that there is an increase in the salaries of PhD's by year. However, in reality, this was very low from the beginning with reference to the cost of living and the value for the degree or how much the same talent is paid in other countries where research is a priority.

Apparently, if India wants to be one among the best for research output, there is a requirement for a substantial increase in financial allocation for research. The 20-year-old trend of 0.6% of GDP is absolutely insufficient for sustaining the increased quality of research(ers).

Saturday, January 19, 2019

Genetic Spying for diagnostics- SHERLOCK CRISPR System: An update

Greetings

There have been so many great microbiology stories that I should have written about in the year 2018, but I was so occupied I never got the time. Well, doesn't mean this blog has died. Of course, the "almost a year gap" in writing has slightly affected my style of blogging. But I will pick it up back in a few weeks. That being said, let us come back to talk some science.

In the field of diagnostics, speed and accuracy of diagnostics is an ever challenging factor. From classic microbiology techniques which takes at least 48 hours to identify and characterise a pathogen, we have come a long way to more modern molecular methods such as PCR which can report in a couple of hours. These days, bacterial diagnostics have become faster with the use of MALDI-TOF instrumentation. For a quick and reliable diagnostics we still mostly rely on PCR and NGS methods. But they come with a high price tag and sophistication which are not directly "Field deployable". An alternative is immunologically based rapid diagnostic tests which take months to develop and validate and have lower capabilities than a genetic test.

On a side note, the CRISPR-Cas system has been widely harvested in genetic engineering. There is a great debate on who owns the intellectual property rights to CRISPR based gene editing technology which is an ongoing legal battle between the Broad and Berkley institute. There is also news of a Chinese research team lead by He Jiankui claiming to have created the first CRISPR-edited twins girls named Lulu and Nana. However, there are strong doubts over the claim and the bioethical aspects of this work have been subject an international discussion (Link).

Cas 13 protein has some interesting properties. Cas13a is functionally comparable to Cas9. In addition to its programmable RNase activity, Cas13a shows a collateral activity leading to non-specific degradation of any nearby transcripts regardless of complementarity to the spacer. Upon recognition of a specific RNA sequence programmed Cas13a is activated which then cleaves the surrounding ssRNA molecules. By using a quenched fluorescent ssRNA which can be cleaved to release the reporter (The idea is similar to TaqMan chemistry in Real-time PCR), the signal can be easily read. This phenomenon was harnessed by a group of scientists from the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University to develop a technique called as "SHERLOCK" in April 2017. SHERLOCK is a fancy acronym for Specific High-sensitivity Enzymatic Reporter unLOCKing. Subsequently the same team in April 2018 reported an improved design further to develop a protocol known as SHERLOCK-HUDSON. HUDSON is an acronym for "Heating Unextracted Diagnostic Samples to Obliterate Nucleases). Originally SHERLOCK presented with a few limitations related to quantification. Subsequently, SHERLOCK V2 which uses a combination of Cas13, Cas12a, and Csm6, was reported which can achieve multiplex nucleic acid detection with enhanced sensitivity. Also, SHERLOCK used fluorescent detectors whereas, in the improved version the reporter was modified such that cleaved reporter could be detected on commercial lateral flow strips.

There are a couple of associated technologies here. HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) platform for nucleic acid detection uses Cas 12 as the enzyme. DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter which was independently developed at Doudna's Lab also uses Cas 12a with small changes in the details.

Fig 1: Overview of the development of the CRISPR systems and their applications. Source

Fig 2: The principle of SHERLOCK and DETECTR detection methods.
SHERLOCK is mainly an RNA detection tool and has been demonstrated to be useful in diagnostics such as Zika Virus detection at attomolar concentrations. In contrast, DETECTR is a DNA detection method and has been demonstrated to be useful in identifying HPV. HUDSON is not a detection method, but rather a processing protocol where the virus particles are lysed and heat treated to work directly with SHERLOCK without the need for separate processing of samples. HOLMES works very much similar to other techniques except that it uses Cas12b in a Loop-Mediated Isothermal Amplification methodology. Figure 2 gives a schematic summary of the working of SHERLOCK and DETECTR.

Feng Zhang, whose lab is involved in developing the SHERLOCK technology commented, "SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method for detecting nucleic acid material — and that can mean a virus, tumour DNA, and many other targets. The SHERLOCK improvements now give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications. The technology demonstrates potential for many healthcare applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer, but it can also be used for industrial and agricultural applications where monitoring steps along the supply chain can reduce waste and improve safety".

References:

Liu H, Wang L, Luo Y. Blossom of CRISPR technologies and applications in disease treatment. Synth Syst Biotechnol. 2018 Oct 22;3(4):217-228. Link

Myhrvold et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 Apr 27;360(6387):444-448. Link

Gootenberg et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28;356(6336):438-442. Link

Friday, January 18, 2019

Mitochondria participates actively in Phagocytosis.

Greetings

Anyone who has studied biology knows about the process of phagocytosis. Phagocytosis, is a process by which a cell engulfs other cells or particles. In context with immune system, phagocyosis is an important mechanism in defense against many different microbes. In fact, the mechanism is so important that several pathogenic microbes have evolved and acquired specialised mechanism to evade killing by phagoctyosis.

Fig 1: Mechanisms of escaping Phagocytic killing
by Microbes. Source
There are basically two broad mechanisms of surviving phagocytosis. First, the microbe can completly evade being phagocytosed for example by using a capsule (Ex: S pneumoniae). Second, after being phagocytosed the microbe can survive being ripped off inside the phagosome either by inhibiting phagosome lysosome fusion (Ex: M tuberculosis), surviving inside phagolysosome (Ex: Coxiella burnetii) or escape from phagosome (Ex: Shigella species). Some microbes can even produce toxins that directly attack phagocytic cells (Ex: Leukocidins by S aureus).  Figure 1 is a summary of microbial tools that allows escape from being killed by phagocytosis.

Though the exact details of phagocytic killing vary, the prinicple is pretty much the same globally. For example, the WBC first attaches the bacteria to its cell wall through a receptor and then internalises the bacteria inside a vacuole (Phagosome). The phagosome then attaches with a lysosome which consists of several enzymes to form a phagolysosome. This process is called as maturation. Simultaneously, the phagolysosome is acidifed by the multi-subunit vacuolar ATPase (V-ATPase), which pumps hydrogen ions into the lysosomal compartment that activates the degradative enzymes of the lysosome. The internal of phagolysosome contains enzymes such as nucleases, proteases, lipases, glucosidase etc which aids in digestion of its content. Figure 2, provides the major steps involved in phagocytosis and autophagy.

Fig 2: Major steps in phagocytosis and autophagy. Source
In classic textbook case scenario, mitochondria doesnt appear in the scene. In 2011, researchers showed experimental evidence indicating that the activation of Toll like receptors- TLR1, TLR2 and TLR4 lead to recruitment of mitochondria to macrophage phagosomes and enhanced mitochondrial Reactive oxygen species (ROS) production. Subsequently a detailed work in 2015 by another group (lead by Mary O'Riordan) discovered that inositol-requiring enzyme 1α (IRE1􏰀􏰀α), a protein from endoplasmic reticulum was an important factor in phagocytosis killing. Further analysis using ROS tracker chloromethyl 2',7'-dichlorodihydrofluorescein diacetate showed that in a normally functioning macrophage ROS migrated from mitochondria to the phagosome to achieve an effective killing. Most probably the process of engulfment turns on the stress signal which leads to mitochondrial synthesis of mROS (mitochondrial ROS),  packed and sent into the phagosome.

Figure 3: MDVs delivers mROS to the phagosome.
Source
Most recently, this understanding has been further explored by Mary O'Riordan and her team. The study followed up the previous findings indicating that the TLR dependent IRE1􏰀􏰀α induced mitochondrial ROS (mostly mitochondrial hydrogen peroxide) that are delivered to phagosomes via mitochondria derived vesicles (MDVs). This study basically identified Sod2 as a key enzyme involved in mH2O2 production in a TLR dependent mechanism. The pathway was also demonstrated to be operational via Parkin/Pink1-dependent mitochondrial stress pathway.

Abuaita (the first author of the paper) comments, "We discovered that macrophages sense invading MRSA and turn on the machinery to increase mitochondrial development of ROS". O'Riordan who has been working on the role of mitochondria in enhancing phagosome function comments on the published study, "ROS are also damaging to our own cells, so we hypothesized that there must be some delivery mechanism. Mitochondria have not traditionally been known to package and deliver substances to different parts of the cell. The immune system is full of redundancies. It has to, by definition; every bacteria, virus, or parasite that we know is a pathogen is one because it has evolved ways to avoid the immune system. The immune system also has a real diversity of purpose and mechanism. Being open to different ways of asking questions about the immune system and understanding the biology of these pathogens helped us to find the right experimental system to use."

References:

Smith LM, May RC. Mechanisms of microbial escape from phagocyte killing. Biochem Soc Trans. 2013 Apr;41(2):475-90. Link

Richards DM, Endres RG. The mechanism of phagocytosis: two stages of engulfment. Biophys J. 2014 Oct 7;107(7):1542-53. Link

West AP, Brodsky IE, Rahner C, Woo DK, Erdjument-Bromage H, Tempst P, Walsh MC, Choi Y, Shadel GS, Ghosh S.  TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature. 2011 Apr 28;472(7344):476-80. Link

Abuaita BH, Schultz TL, O'Riordan MX. Mitochondria-Derived Vesicles Deliver Antimicrobial Reactive Oxygen Species to Control Phagosome-Localized Staphylococcus aureus. Cell Host Microbe. 2018 Nov 14;24(5):625-636.e5. Link