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