Monday, February 24, 2014

Schizophrenia- Ever thought of Microbial involvement?


    A few posts earlier I had talked about Immunology inside the central nervous system (Link). My idea was to highlight how Neuroimmunology is different from what happens in other part of the body. I made a note that immune cells inside CNS has a role to play in cognition and a variety of Neurobehavioral functions. One of my common argument is that microbes are more involved in our biology that is actually thought to be (this one argument that keeps appearing in posts). In a yet another post earlier, I had talked about influence of "Transposable elements" and its influence on conditions such as Alzheimer's, Parkinson's and Schizophrenia. In this post, I explore how infections play a role (if not a major), in some of the neurobehavioral disorders. 

Table 1: Common organisms in context
with Neurobehavioral disorders.
       Many different pathogens have been studied in context with Neurobehavioral and Psychiatric disorders. The list of organisms that has been studied in this regard is shown in Table 1. In theory it is possible, many more organism are involved.

       I believe almost everyone has heard of schizophrenia. It is a mental disorder characterized by a breakdown in thinking and poor emotional responses. In the simplest molecular terminology, it is misfiring and miscommunication of neurons. Patients suffer from Hallucinations, feels like they are possessed, and other qualities of insanity. The actual cause of this horrific problem has not been fully solved.

    Genome wide studies have shown that there is a genetic cause in a small subgroup of peoples. The genes that are linked include- Neuroregulin 1, Dysbindin, Proline dehydrogenase, G72 etc, but most interestingly HLA phenotypes (a major proportion). Schizophrenia is classified into- Catatonic, Paranoid, Disorganised, Residual and undifferentiated. For details please go here. The bottom line is that the patient has had a neural damage during brain development which has lead to false wiring and firing od neurons. That explains the onset age usually around the teenage. However, in a subgroup of patients, there appears to be no risk factors and dont respond to classical therapy. Why?

     In coming, Pathological Neuroimmunology. It has been convincingly shown that increased levels of IL-8 in the mother during pregnancy (especially in second trimester), uncontrolled IL 6 in CSF, blocking NMDA (N methyl D aspartate) and its receptor, other autoimmune disorder etc have all been implicated in schizophrenia. It has been shown that cytokines can directly influence important brain region development (As shown by some MRI studies). For example, IL 6 is activated in case of infection and IL 6 can effect the hippocampus, grey matter etc. This data convincingly shows that immunology has some role. Of all these, NMDA antagonism is a very attractive candidate and has received lot of attention. Studies have speculated that NR1a and NR2b epitopes are important. An important question remains unanswered in this case. What triggers antibody production, or dysregulation of chemokines in the first place? It would be very tempting for people to speculate genes and environmental risk factors as most important important factors. Partly true. But it has now become increasingly clear that infection by itself has an active role. To draw conclusions from studies is very hard in this case, as both sides of the argument exists.

     That maynot be impressive. Especially since i have put in a lot of speculative data there. A landmark paper in JAMA showed that atleast 1/3rd of the people who developed mood disorder and significant association with infection and to a small extent had autoimmunity.

   There are a lot of studies that I could cite. For example, Antibodies in GI tract and contents of normal flora in GIT has a significant effectin bipolar disorder. A condition called as PANDAS (Paediatric Autoimmune Neuropsychiatric Disorders Associated With Streptococcal Infections) that shows up as Obsessive compulsive disorder is very much related to Strepto infection and cross reacting antibody. These are just some of the examples that show that Neuro is not an isolated system.

    It is important to note that infection and inflammation are not the sole cause. But they do form a subtype, which if recognised well enough, should help us develop better treatment options. A word of caution here. Many chronic diseases have been associated with infection and inflammation such as as Obesity, Diabetes, Atherosclerosis, Autoimmune disorders etc (The list keeps growing). We don't have sufficient evidence yet to fully prove the theroy. But remember, Lack of evidence is not evidence of absence. Research is actively on.
Müller N (2014). Immunology of schizophrenia. Neuroimmunomodulation, 21 (2-3), 109-16 PMID: 24557043

Marsland AL, Gianaros PJ, Abramowitch SM, Manuck SB, & Hariri AR (2008). Interleukin-6 covaries inversely with hippocampal grey matter volume in middle-aged adults. Biological psychiatry, 64 (6), 484-90 PMID: 18514163

Benros ME, Waltoft BL, Nordentoft M, Ostergaard SD, Eaton WW, Krogh J, & Mortensen PB (2013). Autoimmune diseases and severe infections as risk factors for mood disorders: a nationwide study. JAMA psychiatry, 70 (8), 812-20 PMID: 23760347

Murphy, Tanya K. (2012-02--1) Clinical Factors Associated with Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections. , 160(2), 314-319. DOI: 10.1016/j.jpeds.2011.07.012 

Monday, February 10, 2014

Protein A, G, A/G, L and Protein M


     Half jokingly I announced one day, "Scientists are normal people who are puzzled by things that most other people don't care about and chase its reality". Well its debatable, in terms of what really that should mean, I leave it for readers to justify. I scan several papers in a week, looking for important research papers, updates, through podcast and RSS feeds. But then usually the one that strikes me are the ones that relates to my routine practice. But every now and then i come up with something that simply blows me of the line, simply cause they are so predictable but haven't been thought about.

Fig 1: Protein A.
     Perhaps everyone is familiar with Protein A (Staphylococcal Protein A; SpA). It is a Type I membrane protein, a surface protein covalently bound to the peptidoglycan layer, which inhibits antibody mediated clearance by binding with IgG1, IgG2 and IgG4 Fc receptors. The structure of Protein A, has been studied in exquisite detail. It is postulated that Z domain (five homologous Ig-binding domains) which contains three alpha helices (arranged in an anti-parallel three-helix bundle), is the most important component that binds the immunoglobulin. The binding has been thought to occur either by 2 possible mechanisms. Rearrangement mechanism or Direct binding mechanism.

       S aureus uses the Protein A to chelate the antibody by binding to the Fc portion abolishing the activity of Fc, leading to Immuno evasion. This mechanism has been classically explained and forms a major part of Staph weaponry. Here's my punch point. A machinery that is too successful, is highly replicated in biology. That means, staphylococcus is not the only organism that produces a protein that can bind immunoglobulins. Protein A is usually used for research purpose to capture antibodies for purification purpose. A better version of this protein referred as recombinant variety, has better properties.

Other well described protein includes- Protein G, Protein L and the latest sensation- Protein M. I have summarized the important features of Protein A, Protein L and Protein G in table 1.

Table 1: Immunoglobulin binding Proteins.
       Protein G, expressed in group C and G Streptococcus with similar binding properties as that of Protein A. Protein G also binds to albumin, which is usually present in large quantities in preparations of blood. It has been modified to remove this additional activity, available as Rec Protein A/G. A domain in Protein G called as G B1 domain (or simply GB1 is known to render a protein soluble. This property has been made use of in research, by fusing insoluble Proteins with GB1 which is important for experiments such as NMR and crystallography. The recombinant variety has also better pH binding range (5 to 8.2).

          Protein L, differentiates itself from others by binding to Light chain (Kappa type) rather than the Fc portion. This property allows Protein L to bind to almost any type of immunoglobulin. It is isolated from Peptostreptococcus magnus, consisting of 719 amino acid residues.

It is important to note that the Proteins A, G, A/G and L are all available commercially for different types of purification based applications.

     So what has led me to write this post is the discovery of yet another molecule in the same pipeline, Protein M (from M genitalium). The surprise is that Protein M doesn't match with other binders in terms of sequence and structure. Instead of multiple Ig like binding folds, this has a large domain of 360 residues (with a very large binding area), binds principally to antibody VL domains. Unlike the Protein L which binds only kappa chains, this can bind both Kappa and Lambda chains. That has a lot of applications waiting to be used. But from bacteria's point of view, it has evolved a defense that is too good. The last I know, the protein is already in high demand to be commercialized like the others.

    Perhaps there are more antibody binding proteins out there in nature with different properties. We may need to look hard enough.
Mitsuru Tashiro, Roberto Tejero, Diane E Zimmerman, Bernardo Celda, Björn Nilsson, Gaetano T Montelione (1997). High-resolution solution NMR structure of the Z domain of staphylococcal protein A. Journal of Molecular Biology, 272 (4), 573-590 DOI:10.1006/jmbi.1997.1265

Sjöbring U, Björck L, & Kastern W (1991). Streptococcal protein G. Gene structure and protein binding properties. The Journal of biological chemistry, 266 (1), 399-405 PMID: 1985908

Akerström B, & Björck L (1989). Protein L: an immunoglobulin light chain-binding bacterial protein. Characterization of binding and physicochemical properties. The Journal of biological chemistry, 264 (33), 19740-6 PMID: 2479638

Grover RK, Zhu X, Nieusma T, Jones T, Boero I, MacLeod AS, Mark A, Niessen S, Kim HJ, Kong L, Assad-Garcia N, Kwon K, Chesi M, Smider VV, Salomon DR, Jelinek DF, Kyle RA, Pyles RB, Glass JI, Ward AB, Wilson IA, & Lerner RA (2014). A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union. Science (New York, N.Y.), 343 (6171), 656-61 PMID: 24503852

Wednesday, February 05, 2014

Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR


      I was going through the statistics of this blog, in context of what is read and discussed the most. I presume antibiotics, HIV and Influenza are the most read and searched topics. It reflects very much on how much the current science craves for a good antibiotic. From my reading (If i understood it correct), we are never going to have the perfect antibiotic, but maybe we will have better. For now, i will talk about a yet another basic of molecular bacteriology, the CRISPR system.

     A long time ago, I had written a very basic post about CRISPR system. This probably is the right time to revisit the topic. CRSIPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRSIPR genetic system is always in association with a set of functional regulators called as cas (CRSIPR associated) genes. Together they are referred as CRSIPR/Cas system. Classically, they are a intracellular microbial defense system, (a type of microbial nuclease) involved in defense against invading phages. The system can be considered as analogous to the RNAi mechanism. A target sequence remembers the sequence of RNA to be attacked, which binds and cleaves the target nucleic acid. Based on the structure, activity and mechanism of functioning, it is classified into 3 types and several sub types.

Fig 1: Summary of CRISPR Cas System.
   In Fig 1, I have included a timeline to give you a glimpse of the developments in understanding of CRISPR. The bacterial immunity was discovered in 1987, but the term was coined in 2002. The CRISPR was thought to be a highly variable genome and was used for genotyping. Its only in last couple of years, its true function has been studied. A breakthrough came when CRISPR system was shown to be useful as a genome editing tool by using RNA nuclease directed editing. There is a great deal of molecular details available in literature, regarding artificial gene editing which I will not visit here. Here, we will see what it biologically means for the bacteria and phage.

Fig 2: Basic Structure of CRISPR system
      The basic structure of CRISPR Cas genome consists of a leader sequence, Cas enzymes, repeating sequence, interspersed with spacer sequences. The basic sequence of events are acquisition, expression and interference. The acquisition of phage DNA happens during the initial phage infection. It is not entirely known as to how this functions. The bottom line is randomly sequence are captured and integrated to the CRISPR system as a spacer sequence. Of note, we can have a pretty good idea of the history of phage infection of the bacteria by knowing the sequence of these spacers. This sequence forms the memory of immunity. The spacer is usually about 30 bp in length. Every time a new spacer is added a repeat unit is also attached creating space for the next integration. The setup of spacers which is due to processed is called as Proto-spacer-adjacent motifs (PAM's). Protospacers are defined as short sequences (~20 bp) of known foreign DNA separated by a short palindromic repeat and kept like a record against future encounters.

     The second step is Expression. The repeat-spacer array is processed to yield small CRISPR RNAs (crRNAs) that contain a full or partial spacer sequence. This crRNA acts as the guide (referred as guide RNA or gRNA in artificial system). This in itself is a multi- step process with formation of intermediates including trans-activating crRNA (tracrRNA). For a detailed step by step process, see here.

Fig 3: Basic Steps in CRISPR Cas functioning
   A little bit of clarification here. Some literature suggests 2 steps- A the highly conserved "Information processing subsystem", which refers to Acquisition (Adaptation), and the "executive" subsystem, which includes the expression and interference stages. This is to demarcate the enzymes involved. The first step uses enzymes that are almost universal in the bacterial population (Cas1 and Cas2), whereas the executive system depends on the organism under question.

       The final step is interference. Once a complete mature crRNA which fits by base pairing to the viral genome that maybe present, is formed, it triggers a series of events (depending on the type of subsystem). The final outcome being degradation of DNA or RNA.

        I want to make an important point here. To have immunity against more phages demands more spacer sequences. Though theoretically you are allowed to have as many sequences as you wish to have, practically, the size itself becomes a limiting factor. Research suggests that, this problem is possibly circumvented by deleting very old memories which is not actively encountered. And that lets the bacteria be immune- erased against that phage. The second important thing to be noted is that the "Nucleic Acid guided attack" is highly sequence specific. This means, a very small change in the phage genome can lead to significant non recognition, defeating the whole purpose. This is how exactly a phage evolves to evade the immunity put up by the bacteria.

    The CRISPR specificity can be used as an advantage point in gene editing. The same editing system can be turned against the bacteria itself, by some neat tricks. It is well known that intentional targetting and disruption of bacterial genome can be very deleterious. A paper by Gomaa etal published in mBio, used this understanding to design a CRISPR system (type I-E CRISPR-Cas system) that could attack E coli selectively in a proof of concept model experiment. The authors suggest that this is an excellent antibiotic design. If a feasible mode of delivery can be invented, this may serve as an important selective anti bacterial strategy.

     Signing of with a take home message. The Zinc finger nuclease and CRSIPR technology (both inspired from natural systems) has come up as an excellent nucleic acid editing tool. But, there is a still lot to be studied about CRISPR, as many of the participant enzymes, and the pathways of this system is not well understood. There is always scope for more.
Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, & Koonin EV (2011). Evolution and classification of the CRISPR-Cas systems. Nature reviews. Microbiology, 9 (6), 467-77 PMID: 21552286

Bikard D, & Marraffini LA (2013). Control of gene expression by CRISPR-Cas systems. F1000prime reports, 5 PMID: 24273648.

Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R, & Beisel CL (2014). Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems. mBio, 5 (1) PMID: 24473129