Tuesday, February 28, 2012

Race between phage and bacteria- CRISPR

        Let me begin with a note “Survival of the fittest”. There would hardly be anyone who doesn’t know this concept. Bacteria make compounds against fungus and other bacteria’s. Fungus makes compound to suppress the bacteria. Phages attacks the bacteria, bacteria mutate to become resistant to phage. And almost instantaneously the phage also mutates so it can again attack the bacteria. And once again the race begins.

Isn’t that amazing?

For quite some time now, we know of some DNA piece in bacteria that is important in an adaptive immunity to the bacteria. Though there are many cellular immune systems such as Restriction endonucleases, gene repression, DNA modification, receptor modification and the much studied CRISPR system.

CRISPR stands for clustered regularly interspaced short palindromic repeats. This gene represents the struggle for existence between the viruses and the bacteria. The sequence length varies from aprox 20 and 50bp. The system runs along with a team mate called cas (CRISPR associated sequence) and are together called as CRISPR-cas immune system of bacteria. In reality it’s also postulated that this system is an operon regulated feature and hence we can definitely expect a very important role for this. Also CRISPRs have been found to be present in about 40% of all sequenced bacterial genomes, 90% of archaeal genomes. That’s a pretty good number.

So what do we know about the mechanisms of action????

The CRISPR sequence is transcribed into a long RNA which is then cut to make crRNA, (Mature CRISPR RNAs). Each of the individual crRNA has a spacer sequence. While in many bacteria CRISPR spacers contain a large proportion of sequences matching fully or partially genomic sequences of phages. The CRISPR system is divided into 3 stages:

1. Adaptation of the CRISPR via the integration of short sequences of the invaders as spacers
2. Expression of CRISPRs and subsequent processing to small guide RNAs
3. Interference of target DNA by the crRNA guides.

For a detailed discussion on the process, click here. (They have explained it in the best possible way).

I want to summarize so….

     The CRISPR sequence contains spacers which are a copy of phage nucleic acid and is inserted into the cell. New phage infections, more spacer registry records are maintained. Each of these new spacer sequences matches some section of the infecting phage genome (called the protospacer). Once a phage enters the cell, if the spacer has the record of infection, immunity is launched by the help of CRSIPR/Cas complex which targets the phage DNA and lyses it. The exact mechanism is not known but appears to be similar to be something like a DNA guided cleavage. 

And when the bacteria laugh’s of having acquired resistance, what does the phage do? Yup, he changes his sequence (Mutation) so that the bacterial spacer is no more in agreement. This was shown by experimental data. Some viral genomes can also interfere by integrating right into the CRISPR sequence and thus disrupting their function. So at the angle of fitness cost the bacteria better have a huge record of sequences. To the best of what I know a record of 18 loci, accounting for 1% of the genome is maintained by Methanocaldococcus jannaschii. 

Finally, I want to conclude saying that the phylogenetic analysis of sequence in this system can tell us a lot about the history of the bacteria. 

Further Reading:

1. Ksenia Pougach and others. Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli. Mol Microbiol. 2010 September; 77(6): 1367–1379. doi: 10.1111/j.1365-2958.2010.07265.x 

2. Rodolphe Barrangou and Philippe Horvath. The CRISPR System Protects Microbes against Phages, Plasmids. Microbe magazine. Link

Monday, February 20, 2012

Plasmodium- A smart parasite

This is a ppt copy of my presentation on Plasmodium in Explore-2012 CME, Manipal.

      The following is the summary of my talk, already published in "Genesis" by MLT department, MCOAHS, Manipal.
     Humans have been known to be effected from Plasmodium species from a very old time. This vector borne disease is transmitted by the female Anopheles group of mosquito. It is believed that this parasite has modified itself, along with human evolution and is with us since human race began. To date, P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi are found to be the etiological agents. It is estimated that, nearly 1 million deaths occur every year which is 1% of the infected population as per the WHO estimates.

     Plasmodium species belongs to Apicomplexa, is morphologically a drop shaped organism in asexual stage with various internal structures, of which rhoptry and microneme has been studied in depth owing to its importance in invasion of erythrocyte. The life cycle has been studied in detail. The parasite attacks the RBC and internalizes itself via a gliding movement, the details of which are still in question. On successful invasion a “Parasitophorous vacuole (PV)” is created and the organism takes control of the RBC machinery. The hemoglobin part is used as a nutrient and broken into components using various enzymes (Such as plasmepsin and others). A special enzyme called as Heme detoxification protein (HDP) is thought to convert the heme portion to hemozoin. This metabolite is a toxic waste and extruded from the PV which produces the ring appearance.

     The laboratory diagnosis is mostly by methods of microscopy. Thick and thin blood smears stained using Romanovsky stain or a QBC preparation is used singly or in combination with RDT (Rapid diagnostic tests). The RDT targets the HRP (Histidine rich protein) or pLDH. Though culture and molecular analysis is available the use in a diagnostic setting is still questioned, thanks to the cost. An expensive but very good method is to look for hemozoin in WBC population using its unique properties of light dispersion (Cell-Dyn automated blood cell analyzers). Speciation is usually by microscopy, which however doesn’t distinguish between P knowlesi and P malariae.

     Control of vector has not been effective. Avoiding exposure to mosquito by use of bed nets and IRS (Indoor residual spraying), or both has been effective. The CSP (circumsporozoite protein) expressed in the sporozoite has been found to contain a unique NANP repeat is being tested by GSK. Now known as RTS, S/AS01 Vaccine is under evaluation in a phase 3 trial. The preliminary results are showing a 50% protection, good leap from nothing. PfRH5 which has a strong affinity to CD147 has also been projected as possible vaccine candidate for falciparum species. 

     In nutshell, the pragmatic aspect of controlling malaria is good but elimination is not a possibility, at least as per the existing knowledge on plasmodium biology.

Tuesday, February 14, 2012

Prion- The Enigmatic Protein

     Prion diseases, also known as transmissible spongiform encephalopathies (TSE), are a group of rare, rapidly progressive, and fatal neurologic diseases. Precisely prions are proteinaceous infectious particles that lack nucleic acids. This field has been intense areas of research owing to the capacity of the molecule to self proliferate without a nucleic acid. Very little is known about this molecular enigma though a small amount of information is available. Dr. Stanley Prusiner was awarded the noble prize for his work on prions.

     The most earlier assumption that they are viruses was contradicted by the failure to directly demonstrate such a virus (or any immunological response) and by evidence indicating that the transmissible agent showed remarkable resistance to treatment expected to inactivate nucleic acids but susceptible to protein inactivation procedures. It is believed that most of the species have their own prion infections. Prions against bovine and many other species are well known.

     It is now widely agreed that the prions are structural abnormal isoform (PrPsc) of host proteins designated as PrP through a post translational mechanism. PrP is a protein found in maximum concentration in the central nervous system of many different vertebrates and highly conserved. The protein is found predominantly in synapses at cholesterol rich microdomains or caveolae and also in immune system. A complete analysis of prion structure with Fourier transform infrared (FTIR) spectroscopic methods reveals that the beta pleating of the protein molecule is abnormally high. (well that explains a lot!!!!)

     Under experimental mixing conditions the PrPsc is used as a seed to convert the PrPc to form protease resistant forms. However the concentration of seed used for the experiment is high which makes the assay of new infective forms very difficult. For experimental protocols swainsonine which is an inhibitor of complex glycosylation is used to block prion formation, though all strains are not equally susceptible. The general idea here is the biochemical amplification of protease-resistant PrPSc-like protein (PrPres) using a protein-misfolding cyclic amplification method. 

     So our basic understanding is that the prions are misfolded proteins that have an unusual high tendency to resist degradation and its subsequent accumulation. In addition they cause conversion of other normal proteins to abnormal phenotypes. Now thats really a rogue molecule. 

Creutzfeldt–Jakob disease

     CJD is a neurodegenerative disorder, which is the most common of prion diseases. It was first described by German neurologist Hans Gerhard Creutzfeldt. The prion molecule involved is human PrPsc from PrPc encoded by a gene PRNP. The human PRNP gene is located on the short (p) arm of chromosome 20 between the end (terminus) of the arm and position 12, from base pair 4,615,068 to base pair 4,630,233.PRNP has also recently been designated CD230. CJD can be subdivided into three different subtypes – genetic, acquired and sporadic. The PrPsc molecule is resistant to normal cellular degradation process. This accumulates to form an amyloid aggregate. PrPsc also forms tiny fibers called scrapie-associated fibrils (SAFs). 

Laboratory Diagnosis of Prion diseases

     Prions are simply proteins and the diagnosis implies finding the unique pathotypes in the specimens. Almost universally the prions are found in the nervous tissue (probably because most of them arise from PRNP exclusively active in nervous tissue.) Though a few studies have reported isolation and detection of prion particles from other sites they are less diagnostically sensitive, (except in vCJD tonsilar biopsy is highly diagnostic). The Prions are identified for its presence by resistance to proteinase K. An extracted material is subjected to enzymatic digestion to proteinase K and the remaining tissue is extracted for proteins. They are then subjected to a western blot. A further identification is possible by Proteomic analysis and Protein sequencing. 

     Culture of the prions is a Hercules tool for research. This can be done by seeding prion protein (from the sample) to a susceptible tissue possibly into a monolayer maintained by the tissue culture methods. This however requires time and the process may be very slow. The sensitivity and specificity of the method is quite low. The newer innovative technique is by use of PMCA (Protein Mis-fold cyclic amplification). PMCA is an amplification technique (conceptually like PCR without use of nucleotides) to multiply misfolded prions. The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasound, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into mis-folded prion.

Further reading:

1. Stanley B prusiner. Prions, PNAS, Vol. 95, pp. 13363–13383, November 1998. Nobel Lecture.
2. Alison etal. 14-3-3 in the cerebrospinal fluid of patients with variant and sporadic Creutzfeldt–Jakob disease measured using capture assay able to detect low levels of 14-3-3 protein. Neuroscience Letters 324 (2002) 57–60.

Monday, February 06, 2012

C diff- The poop rescue

        C. difficile (Often known as C diff!!!!), is a commensal bacterium in nearly 2-5% of population. The C. difficile is known to occur in a spore form (Non infectious environmental resistant form) and a vegetative infectious form. This organism is commonly found in the large intestine and inherently resistant to various forms of antibiotic. So what happens when a person gets heavily treated with oral antibiotics? Straight forward answer is the normal flora of the gut gets disrupted. Now that the C diff gets a chance (Selection pressure they say!), the organism pre dominates in the gut and plays smart. This causes a condition called "antibiotic-associated diarrhea (AAD)" or "Clostridium difficile associated diarrhea (CDAD)".

        The pathogenic strains produce 2 toxins- TcdA and TcdB. These two toxins, TcdA and TcdB, are encoded on a pathogenicity locus along with negative and positive regulators. That means a fully functional pathogenicity island. Following expression and release from the bacterium, TcdA and TcdB reaches the cytosol of target cells and inactivate small GTP-binding proteins, which include Rho, Rac, and Cdc42. Inactivation occurs through monoglucosylation of a single reactive threonine, which lies within the effector-binding loop and coordinates a divalent cation critical to binding GTP. Ultimately this cause actin condensation and cell rounding, which is followed by death of the cell. TcdA elicits effects primarily within the intestinal epithelium, while TcdB has a broader cell tropism.

Fig: Taken from Daniel E. Voth and Jimmy D. Ballard (Further Reading 1)

        Pseudomembranous colitis (PMC), which is an infection of the large intestine (colon) with an overgrowth of Clostridium difficile bacteria is a very difficult to treat devastating problem. PMC is often associated with Ciprofloxacin, Levofloxacin, Amoxicillin, Ampicillin, Clindamycin, Cefixime and Cefpodoxime use. The condition often presents with bloody diarrhea and abdominal cramps. Unless treated vigorously the condition can worse clinically. 

Now that you understand the C diff tricks so well and given the fact that it’s very difficult to treat the organism what do you do?

        50 years ago when we didn’t have a good knowledge of what an intestinal micro flora is like and C diff was not properly known fecal flora was administered. The doc’s used to take a fresh healthy fecal sample and do some treatment of it and made the patient eat the stuff. I don’t know how exactly how they did it but for sure they treated the fecal samples in such a way that patient didn’t know what he was made to consume. 

       Now that was exactly what was replicated by Kelly etal in now what is called as “Fecal Microbiota Transplantation”. They studied 26 patients with relapsing C. difficile infection, who underwent FMT over a 28-month period. FMT was performed during colonoscopy by direct infusion of minimally processed donor stool. The results show about 92% effectiveness. Now what’s interesting to me from this study is the use of fecal samples. As per my understanding, the FMT is transferring the normal flora to a person who has depleted flora and restoring gut health. This could have also been done with a probiotics also. If there is a study with parallel controls, with probiotics and then results compared we will have a more reliable data. Of course this current study is just a case series and more needs to be studied.

        So don’t be surprised if the pharmaceutical company asks for fecal donors and process it and markets it tomorrow. May be someone will eventually find the right cocktail of bacteria’s that can be incorporated into the gut to restore floral balance and ask the C diff to shut up.

Further reading:

1. Daniel E. Voth and Jimmy D. Ballard. Clostridium difficile Toxins: Mechanism of Action and Role in Disease Clin. Microbiol. Rev. April 2005vol. 18 no. 2 247-263.

2. Thomas J, Borody & Alexander Khoruts. Fecal microbiota transplantation and emerging applications. Nature Reviews Gastroenterology and Hepatology. doi:10.1038/nrgastro.2011.244

3. Kelly, Colleen R. MD; de Leon, Lauren MD; Jasutkar, Niren MD. Fecal Microbiota Transplantation for Relapsing Clostridium difficile Infection in 26 Patients: Methodology and Results. Journal of Clinical Gastroenterology. February 2012 - Volume 46 - Issue 2 - p 145–14. doi: 10.1097/MCG.0b013e318234570b.