Thursday, December 19, 2013

Fighting tuberculosis


      A few posts back, I was discussing about the problems associated with tuberculosis (Link) and how scientists have come up with a novel strategy of attacking ATP pathways. The need for an effective drug is reflected in the rapid FDA approval of Bedaquiline (diarylquinoline anti-tuberculosis drug). As a matter of fact we badly need a Anti-tuberculosis drug. The second aspect of tuberculosis fighting is vaccine. BCG vaccination is faithfully given to a huge number of population, and yet TB is rampant in country like India. Indeed, BCG is reported to be the most widely used vaccine worldwide (administered to more than 4 billion individuals) with unmatched safety records. But how far has this whole scenario taken us into the fight against TB, is the question.

Photo 1: AFB (TB) in sputum sample
     A little bit into the history. The organism was discovered in 1882 by Robert Koch. Later, Albert Calmette and Camille Guérin, working at the Pasteur Institute (Lille), studying basic biology were trying to get a homogenized suspension of culture in a glycerin and potato medium. This couldn't be directly achieved. Trying a variety of chemicals they stumbled on the use of bile which incidentally impacted the virulence. Based on the research, they sub-cultured TB for about 11 years (Nearly 230 subcultures), a vaccine strain emerged which was successfully tested on a variety of animal models. Originally known as Bacille Bilie Calmette-Guerin, was latter renamed as Bacillus Calmette–Guérin (BCG). The story goes that the vaccine was first given orally by Weill-Halle assisted by Raymond Turpin, on 18 July 1921. The practice was continued for a significant longtime, before it came to the present day form.

       Most often am asked a question. What are the genetic changes in BCG vaccine strain? Wish I could give you a simple straight forward answer. Right from the start, there were different versions of BCG- such as the Copenhagen strain, Tice strain etc. The strains vary by several laboratory features, which has significant effect in vaccination outcome. We are really looking forward to a more robust vaccine.

Photo 2: Fiona Smaill and Zhou Xing
      An improvement has come in the form of immunity boosting vaccine.AdHu5Ag85A is a recombinant human type 5 adenovirus (AdHu5)–based TB vaccine that has been deigned at McMaster Lab (Link). The phase 1 study showed that administration of vaccine boosted polyfunctional CD4+ and CD8+ T cell immunity in previously BCG-vaccinated volunteers. According to the WHO, the vaccine is one of about 10 that are currently in the works worldwide. The lead author Zhou Xing comments "It’s critically important for us to push forward multiple vaccine candidates in order to determine which one will be the champion". Smaill added: "As a doctor who looks after patients who have tuberculosis, including those who are HIV infected, I realize how important it is going to be to control this infection with a good vaccine. We are probably one of a few groups in the world who are actually doing bench-to-human tuberculosis vaccine work, and we are excited to be part of this and thrilled that it started at McMaster." Source

Fig 1: Proposed model of Mycobacterial
heme uptake. Source
      The second wing of TB research is busy building antibiotics. A massive genomic, Proteomic and integrated bioinformatic approach, has suggested for lead candidates as antibiotic targets. AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. Of these, MmpL3 is a component of the heme uptake system. Any microbiologist would tell you iron is a very important component of the bacteria functioning and a bacteria would go to great lengths to get it from the host (By such as producing hemolysins and subsequent extraction of iron, or by producing high affinity iron binding proteins such as enterochelisins). That is a potential target to target. A recently published science article has shown 2 compounds NITD-304 and NITD-349 (Indolcarboxamide compounds) showed promising pharmaco-kinetic, and favorable toxicological profiles in a mouse model. From the preliminary studies by Genetic and lipid profiling studies MmpL3 has been suggested as a likely candidate attacked.

      Scientists are often puzzled about the absolute dormant state that the MTB can achieve, a key reason for resistance. Though newer TB antibiotic development is focused on pathways that couldn't be shut down (Such as ATP synthesis), developing these drugs will take its own sweet time. Now answer for how has shown up in research. It is postulated that a MTB internal toxin called VapC20 (An endoribonuclease) can be switched on in presence of Antibiotics and off when antibiotics are not around. The VapC20 inhibits translation by cleavage of the Sarcin–Ricin loop (SRL) of 23S ribosomal RNA. This tops protein translation and induces dormancy. The cleavage is more of a loop structure dependent chemistry rather than sequence. The point is if we could make a chemical that can stop this process, may be we can do more with current antibiotics.

     In conclusion, we have still more a lot to research on TB. We are currently heading into a possible improvement in BCG vaccination and better antibiotic targets. Indeed basic biology of this organism is still left to be understood.
Simona luca, & Traian Mihaescu (2013). History of BCG Vaccine MAEDICA – a Journal of Clinical Medicine, 8 (1), 53-58 : PMC3749764

Behr MA (2002). BCG--different strains, different vaccines? Lancet Infectious diseases, 2 (2), 86-92. PMID: 11901655

Smaill F, Jeyanathan M, Smieja M, Medina MF, Thanthrige-Don N, Zganiacz A, Yin C, Heriazon A, Damjanovic D, Puri L, Hamid J, Xie F, Foley R, Bramson J, Gauldie J, & Xing Z (2013). A human type 5 adenovirus-based tuberculosis vaccine induces robust T cell responses in humans despite preexisting anti-adenovirus immunity. Science translational medicine, 5 (205) PMID: 24089406

Ioerger TR, O'Malley T, Liao R, Guinn KM, Hickey MJ, Mohaideen N, Murphy KC, Boshoff HI, Mizrahi V, Rubin EJ, Sassetti CM, Barry CE 3rd, Sherman DR, Parish T, & Sacchettini JC (2013). Identification of New Drug Targets and Resistance Mechanisms in Mycobacterium tuberculosis. PloS one, 8 (9) PMID: 24086479

Rao SP etal. (2013). Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis. Science translational medicine, 5 (214) PMID: 24307692

Winther KS, Brodersen DE, Brown AK, & Gerdes K (2013). VapC20 of Mycobacterium tuberculosis cleaves the Sarcin-Ricin loop of 23S rRNA. Nature communications, 4 PMID: 24225902

Tuesday, December 10, 2013

Bacteria count each other using homoserine lactones or peptides- Quorum sensing


      Imagine you are a pathogenic bacteria (ughh..). You are interested in messing up the host system so that you could manipulate. How would you do that. Here's one important point, strategy. Any army commander would tell you that the best strategy in war, is not letting the other person know you are in. In other words stealth. And when you are enough in number you erupt with a surprise attack. So you enter the system and try to make space. For argument sake, say you are 100 in number. You get up and say "Wow, lets attack". Even before you knew it, host immune system would have destroyed your puny army. Even if you had throw very potent toxin, thats not going to be good enough. Cause you are less in number.

    In reality, bacteria avoids this problem by keeping quiet and not blowing the immune alarm system. The bacteria silently replicates till there is enough number of cells in the army and everyone in single shot throws up everything that it has- toxins, enzymes etc.. Now the number being high, and taken by surprise the immune system is thrown off balance temporarily. Consequence is Infection. But did you notice, the bacteria needs to count each other and coordinate their strategy. This is the concept of Quorum sensing. Before you actually launch an attack, you need to know how many of you are in the vicinity. It will also be good to know how many of others are there. This post is about how such bacteria can actually work this out and how could we use this idea in the field of Clinical Microbiology.

Table 1: Autoinducer signals
     The chemicals used by the bacteria to generate signals of counting are referred to as Auto-inducers. Chemically they are of 2 types. Short peptide derivatives, commonly utilized by Gram-positive bacteria and fatty acid derivatives, called homoserine lactones (HSLs) utilized by Gram-negative members. This is not a absolute rule, as there are many other molecules used that are more recently discovered such as Butyrylactones that stimulates antibiotic synthesis in Streptomyces species and amino acids inducing swarming in Proteus. The basic idea is that when the bacteria secrete molecules if the cell count is too less, the molecule would simply drift away and the chances that it is detected is very low. But if the count is increased, the chances that the molecules are received and intercepted is much higher. When a sufficient level of autoinducer concentration is reached (Threshold level) a critical cell mass is inferred leading to activation or repression of genes as per requirement (But in a coordinated fashion).

Fig 1: N-Acyl Homoserine Lactone
      There are 2 types of autoinducers. First type is involved in surveying the whole population, in other words a universal language. This type can bind to a universal receptor and signals the total number in terms of microbes, and not sequence specific. The 2nd type can bind species specific receptor only. This maybe an oversimplification, as strain specific autoinducers are demonstrated to exist. Whatever, is the case, it is a signature sequence and hence surveys only the specific population. This can be shown by the fact that there is no response produced in experiments when the chemicals are crossed between species.

Quorum sensing in Gram Negative Bacteria:

Fig 2: Quorum sensing in Gram Negative Bacteria
   The Gram negatives, produce homoserine lactones, most of them belonging to N-acyl homoserine lactone group. The classical sensing system is regulated via a gene complex containing two regulatory components: A transcriptional activator protein (R protein) and the AI molecule produced by the autoinducer synthase. R protein consists of two domains: the N terminus of the protein that interacts with AI and the C terminus that is involved in DNA binding. The AI molecule is synthesized and secreted. When a sufficient threshold of signal is detected, these molecules can bind to and activate a transcriptional activator, or R protein, which in turn induces expression of target genes. A variety of chemicals has been studied in this category (Link), showing the same backbone structure as shown in figure 1, with differing side chains.

Quorum sensing in Gram positive bacteria

Fig 3: Quorum sensing in S aureus
     Gram positives use a more variety of molecules which are often post translationally modified peptides. The peptide signals interact with a histidine kinase two-component signal transduction system. One of the best studied gram positive QS is the "Arg" system in S aureus. A small 7-9 aa peptide referred as an auto-inducing peptide (AIP) is secreted, which is a transmembrane receptor histidine kinase. The activation of kinase phosphorylates and activates ArgA, which regulate "arg" operon RNA polymerase- III, which in turn regulates gene expression.

     Here's where it gets more interesting. Different strains of S aureus are shown to produce variants of AIP (Such as AIP-1, AIP-2, AIP-3, AIP-4 etc). Some variants can actually inhibit other types. This Cross-inhibition of gene expression represents a type of bacterial interference.

Fig 4: Secreted oligopeptide regulation
of bacterial quorum-sensing
receptors. Source
    The molecules involved in quorum sensing system in gram positive are classified under a group of proteins called RNPP according to the names of the first four identified members: Rap (RNAIII-activating protein), NprR (neutral protease), PlcR (Phospholipase C Regulator) and PrgX. Rap proteins are phosphatases and transcriptional anti-activators, while NprR, PlcR, and PrgX proteins are DNA binding transcription factors. Rap proteins consist of a C-terminal tetratricopeptide repeat (TPR) domain (Consisits of seven similar helix-turn-helix repeats) connected by a flexible helix-containing linker to an N-terminal 3-helix bundle. The Rap can act as a on-Off switch, depending on the binding of signaling peptide. The actual molecular detail of the process is still under studies but a possible mechanism is shown in fig 4.

       Why are we interested in knowing all these? Quorum sensing is a highly specialized phenomenon. The effects are highly selective, with a variety of effects. The most surprising example is in V cholerae. Quorum sensing negatively impacts the CT (Cholera Toxin), TCP (Toxin-Coregulated Pili) and HlyA (Hemolysin) the quorum-sensing-regulated transcription factor HapR. Vibrio cholerae autoinducer CAI-1 can interfere with Pseudomonas aeruginosa quorum sensing and inhibits its growth. Cross-species and cross strain bacterial interference helps in competing for the bacteria. Our silver lining is designing antibiotics. We can make synthetics which can target this specificity. This approach is much more specific than any existing antibiotic could achieve.

    Antibiotic designs against the quorum sensing can be considered under following categories- (i) Chemical blockers of sensing which can directly inhibit the specific quorum molecule sensing by use of competing analogues or biniding inhibitors or (ii) Quorum quenching where the mediating molecule is quenched out. Organisms such as Bacillus sp. 240B produce lactonase, cleave the lactone ring from the acyl moiety of AHLs and render the AHLs inactive in signal transduction. Other organisms that can do the same (Break down intermediates of signaling) include Variovorax paradoxusRalstonia sp. XJ12B, A. tumefaciens producing AttM and AiiB, Arthrobacter producing AhlD, K. pneumonia producing AhlK, Ochrobactrum producing AidH, Microbacterium testaceum producing AiiM, Solibacillus silvestris producing AhlS, Rhodococcus strains W2, LS31 and PI33 producing QsdA and certain Chryseobacterium strains. As you can see many bacteria have evolved mechanism to cheat or destroy other's signalling molecules.

    The rational of designing an antibiotic targeting Quorum sensing is that unlike our traditional antibiotic that attack some key process in the survival of organism (and hence evolutionary pressure is laid on the organism), these antibiotic simply block the expression of their virulence, giving our immune system a chance to fight. This is expected to bypass the resistance emergence. However, my perspective is that if immune system will kill the bacteria, then there still had be a low evolutionary pressure on the organism to evolve and resistance will still develop, though less faster than it currently is.
de Kievit TR, & Iglewski BH (2000). Bacterial quorum sensing in pathogenic relationships. Infection and immunity, 68 (9), 4839-49 PMID: 10948095

Ji G, Beavis R, & Novick RP (1997). Bacterial interference caused by autoinducing peptide variants. Science (New York, N.Y.), 276 (5321), 2027-30 PMID: 9197262

Parashar V, Jeffrey PD, & Neiditch MB (2013). Conformational change-induced repeat domain expansion regulates Rap phosphatase quorum-sensing signal receptors. PLoS biology, 11 (3) PMID: 23526881

Tsou AM, & Zhu J (2010). Quorum sensing negatively regulates hemolysin transcriptionally and posttranslationally in Vibrio cholerae. Infection and immunity, 78 (1), 461-7 PMID: 19858311

Ganin H, Danin-Poleg Y, Kashi Y, & Meijler MM (2012). Vibrio cholerae autoinducer CAI-1 interferes with Pseudomonas aeruginosa quorum sensing and inhibits its growth. ACS chemical biology, 7 (4), 659-65 PMID: 22270383

Chen F, Gao Y, Chen X, Yu Z, & Li X (2013). Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection. International journal of molecular sciences, 14 (9), 17477-500 PMID: 24065091

Wednesday, December 04, 2013

Isolated Immunology inside Central Nervous system


    This blog has focussed quite a lot on the concepts of core microbiology. As an occasional drift, today I want to talk about a topic that is one of the fields with very less literature available on hand. Don't get me wrong. Am not going to talk about extreme geeky stuff, but just the basics. A rare field of Microbiology, Neuro-Microbiology and its counterpart, Neuro-Immunology. So here's a question for you to gaze at. The popular view that used to exist in the field of medicine is "Neuro" is a Immunoprivileged site. If thats the case, someone once asked "How the antibodies and cellular Immune response to Neural infections if there is very little exchange and immunologically inert?"

     The popular scientific view was that CNS (Central nervous system), is a highly protected area and there is a very little exchange of molecules (very tightly regulated exchange) from the other parts of the body compared to CNS. In reality, this holds true for many molecules. The molecules found in the CSF (Cerebrospinal fluid), which baths the CNS is in equilibrium with serum molecules. The ratio of molecules (generally) in CSF being approximately, 1/3rd of that in serum. Of course there are exceptions.

Fig 1: Experiment demonstrating the BBB
      In 1880's Paul Ehrlich experimentally observed, intravenous administration of dyes stained all organs except the brain and the spinal cord. In 1913, Edwin Goldman, demonstrated the same dye when directly injected into the CSF, readily stained nervous tissue but not other tissues. The experiments for the first time indicated that there was a barrier that separated the two anatomical regions. However, it was Lewandowsky, while studying potassium ferrocyannide penetration into the brain, was the first to coin the term blood-brain barrier. Later experiments used basic lipid soluble dyes, which could stain all parts including CNS, showed that there was a direct transport of the dyes across the cerebral microvasculature. Further studies by Broman concluded that it was not a single system. The barrier is a two component system, Blood-CSF barrier (BCB) at the choroid plexus and the blood-brain barrier (BBB) at the cerebral microvasculature. The final confirmation came from EM studies by Reese and his team demonstrating the barrier to the capillary endothelial cells within the brain by electron-microscopic studies. For source and more details, refer here.

Fig 2: The BBB and BCB.
     The BBB and BCB maintain the cellular and chemical contents of CSF within strict limits. Lipid soluble substances within blood can diffuse across. However, passages of fluids, ionic and polar substances requires facilitated transport. Na+ an important component nerve firing, is transported via passive diffusion and Na-K pump. Potassium is however, actively removed from CSF circulation. Interesting to note that the substances as important as glucose, amino acids, certain hormones (such as insulin) requires specialized transport. Chloride (Cl) represents a major anion in the CSF, and its concentration is 15-20 mEq/L higher than in serum. Earlier papers suggested that in Tubercular meningitis (TBM), Cl concentration was lowered and the test was used to predict TBM. This was thought to be due to a breach in BBB. However, we now know that it is simply a reflection of lower serum values. What I mean to say is CSF chloride levels is no more considered a diagnostic or prognostic marker for TBM. The Acid- Base balance is also maintained by the choroid plexus. It can remove weak organic acid and antibiotics such as Penicillins, cephalosporins, aminoglycosides from CSF.

      Give this some thought. CNS is THE MOST important part to protected. Theoretically speaking, we should have had an immune system, that is more aggressively active in this part. But the truth is, it isn't. I understand given the importance, to have a specialized gate mode of entry but why immunologically less active? The restriction of movement is so much that even molecules such as IgM is not allowed to cross. There is vritually no lymph node (Although Virchow Robin space is considered as a analogus version of Lymph node of brain). The possible answer is, Immunity is a double edged sword. The battleground of immunity often leads to damage to neighbouring cells, through inflammation, a risk that cannot be taken easily in the nerve environment. Maybe thats why we have evolved our CNS to be preferentially previleged.

     The concept of Neuro-inflammation is quite complex. When warranted (physiological and pathological) the CNS can respond to a variety of factors, such as pathogens, toxins, degeneration etc. Surprisngly the neuronal activity can itself activate the immune system of immunity. A recent publication, suggests the term "Neurogenic Neuroinflammation" for inflammatory reactions in the CNS in response to neuronal activity.

    Digressing from the above, I will put forth a question. Is autoimmunity bad? Conventional scientific wisdom is "Of course bad". Autoimmunity is not always pathogenic. A certain degree of autoimmunity is required to Destroy abnormal, dead cells, Tumor immunity and the more recent researched field- "Protective autoimmunity", which pertains to role of Neuro-inflammation for repairs in CNS. I have put forward this idea here just to illustrate it to you one of the lead roles of tight regulation of immune cells, that is served by the selective barriers of CNS. That should hint you a connection between Neurogenic Neuroinflammation and Protective autoimmunity

      Let me put the whole thing in a perspective. Yes, Neuro-immunology is a total different way of operating of immune cells in context to CNS. There is a special barrier that possibly excludes a large subset of leucocytes from accessing the brain microenvironment. However, certain subset of cells can involve in the CNS immunosurveillance. In other words, CNS controls its own immunosurveillance (by selectively regulating cell trafficking), a luxury not available at any other part of the body. The immune functioning (Inside the CNS) plays significant role in supporting normal stem/progenitor cell renewal and neurogenesis, hippocampal-dependent cognitive ability and attention, and they are crucial for containing mental stress by enabling its resolution, and for fighting off depression. Reference

ResearchBlogging.orgBallabh P, Braun A, & Nedergaard M (2004). The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiology of disease, 16 (1), 1-13 PMID: 15207256

Abbott NJ, Rönnbäck L, & Hansson E (2006). Astrocyte-endothelial interactions at the blood-brain barrier. Nature reviews. Neuroscience, 7 (1), 41-53 PMID: 16371949

Xanthos DN, & Sandkühler J (2013). Neurogenic neuroinflammation: inflammatory CNS reactions in response to neuronal activity. Nature reviews. Neuroscience PMID: 24281245