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

1 comment: