Monday, July 16, 2012

Shigella and salmonella messing with mTOR

Good to see you back,

Fig 1:Vaccines combine to produce new virus strain (Image from science news)
        As always, this week too had a lot of microbiology going on. The one which has beaten the hell out of people is a report from University of Melbourne, that a veterinary vaccine has backfired. The paper is by Sang-Won Lee and others titled "Attenuated Vaccines Can Recombine to Form Virulent Field Viruses". This would have been an excellent topic for me to talk about, but i couldn't get hold of the paper from Science. However, i could find an interesting story on the same well written in science news. And since its well covered by the science news, i guess i could just leave this matter for the time now, with a link to the story.

Fig 2: TEM of GFAJ-1 (Source)
            A yet another news that am not blogging about here, is the debate on arsenic based life forms. But that's still worth a mention here. The paper first published in 2010 reported that  bacteria GFAJ-1 can survive in high arsenic concentration by using arsenic instead of phosphorous met with strong controversy. The study was challenged by yet another study which argued the other way. Though most of the scientists have concluded that phosphorous cannot be replaced, the original contenders of the story are still trying to latch on to their claim. (Link to the controversial story here and here). Maybe we have more twists and turns to come up in this story. Anyways, keep your senses open for any news.

      The third story that i thought i will post in the blog is about "Galectin-8", and its association with autophagy. But then I came across a post in ASM blog that talks about it. (Oh man, that cover story also well covered up). In fact only after reading that post i came to know there's a journal attributed to discuss only Autophagy. That means, the topic has incredible amount of importance more than what i had thought about. But then i haven't lost a blog story totally after all. At least not after reading the article from "Cell Host & Microbe". I didn't find a blog on that so guess i can talk about it.

So here goes the story.

     Let me give you a analogy of autophagy. The best way to evade an intruder from getting into a house is to guard it well with defence (Immune system in this case). But with modern weaponry (Virulence factors), the intruder just manages to get into the system and takes good control. Now you are left with 2 choices, one abandon the battle (Don't expect the immune system to do that) or set of a detonator, that blows up itself from inside the house and blows up everything (Not a bad option, when it comes to cells. You can regenerate one).

  Interruption!! Did i forget to tell you what paper am i talking about? The paper is titled "Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program" by Ivan Tattoli and others.

      The innate defense mechanism of autophagy is regulated by a molecule called as mTOR which is a metabolic checkpoint kinase. The authors main aim of investigation was to give  deeper look into the how of the whole situation. Autophagy is a very conserved process that allows the cells to remove old non functional or highly damaged tissue elements to be digested and then removed from existence, that paves way to new ones. The process is highly regulated. The Fig 3 shown below depicts how the mTOR is invloved with autophagy. However it should be mTOR has many identified roles such as its importance in Influenza replication (Source) and in normal cellular translation process (Shown in Fig 4; Source)

Fig 3: Coordinated regulation of nutrient sensing by mTOR and AMPK (Source)

Fig 4: Importance of mTOR in normal cellular process
     The first thing that the authors did was speculate. They argued that probably the intracellular infections messed up with cellular metabolism and thereby with mTOR signaling.

      The study used Shigella (They didn't talk about the species or type, but still makes sense cause all 4 are nearly the same) and infected the HeLa cells, and they were able to demonstrate that there is a down-regulation of the phosphorylation of S6K1 and 4EBP1, with important interaction with mTOR. Rapamycin which is a potent inhibitor of mTOR (Source) was also used to demonstrate the reduction of phosphorylation. It was also noted that Shigella infection resulted in a strong induction of AKT phosphorylation.
Fig 5:  HeLa cells left unstimulated (CTR) or infected with Shigella for 4 hr, analyzed by IF with antibodies against mTOR and LAMP2.
Fig 6: Stress response
    Then to give a more detailed look, like all other scientists would do, they looked directly (I mean to say fluorescence based studies). With IF (immunofluorescence), it can be seen that Shigella infection resulted in a cytosolic relocalization of mTOR in infected cells (See Fig 5 on right). If you are thinking why they labelled LAMP 2 (Lysosomal-associated membrane protein 2), the full form should tell you why. Then by playing around a bit with raptor they could conclude that "Shigella infection down regulates mTORC1 activity by displacing mTOR from LAMP2+ LE/Ly vesicles, at the level of Raptor targeting to Rag GTPases".

    They could show that intracellular amino acid sensor GCN2 was rapidly phosphorylated in Shigella-infected cells mean to indicate that there is a Amino acid starvation. To confirm that this was actually the case a liquid chromatography/mass spectrometry (LC/MS) analysis was done on 2 amino acids L-leucine and L-isoleucine (Since they are supposedly highly influential with the mTOR pathways). Result? Shigella caused a reduction of cytosolic L-leucine/L-isoleucine levels. With these, they were able to elucidate and support stress response cascade (See Fig 6).

      The authors set to identify at genetic level. By comparing RNA expression patterns, from the one's in Shigella-infected cells and rapamycin-stimulated cells a large group of 87 genes were found. But as they looked for high expression patterns they found transcription factor ATF3 and its target CHAC1, were highly upregulated. The kinetic studies showed that ATF3 was induced as early as 1 hr. So thats how they came across the Fig 6, a classic GCN2/ eIF2a / ATF4/ ATF3 integrated stress response (ISR).

     When i had finished reading this much the first thing that struck me was "This should be a broad phenomenon". And to answer this ? (I mean the authors also had the same question) they did the study with  Salmonella Typhimurium. But they found that in this case the Salmonella-induced amino acid starvation was transient. And they did many other studies such as lentiviral-mediated RNA interference to look into other aspects (It didn't grab my attention much).

From these points i can conclude one thing. The amino acid starvation response, TOR pathway, and autophagy are highly audited metabolic connections that seems to define our understanding of the importance of battle inside the cells.

*Please note that the entire paper has not been discussed. I have taken only the upper half that i felt had enough interesting catch. But the paper dwells more deeper into various aspects and interested people should read the paper. The theme of this blog was to generate interest and i guess by leaving some part undiscussed here am giving you a room to explore certain areas yourself. Or maybe with enough request a episode 2 of this will be written.


ResearchBlogging.org
Ivan Tattoli, Matthew T. Sorbara, Dajana Vuckovic, Arthur Ling, Fraser Soares, Leticia A.M. Carneiro, Chloe Yang, Andrew Emili, Dana J. Philpott, & Stephen E. Girardin1 (2012). Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program Cell Host & Microbe DOI: 10.1016/j.chom.2012.04.012


Further Reading:
  1. Qian Yang and Kun-Liang Guan. Expanding mTOR signaling. Cell Research (2007) 17:666–681. doi: 10.1038/cr.2007.64 (Link)
  2. Mathieu Laplante and David M. Sabatini. mTOR signaling at a glance. J Cell Sci October 15, 2009 122, 3589-3594. doi: 10.1242/​jcs.051011 (Link)

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