This Week in Microbiology
With Vincent Racaniello, Michele Swanson, Michael Schmidt
Episode 175: Neomycin is antiviral
Aired May 2, 2018
Vincent: This Week in Microbiology is brought to you by the American Society for Microbiology at asm.org/twim.
(MUSIC)
Vincent: This is TWiM, This Week in Microbiology episode 175 recorded on April 26, 2018. I’m Vincent Racaniello and you are listening to the podcast that explores unseen life on Earth. Joining me today from Ann Arbor, Michigan, Michele Swanson.
Michele Hello!
Vincent: How’s everything?
Michele Everything is peachy! It’s graduation here so the streets are full of vans being loaded with things and vans being loaded up and people dressed up and the occasional cap and gown.
Vincent: You got nice weather for that?
Michele: Yeah I think right now it’s in the 60s. It’s sunny.
Michael: It’s spring!
Vincent: So joining us from Charleston, South Carolina, Michael Schmidt.
Michael: Hello everyone! And it’s spring here!
Vincent: 70s, 80s?
Michael: 70s. We haven’t gone into full blown summer yet so I am very delighted that it is staying in spring.
Vincent: It’s still chilly here but today it’s kind of nice outside. Nice blue sky. The plants are blooming, all the daffodils are out here. That means it’s spring.
Michele: Hooray!
Vincent: Now, today being the 26thof February, today is Elio’s birthday.
Michael: April! 26thof April!
Vincent: Where am I getting February from? Is that what I want it to be?
Michael: It’s chilly! It’s chilly!
Vincent: 26thof April, it’s Elio’s birthday, happy birthday Elio!
Michele: Happy birthday!
Vincent: Ninety.
Michele: Woo. Who would have guessed.
Vincent: That is amazing. He must be the most advanced in age person doing a podcast in the world. Now someone will write in and tell us—
Michele: Most experienced.
Vincent: Most experienced. We are grateful that he is joining us. Anyway, he’s away for today. He asked for the day off to celebrate his ninetieth.
Michele: He’s having a wonderful family celebration.
Vincent: That’s great. He’ll be back next time, but boy that is some milestone.
Michael: And his collaborators on his blog site, Small Things Considered, wrote a tremendous tribute to him. Very with tongue planted firmly in cheek, and it’s really quite spectacular on the Small Things Considered blog. If you have not checked it out, you really need to see what it says and the title of that particular blog post is A Whiff of Taxonomy — Eliobacter Woosyi. And it’s really quite spectacular, describing some of his phenotypic traits.
Michele: So should we link to that? Is it appropriate for a birthday?
Vincent: We will. Sure, it’s totally appropriate. We’ll put that in. I’ll paste it in now to remind me. That’s good, thanks for bringing that up. We have a followup that is related to Elio, it is from Thao and the TWIM fans on the BBM organizing committee. BBM is the Boston Bacterial Meeting. And they write:
We wanted to write to sincerely thank Elio for his endorsement of the Boston Bacterial Meeting during last week’s episode. We enjoyed having him as a guest two years ago and we were pleasantly surprised to hear him mention us. Would you be willing to provide a few additional details about our conference to your listeners?
Sure!
Boston Bacterial Meeting invites you to join a quorum of fellow bacterial researchers in New England and beyond. Visit bostonbacterial.org to register. It is a 2 day conference from May 31stto June 1st. It is organized by grad students and post docs. Attendance is now at about 550 researchers from hundreds of labs. There are opportunities to attend poster and oral sessions as well as breakout discussion sessions. The keynote speaker is Dianne Newman, professor of biology and geobiology at CalTech. There you go.
Michele: Wow. Can I add, I did both my doctoral and postdoctoral training in Boston and I had the privilege of giving a talk at that meeting many years ago and it was right about as I was going to go on the job search, so it was really a great opportunity to present my post-doc work to a group and get some feedback. So I highly recommend it.
Vincent: Yeah, and it’s not very expensive. A hundred-twenty dollars for a two day conference. This year it is going to happen at the Science Center at Harvard. So they do have that website bostonbacterial.org. So if you are in the area, check it out! Thank you Thao and the BBM organizing committee. And we also have some sad news for you. Allan Campbell has passed away.
Michael: Yeah, it’s really…I had the opportunity to meet him at the general meeting of the American Society for Microbiology a number of years ago when it was in Los Angeles. Jeff Miller, who I guess he was chair of the meetings committee at the time, was gracious enough to introduce me to Dr. Campbell. If you know anything about phage biology or have ready any of the papers on lambda, you really are in awe when you encounter the author of the Campbell model of DNA integration into the bacterial chromosome. It’s what we do, it explains everything, and for many years it was the great frontier.
Vincent: He was the one that proposed that idea that lambda DNA circularizes and then integrates into the E. coli chromosome. That is something that everybody learns and he proposed it in 1962.
Michael: It was in a paper, the Advances of Genetics, and it was simply titled Episomes. And he was the sole author. It was much smaller science back then.
Michele: But so influential because we read it now and just assume it was handed down on a stone tablet.
Michael: Absolutely!
Michele: But there was actually a group of scientists that had to deduce this mechanism from the elegant genetics that they did back then.
Vincent: So we have a little piece on his work in our textbook, Principles in Virology, which was written by Anna Skalka who worked with him. She writes, although the model seems obvious today it was not so in the 60s. It was an alternative in which linear DNA integrated. But that was shown not to be true. And she writes, lambda DNA integration remains an important paradigm for understanding the molecular mechanisms of DNA recombination and the parameters that influence the joining of viral and host DNAs. He was a professor at Stanford since 1968. Wow.
Michele: But he got his start, he was a graduate student at the University of Illinois in Urbana-Champaign, and trained with Sol Spiegelman but also took a genetics course from Salvador Luria there. And I was fortunate to be a seminar speaker at Illinois just last week and they are a proud host of the Milestones in Microbiology designation. So they have been recognized for their longstanding contributions to the field of microbiology.
Michael: And in 2004, the American Society for Microbiology honored Dr. Campbell by awarding him the Abbott ASM Lifetime Achievement award and in honoring Dr. Campbell in his award citation, they cite his exceptional insights and achievements in the field of molecular genetics. I guess he’s one of the founding fathers of the field of molecular genetics along with Luria and Delbruck. And a career of groundbreaking research, it says, that has had a profound influence on several fields including molecular cloning and gene therapy. Dr. Campbell’s research has concentrated on the genetics of bacteria and their viruses, especially the integration of viral DNA into host chromosomes. And as Vincent already alluded earlier, his most prominent discovery was the proposal of the Campbell model of virus insertion, where viral DNA is inserted into the host chromosome becoming covalently bonded to the bacterial DNA. It remains dormant until activation, so it’s just an incredible body of work that he has left for us all, and there is still much to be learned about phage lambda. It’s not completely put to bed.
Michele: What I found really fascinating is as I was doing some reading about his life I came across a piece he wrote in 2012 in the journal Bacteriophage called Life in Science, and he tells a story that after graduating from Berkeley with a major in chemistry, he really wanted to be a novelist. He grew up in a house where his parents had studied English and journalism, but they pushed him towards science. But he wrote that ‘I had no ambition to do research’, he said his main motivation for applying to graduate school was mainly a desire to postpone getting a real job.
Vincent: (laughs)
Michael: Oh my.
Michele: He talked about his thesis project and said that he really admired his adviser, Sol Spiegelman, because he kinda tackled really difficult questions and he wrote that his thesis adviser’s efforts from that period ended up having virtually no impact and neither did my thesis research. (laughs) So I think it’s a lesson to all of us that don’t think you are behind if you have not already published some major paper. People move through science and their careers at different stages, and here he is obviously a great in our field and he did not have a direct path to that. He was here at Michigan for four years by the way.
Michael: Really? That I did not know.
Michele: I didn’t either, but it’s here in this little bio. He was here as a lecturer in the department of bacteriology.
Vincent: As of this recording, there is not much on the internet about his passing. It has appeared on Facebook. I confirmed it with Anna Skalka yesterday, but there’s no obit yet. So I assume that will be coming in the next few weeks. I also misspoke, Anna Skalka did not work with Allan Campbell, she worked with Al Hershey, who was the same era, more or less, in that group.
Michael: Oh and one other thing. If you are interested in phage lambda, the virus that he worked with, there is microbiology and molecular biology reviews that are in the public domain called Little Lambda: Who Made Thee by Max Gottesman and Robert Weisberg, and it really highlights how lambda—it’s really a nice treatise on lambda biology that highlights some of Dr. Campbell’s work in it, and actually the contributions that he has made deciphering it. And it gives a little historical perspective of how people did the science in order to work out the intricacies of how this prophage actually does its thing in E. coli. So I’ll put that in the show notes for you.
Michele: Yeah. Michael I also appreciated you pointing out this piece he wrote for Nature Reviews Genetics in 2003, where he talked not only about the basic science that we have learned thanks to the phage model, but also how phage are now being used as tools as an alternative to antimicrobial therapy for example, and in phage displays, what techniques we can use in the research lab and other things. It’s a really great essay published 2003 Nature Reviews Genetics. I’ll link to that as well.
Michael: And Stan Maloy posted on his Facebook account. Stanley of course is very much a gadfly of all things cool in science. He posted yesterday that the FTA is looking to clear phage therapy for the treatment of Crohn’s disease. And this is of course out of the group out of Georgia, specifically out of the group that was originally founded by D’Herelle at the Eliava Phage Institute. So it’s really, we are seeing this renaissance and it is a little bit different than our traditionals, but I think when one of the founding fathers leaves us, we should pay special tribute to the contributions that they made to our particular field.
Vincent: Mark Martin says Allan was on my PhD Committee, Mark Martin from Puget Sound. Was the smartest person I ever knew fairly well. He was so patient with my own 25 year old self. We were not friends but I miss him as a person as well as a one of a kind scientist. Cool. 88 years old.
Michael: Yep.
Vincent: I want to tell you about the upcoming meeting called ASM Microbe, the annual meeting of the American Society for Microbiology. This year it is in Atlanta, Georgia, and ASM has a special opportunity for our podcast listeners. Get 50$ off registration for Microbe 2018, which is June 7-11thin Atlanta, using the promo code ASMPOD. ASM Microbe 2018.
And next, scientists with their science showcases the best microbial scientists in the world. Delve into your scientific niche in eight different tracks. Don’t miss this opportunity, visit ASM.org/microbe. That is asm.org/microbe and use the promo code ASMPOD. All one word for fifty dollars off registration.
We have a paper for you today which comes from Nature Microbiology. The title is Topical application of aminoglycoside antibiotics enhances host resistance to viral infections in a microbiota-independent manner. And this comes from Yale University and the Wakayama medical university in Japan. The first author is S Gopinath, the last author, the PI, is A Iwasaki.
Aminoglycoside antibiotics, now I think what they were doing here in this paper, they have a model of herpes simplex virus type two infection of mice. And this is a genital herpes simplex virus, and they have a model where they inoculate mice intravaginally and they can watch the virus replicate and spread to dorsal root ganglia and replicate there and spread to other sites and develop disease. And I think they were trying to figure out the role of the microbiome in this infection. They were treating mice intravaginally with a cocktail of antibiotics including ampicillin, neomycin, and vancomyicin. And they found an inhibition of viral replication because they don’t put it that way, but they say to investigate the effect of local antibiotic treatment of genital herpes infection, we treated mice with this cocktail and to their surprise the antibiotic treated mice had decreased vaginal viral titers.
Michele: And to my surprise.
Vincent: I have to tell you a little bit about neomycin because it was discovered by Selman Waksman and last Friday I was the 65thSelman Waksman lecturer at a Theobald Smith conference at Seton Hall university.
Michael: Well, how about that!
Michele: That’s great, congratulations!
Vincent: For coincidence. So he discovered, his laboratory discovered it in 1949. And I know neomycin because I have a tube of it right here on my desk because when you get a cut you put it on it and it really prevents infection. It is mostly a topical aminoglycoside that binds the 30s ribosomal subunit of bacteria and inhibits protein synthesis. So you would not think that this would have any antiviral activity, but it does as you will see in this paper. It’s indirect antiviral.
So they have this treatment of mice with this cocktail, they find it reduces viral titers, herpes simplex type two vaginal viral titers. So they treat the mice, first they pre treat them for about a week and then they infect them. That’s the protocol. And then it also reduces disease and antibiotic treated mice have much lower titers and very little disease pathology. So then they say which of these three antibiotics does this? And they narrowed it down to neomycin by doing infections with single antibiotics. The first question in my virology class is a question designed to trick students.
Michele: No! Say it’s not so!
Michael: And now they’re gonna be able to litigate it, Vincent, with this podcast!
Vincent: I know, they’re gonna say but you said this, let me tell you what the question is, because I say: which of the following statements is true? One. All viruses make us sick and can be lethal, so that is obviously not true. Two. Our immune system can manage most viral infections, that’s not true. Three. Humans are usually infected with one virus at a time, that’s not true. Four. The press is usually correct, oh that’s not even in this one. That’s—anyway.
Michael and Michele: (laugh)
Vincent: I ask them a question about antibiotics are used to treat virus infections, and of course I want them to realize that an antibiotic is not used to treat a virus infection. Of course they could now find this podcast and say but you’re wrong. We’ll see, we’re not going to treat virus infections with this.
Michael: The authors even basically end their paper by telling us hey, we don’t want you to use aminoglycosides to treat viral infections.
Michele: Speaking of the authors, did you have a chance to say who published this paper?
Vincent: I did.
Michele: Oh, okay.
Vincent: You must have been running away somewhere.
Michele: I must have been distracted momentarily.
Vincent: Yeah I mentioned the first and last authors and the institution. What’s going on here? The rest of the paper is trying to figure out what is the mechanism. The first they say does it have anything to do with the microbiota? Does wiping it out with neomycin or lowering it have the effect? So they take germ free mice and they treat them with neomycin and then they infect them with herpes simplex virus type two and those mice also have very little virus replication and no disease pathology.
Michele: I’ll bet that was a surprising day.
Michael: And they also do the flip experiment too, where they infect and they do the flip experiment where they actually so the drug and the germ free animals that the virus can actually be treated by the neomycin.
Michele: Infect first.
Vincent: Yeah, in previous experiments they treat it for a week to deplete the microbiota, right? And so here the microbiota does not matter because the germ free mice do not have them and you can add the drug after infection. It is not as good after but it does work. So what’s going on here? What they did was they extracted RNA from the vaginas of these mice, either treated or not treated, and asked what genes are differentially transcribed? Maybe we will get an idea of what is going on. Neomycin is doing something. And they find a lot of genes in the type 1 interferon pathway are enriched. They go up when you treat the mice with neomycin. So the interferon type 1 pathway, interferons bind receptors on cells and then they turn on hundreds and hundreds of interferon stimulated genes or ISGs. So these ISGs are what go up, and they say even in germ free mice treated with neomycin the ISGs go up as well. So how about that.
Michele: And it was not a subtle signature, I was impressed that 30% of the genes that were upregulated fell into the class of these interferon induced genes.
Vincent: It is quite clear. So somehow this neomycin is turning on interferon pathways and these interferon stimulated genes, those are the ones with antiviral properties. So an example was in the old days we used to treat hepatitis C infections in humans with interferons because we had nothing else and of course that turns on interferon stimulated genes, which have the antiviral effect. Interferon itself is not antiviral but it turns on other genes. Just like now, we see neomycin is not antiviral but it turns on genes that are antiviral. But you don’t want to treat people with interferon because–
Michael: It’s toxic. It becomes toxic.
Michele: The dosing is tricky.
Vincent: You feel very badly and for Hep C only a fraction of the people it worked in and it made everybody feel very badly so people would stop taking it. So it’s not good.
Michael: We had to give it with antifreeze as well.
Vincent: Antifreeze?
Michael: Yeah, you used polyethelyne glycol in concert.
Vincent: Oh yeah, they would pegylate the interferon. It’s funny the way you said it.
Michael: It’s antifreeze!
Vincent: Yeah, they pegylate the interferon. They chemically conjugate it, it makes it last longer, that’s right. But I don’t think, the way you said it, I thought they were (laughs)
Michael: Oh no, but it’s how I remember they were pegylated. It’s how I remember.
Vincent: It prolongs the half life of the interferon in the system. Right. So here the neomycin is upregulating ISG expression. Now what’s interesting is they did a couple of other experiments. This ISG induction by neomycin is maintained up to three days after treatment and is only partially lost after a week, so it’s really sustained.
Michele: I was surprised by that.
Michael: And you don’t need much.
Vincent: You don’t but it is restricted to the site of application because they did not see ISG regulation in the lungs.
Michele: So this was a topical application. That’s good news.
Vincent: Tell me, either of you, I was reading a little bit on neomycin. It’s not well absorbed, right?
Michael: No, and it never was made to be well absorbed because of all of its associated bad side effects that it has in people, so it’s not all that well absorbed. But there are other aminoglycosides out there that are in clinical use that are well absorbed. But they also have all the bad side effects of aminoglycosides.
Vincent: Yeah. So then as Michael said there are other aminoglycosides. So they looked at a panel of different ones and they do the same experiment. They ask if they apply to the vaginal mucosa of these mice do they induce ISGs? And 5 of the 7 do induce or increase ISG expression. The two that do not are streptomycin, which is another Waksman product, right?
Michael: Absolutely. Won the Nobel Prize for it.
Vincent: There was a guy at this meeting in Seton Hall last week who was trying to get the New Jersey legislature to name the state microbe Streptomyces.
Michele: (laughs) How many states have state microbes?
Vincent: Two, Wisconsin has lactococcus is for cheese, and Oregon has saccharomyces for beer.
Michele: For beer! Wow.
Vincent: If Michigan had one, what would they have?
Michele: I’m gonna need to give that some thought. Let me get back to you on that.
Vincent: How about South Carolina?
Michael: South Carolina would probably have a mycobacterium because there are lots of mycobacteria wandering our pine trees.
Vincent: So the aminoglycosides, getting back to this, two of them do not induce and they have a chemical core that is different. So that may explain that. And then they say that these antibiotics bind ribosomal RNA and there are other antibiotics that target the ribosome like tetracycline and chloramphenicol but they do not induce ISGs when topically applied. So that is very interesting. So binding the ribosome is not all you need to induce ISGs. And now, is this ISG expression actually what causes the antiviral effect? I would say for sure, but they did an experiment to show that they treated mice with two ISG inducing and two ISG non inducing aminoglycosides. They pre treat the mice, they infect with herpes simple type two, and only the mice treated with ISG inducing aminoglycosides were protected from infection. So it looks like ISG induction is what is protecting them. They also do a cool series of experiments to ask would topical application of neomycin protect you at a different mucosal surface? So that’s cool. They use influenza infection of mice to do this. Intranasal infection and what they do is they pre treat mice with neomycin and in fact it’s substantially decreases viral replication and increases survival time in mice.
Michele: I forget. How did they deliver—oh, intranasal.
Vincent: Intranasally.
Michael: They probably just topically applied it to the nose with a Q tip, is my suspicion.
Vincent: Yeah, I’m looking here.
Michael: I’m scrounging the methods as well.
Vincent: How about other viruses? They check, so so far we have herpes simplex and influenza. They checked Zika virus which replicates in the vagina of mice, and it’s known to be controlled by interferon. And they find the two ISG inducing aminoglycosides do reduce viral replication as well.
Michele: And these are very different viruses, aren’t they?
Vincent: They are, they’re all pretty different. Herpes simplex is a pretty large double stranded DNA virus and influenza–
Michael: It persists, as well.
Vincent: And influenza is an RNA virus, a negative stranded RNA virus. And Zika virus is a positive stranded RNA virus. And they are all inhibited so that is the power of interferons. They have broadly antiviral activity because you have so many hundreds of ISGs that are induced. So far all this is done in mice. What about human cells? They take peripheral blood monocytes from donors which you can just get from someone in your lab if you have the right protocol, and I remember Howard Chune used to do that all the time, Michele you must do that too, right?
Michele: Well, when I was a technician in Sam Silverstein’s lab at Rockefeller and Columbia, that’s what we did.
Vincent: Did you have to pay them?
Michele: I don’t remember paying them.
Vincent: I know Howard used to pay them all the time, he said that was required. You can get blood and then you can purify monocytes and you can infect, well what they did here is they treated these monocytes with these aminoglycosides and it increases ISG expression in the monocytes and the control here is polyI:C which is a synthetic double stranded RNA, which is known to trigger ISG responses. And that also does that in these cells. They also found that this treatment in monocytes will reduce influenza virus replication. So it works in human cells as well as in mouse cells. Now, what’s going on? What is sensing the neomycin? We know that interferon is turned on by any one of a number of sensors like Toll like receptors and cytosolic helicases and the cool thing is if you are at Yale, you have access to mice that lack the genes for every one of these sensors, because that is the home of innate immunity.
Michael: Ah, yes.
Michele: They’re certainly leaders.
Vincent: What was his name, Richard Flavell started the field, they’re leaders. He had this idea years ago and he put together a department including Akiko Iwasaki who works on innate immunity. So they have mice lacking RIG-I which is a cytosolic nucleic acid sensor. They have mice lacking DNA sensors, CGAS and STING, so they could take these mice and treat them with neomycin and ask, do we have an antiviral effect? Do we have ISG induction? And in fact, the mice lacking the DNA sensors, they have no problem responding to neomycin. But if they use mice lacking a protein called MAVS, which is downstream of these cytosolic receptors, then you have reduced ISG production and reduced protection. So it looks like neomycin is going through typical RNA sensor pathways. So that’s pretty interesting.
So next they say, what is exactly the sensor involved? And they have mice lacking Toll-like receptor number 7 and Toll-like receptor number 3. These are endosomal RNA sensors, they are proteins on the membrane in the endosome, so when a virus comes into the endosome and the RNA is exposed, these sensors will sense it and the cell will start producing interferon. They found that the TLR7 is not involved in the neomycin effect but TLR3 is. So somehow neomycin is tickling TLR3 and turning on the interferon stimulating gene pathway. So that’s pretty interesting, that neomycin would do that. You could not predict that (laughs) that’s the way research is, right?
Michele: Yeah. Although we do know that the aminoglycosides bind to ribosomal RNA, so to think about nucleotides was not so far off.
Vincent: No, I suppose, yeah. As we found before though, binding ribosomes is not enough, interestingly, because chloramphenicol etc do not have this effect.
Michele: Right, right.
Michael: Well the chloramphenicol hits the 50S and remember what the hallmark of the aminoglycosides is that they interact with the ribosome shape at the same time as they are interacting with the mRNA codons. And they are effectively doing two things at once, if you will. And that is the magic of it and so it’s, as you are gonna get to the polyI:C stuff, and the TLR3 receptor again, it will come into greater clarity as you begin to unfold this wonderful story.
Vincent: So far, we know that TLR3 is involved, Toll-like receptor number 3 is involved in the neomycin effect. They also show that proteins downstream of TOL3 including TRIF and IRF3 and IRF7, these are transcription factors that are activated by sensing of nucleic acids. They go in the nucleus and they turn on the synthesis of these ISGs. And they have mice that lack every one of these. It’s just great to be in a place that we have all these mice.
Michele: Yeah, just pull the reagent right off the shelf and do the experiment. This is a lot of work they did, my goodness.
Vincent: And they show that, basically neomycin is working on this Toll-like receptor 3, TRIF IRF3,7 signaling pathway and that is what turns on the ISGs. So that is the molecular part. Now we get into what Michael would say the immunology. What cells are responsible for this? And in the vaginal mucosa you have of course epithelial cells, you have resident white blood cells, and you have circulating white blood cells. So they ask whether the induction of ISGs by neomycin requires tissue resident cells or circulating leukocytes. So they can actually block the recruitment of circulating white blood cells to the vaginal mucosa by pertussis toxin. Basically, pertussis blocks receptor signaling which prevents chemokine mediated recruitment to the vaginal mucosa of these cells. It’s amazing, right?
Michele: Yeah. Amazing approach.
Vincent: Treatment with both neomycin and pertussis toxin, and this ablates the protective effect of neomycin.
Michael: This also illustrates the insight of having all these knockouts affords these investigators, because if you didn’t have all the knockouts at your disposal you would not have appreciated the subtleties of pertussis toxin, what it’s actually going to do, unless you have the opportunity to use the reagent and then be able to ask a binary question.
Vincent: Yeah. Now what’s interesting here, you treat it with pertussis toxin and you ablate the effect of neomycin, in other words there is reduced ISG induction, and you get higher viral replication. But what that tells you is that recruitment of white blood cells to the vaginal mucosa is important, and that apparently is done just by topical treatment with neomycin. So I’m surprised by that because this is not penetrating the mucosa, but I guess just being on the mucosa is enough to recruit these cells.
Michele: Could it be taken up by endocytosis?
Vincent: Maybe. It could be, yeah. Maybe that’s it. But it’s clearly these recruited cells that are doing this. Now, what kind of cells? What specifically are they? There would be a number of different cells that would be recruited by neomycin. Monocytes, monocyte derived dendritic cells, or classical dendritic cells, all of which have cell surface markers you can use to distinguish them. And they find that the classical dendritic cells are increased on neomycin treatment. So these are being brought into the mucosa by neomycin. So what they do is they isolate these classical dendritic cells in the vaginal tissue of mice, either treated or untreated with neomycin. And they show that the ISGs are increased in those cell types. And again, these are dendritic cells being recruited to the mucosa by treatment with neomycin, and those, Michele, those are very good at taking up things at phagocytosis, right?
Michele: Right.
Vincent: So maybe they are taking up the neomycin and its tickling TLR3 in the endosome. They treat mice, so they have mice that you can treat with diphtheria toxin and ablate the classical dendritic cells specifically, which is another beautiful mouse reagent to have.
Michele: They’ve engineered that molecular trick.
Vincent: So then you give those mice diphtheria toxin, you deplete the dendritic cells, then you treat with neomycin. Guess what? No ISG expression. The absence of classical dendritic cells. So another piece of the puzzle, classical dendritic cells are essential for this neomycin mediated antiviral effect. In the classical dendritic cells, there are different subsets of course. What would immunology be without subsets?
Michael: and Michele: (laughs)
Michael: More intelligible.
Vincent: Within each cell type, and the are defined by cell surface markers. And they look for within the dendritic cell subsets ones with Toll-like receptor 3 expression and they find a single subset with ITLR3 expression. These are typically found in the thymus, in the spleen. And they find that this subset is the one that is recruited to the vaginal mucosa by neomycin. It’s the one that is blocked with treatment by pertussis toxin. And they can deplete these from mice specifically this specific subset of dendritic cells, they can deplete those when they treat with neomycin, you don’t get induction of ISG. So this is beautiful, they’ve teased their way through to a specific subset of dendritic cells that come into the vaginal mucosa upon treatment with neomycin, they make ISGs and they help clear the infection.
Michele: It really is an impressive, rigorous, logical body of work in one paper.
Vincent: Yeah. What else do we have here, now what is the mechanism? That’s the last part of this paper. They say well, the TLR3 is a sensor of double stranded RNA and they are thinking maybe these aminoglycosides make host RNA more visible to TLR3 in neighboring dendritic cells. So the idea here is in a host RNA is not supposed to be recognized by TLR3 itself. TLR3 only recognizes foreign RNAs. So what they do is they take splenocytes which have these dendritic cells. They treat them with their aminoglycoside and they wash them to get rid of the aminoglycosides and then they incubate them with dendritic cells of the same subset that don’t have the aminoglycoside in them, and from wild type and TLR3 knockout mice, they measure ISG expression. So you’ve got neomycin treated dendritic cells, you mix them with untreated dendritic cells from wild type or TLR3 knockout mice, and you measure ISGs. And interestingly, if you mix the neomycin, it’s not neomycin but I’m using that one because its easier to say than kasugamycin, kasugamycin treated DCs with untreated DCs wild type, you get ISG induction in the cells.
So they must be taking up something from the other cells, but if you use TLR3 knockouts you don’t get that induction. So they are saying, this suggests that the aminoglycosides are binding host RNA and making it better to be recognized by TLR3 and to sort of confirm that, they ask, will synthetic double stranded RNA augment the activity of these aminoglycosides? And they find that if you add enough double stranded RNA you get a synergistic induction of ISGs with the aminoglycoside and the double stranded RNA. So their idea is that somehow the aminoglycoside is binding to the double stranded RNA and allowing it to be better recognized by TLR3.
Michele: But there are some topological mysteries here.
Vincent: Of course!
Michele: Where is this double stranded RNA normally?
Michael: Well remember, RNA likes to pair with itself. So it may not necessarily be double stranded RNA. It may be just mRNA that happens to be pairing with itself, forming a partial duplex.
Vincent: There is also a whole set of RNAs in eukaryotic cells that are transcribed by these highly repeated regions called Alu regions in the genome. And they can form double stranded RNAs. They are typically modified by an enzyme in the so called ADAR, adenosine deaminase, and it changes A to I and so that makes the cell blind to that double stranded RNA.
Michele: Nice.
Vincent: Maybe the aminoglycosides are reversing that blindness. So that’s something they could actually explore. Okay. So that’s the story. These aminoglycosides hit a Toll-like receptor and turn on ISGs and they think it makes cellular RNA more visible to TLR3. Now, they do mention that other antibiotics have been shown to have antiviral activity and that includes azithromycin which inhibits Zika virus replication. We don’t know the mechanism but of course when Zika appeared everyone tested everything that was FDA approved for antiviral effects. Now Michael, isn’t azithromycin the one that is used to treat typhoid that’s left?
Michael: Yes, and it, remember, it concentrates in the phagocytic cell. Its concentration is much much higher, like fifty fold and higher, in the phagocytic cell. So when the phagocyte is effectively cleaning out the environment of salmonella in the case of typhoid fever, it of course will grab onto it and because the concentration is so much higher they dispatch the microbe right quick. So again this is another mechanism where we are beginning to rediscover antibiotics in this whole new world of eukaryotic biology where it is beginning to teach us something, these are not the antibiotics your father used. These are the same antibiotics but we are now beginning to understand they are doing something more than just killing bacteria.
Vincent: So azithromycin and Zika. Another one, nonchonmycin. Haven’t heard of that one, but it blocks entry by inhibiting endocytosis of flaviviruses. And others, macrolides, antibiotics have been shown to induce interferons and there are more. The point is that as Michael said, these antibiotics can have other activities, as well. And what I wondered, which they did not mention. Do either of you think that this ISG expression by neomycin, do you think it plays any role in bacterial infections by stimulating inflammation for example?
Michael: By bet is any time you have cytokines being aggravated, inflammatory response kicks up, it brings the full force of our adaptive immune system into play and it can facilitate the clearance. Because one of the great mysteries that you should scratch your heads about is how do bacteria static antibiotics resolve an infection? Because eventually the bacteria static antibiotic is diluted out just simply, the normal physiological process of clearance. But yet bacteria static antibiotics like tetracycline clear infections.
Michele: How quickly? So quickly that it can’t be the adaptive immune response?
Michael: Well, my suspicion is they are working in synergy with each other.
Michele: As we see in this paper.
Michael: Yeah, as we see in this paper because you have to dose yourself more than once a day with tetracycline in order to maintain the blood levels. And it has all to do with the area under the curve, but I think this is the brave new area of revisiting our antimicrobials, especially now that we have all this knowledge about immunology that we did not have when the antibiotics were discovered and licensed.
Michele: True, but we also have all this knowledge of the microbiome and how protective it is, and so it’s interesting that they do close their paper as you’ve put it out, Michael, that they do not advocate the use of these compounds as antivirals. And I don’t think we talk specifically about what their toxic side effects are, but they cause some damage in our ears by killing the little sensory cilia, I believe it is.
Michael: The hair cells, yeah.
Michele: There is some other liver toxicity, or is it kidney, I forget.
Michael: It’s kidney toxicity that’s pretty horrific.
Michele: But in thinking about that, they did say that it stays local, so if you administer it just topically and they found that it works a different tissue site, a different mucosal surfaces and on different viruses. So Vincent, what do you think about using Neosporin for cold sores? Do you think that activates this pathway locally?
Vincent: That’s a good question. A cold sore would be on your lip or your nose, so.
Michele: And it would just be one.
Michael: It’s a recurrent herpes infection.
Vincent: It could, but we have a really good drug for that, acyclovir, and that works great. I don’t know that nay company would try and get this licensed for cold sores. You would have to go through clinical trials to do that. I know that when you already have a drug that works really well it is hard to get a new one licensed. I don’t know if it would work, but based on this–
Michele: Neosporin is already safe, it’s on the market.
Vincent: Yeah. That’s an interesting question. I don’t know, I would predict it would work because its herpesvirus, it’s type one but that shouldn’t matter. It’s an interferon induction. Are you worried about, we put Neosporin on our skin all the time for cuts, we don’t worry about the skin microbiome being depleted because it’s a local.
Michael: There’s not very many of them, either.
Michele: It’ll be replenished easily.
Vincent: That’s the point, if you don’t have acyclovir, I think now it’s over the counter, you could try neomycin.
Michele: But acyclovir is oral.
Vincent: You can get topical acyclovir.
Michele: Oh, right.
Michael: And that’s what they probably recommend for cold sores to cause them to go in more quickly.
Vincent: There are a number of over the counter cold sore medications. I wonder if any of them just have neomycin in them because people have found that it works. Who knows. Well I don’t get cold sores, so I can’t try it. So anyone out there wants to try it, but don’t blame me if you have a problem.
Michael: and Michele: (laughs)
Michael: You’re not licensed to practice medicine.
Vincent: I am not, nor to do clinical trials. That is interesting. They say we do not advocate this use of this because it will, especially vaginal will affect the commensals. But skin might be okay. However, in a broader sense, inducing interferon stimulated genes, we know is toxic with Hep C. So I don’t think, that’s not the real message of this paper. I think that the really interesting thing is that these aminoglycosides can do something to activate TLR3 and figuring that out is really interesting, to find out what the mechanism is. We’re clearly not going to use neomycin for treating viral infections unless as Michele suggests a cold sore.
Michael: And you have it already in the house.
Vincent: You have it already in the house, yeah. There are two more things I wanted to mention, which maybe were not clear. First of all, they suggest that dendritic cells which come into the vaginal mucosa take up aminoglycosides by endocytosis. Michele mentioned that earlier but they actually mention that in the paper here, and that’s how it would tickle TLR3. But where we encounter RNA, we don’t know. And they also say the specific RNA is unknown as you said also, Michele.
Michele: Yeah. They do suggest that the epithelial cells in the mucosa might phagocytose or might take up the aminoglycoside and then if the DCs then ingest one of those epithelial cells that is loaded with aminoglycoside then that interaction can occur in the DC endosome.
Vincent: Yeah. Michele, they did that experiment where they mixed two different DCs, one had been pretreated and the other not, and they could see ISG expression. But I don’t know how they could see which cell was making the ISGs in that experiment, right.
Michele: Right.
Vincent: I’m not sure what that means except that its TLR3 dependent.
Michele: Right. And when they say splenocytes, is that a mixed population of epithelial cells and DCs in the spleen?
Vincent: Probably a mixed population of lymphocytes and monocytes, I would think, yeah. Most, not a lot, I don’t think it’s a lot of epithelial cells. Mostly lymphocytes and DCs.
Michele: The authors do point out that they have more work to do to try to understand where the presentation is happening.
Vincent: I think that is the interesting part. How does this mechanistically work? I don’t think there is an antiviral here, or even an antiviral approach, because as I said many times already, interferon is not really good because it turns on too many genes that are toxic. So the basic science here is what is interesting, I think.
Michael: Yeah, interferon is good for the cell that’s being attacked. It’s not good systemically.
Vincent: Yeah, that’s the problem. It goes systemic and you get all kinds of side effects, including fever and so forth. Anyway, that’s that paper.
Michele: It’s very cool and surprising.
Vincent: It is surprising and it’s always good to be surprised.
Michele: And very thorough.
Vincent: It’s very thorough, yeah. It’s great. Alright, let’s do two emails. We have two, one is from Dallas.
In your discussions about the hemosynthetic bacteria you mentioned metabolism of hydrogen carbon monoxide and methane. That was a couple of TWIMs ago. In terms of energy available, methane has both the highest concentration, up around 1.7 PPMV versus 0.5, I don’t know if that V is right there. PPM, right?
Versus 0.5 for hydrogen and 0.1 for carbon monoxide. Note that these are molar amounts in air and the weight concentration ratios are even high in favor of methane along with the energy ratios. CH4O2H is about 3.5 on energy. The H/C ratio on methane as the food supply is higher than normal lifeforms with far more C and O in their makeup, and that must come from carbon dioxide providing a carbon supply, it gives a link for that.
It gives a link to an article which is called Soil Microorganisms as Controllers of Atmospheric Trace Gases, which was published in 1996. This was the paper we covered in Antarctica, where they find that these trace gases are made by the soil microorganisms which are very sparse because it’s a very harsh environment.
Dallas continues, I didn’t bother reading the article so it may have stressed methane plus oxygen metabolism, but I do know that methane would be the dominant food supply. Methane is highly soluble in membrane lipids and is chemically concentrated, allowing even more efficient metabolism. That is why I consider all the environmental activists worried about increased methane emissions from fracking pure activist nonsense. To get the methane concentration high enough to be a significant greenhouse gas, the bacteria in soils and water that run on methane plus oxygen as an energy supply would dramatically increase the bacterial concentration and eliminate the methane. They left out methane metabolism, perhaps they were just being PC, as they know the concentrations and energies. Science shouldn’t be PC, it should be what is true, even if it says the methane emissions may be self limiting with increased emissions just being eaten up by the increased bacterial biomass. By the way, a factory producing single cell bacterial protein is under construction in Louisiana, I think, using methane and oxygen. It is going to be used in fish diets, especially targeting substituting fish meal used in farm salmon diets. Instead of using food to produce fuel with little CO2 reduction and lots of profits for cronies, the mandatory ethanol production system, this process uses fuel, methane, to produce protein for fish feed, providing human food. It is fully approved feeding ingredient in Europe where they have actually permitted offshore aquaculture to exist as a food production method. With our fracking, we have cheap methane to feed the bacteria energy and food are fungible.
That’s very interesting. Do you get any of that Michael?
Michele: Food is fungible.
Michael: I did, and one of my colleagues here at MUSC, Hal May, is actually working on a process rather than taking methane out of the ground, he’s actually taking electricity and feeding electrons to the bacteria that is instead making acetate, which he in turn converts into food for salmon. And it’s because given the amount of solar that we have available and given the amount of wind that’s available in very windy countries like Chile as it goes around the end of South America, it is extremely windy. It’s a good way of storing energy because if you convert the electrons to acetate you of course have a much better battery than anything that you could possibly make and since you are converting free electrons that you get from the wind which is effectively the ultimate solar energy because that’s what makes wind. He’s looking at rather than sinking a hole in the ground and pulling out methane from a couple of billion years ago.
Vincent: Neat! Cool stuff.
Michael: So he has a very good point. And PPMV is parts per million volume, which is a way they measure gases. It’s a volume percent.
Vincent: So he’s basically saying you cannot have too much methane, because bacteria will eat it up or metabolize it.
Michael: Well, energy is energy. It’s like leaving gold coins on the subway. They don’t stay there long.
Vincent: (laughs) Michele, were you going to say something?
Michele: I’m just trying to think about the gas being emitted from cattle and fracking and the bugs being in the soil and what concentration of methane is available locally to the bacteria. I’m just having a hard time thinking about this. I could see using the bacteria that can chew the methane to create biofilters coming out of manure piles and fracking operations.
Vincent: So you’re saying that atmospheric methane would not be available to soil bacteria?
Michele: That’s what I’m trying to, that’s what is puzzling me.
Vincent: Yeah, because there’s no way it would be circulating right? Whatever is close to the ground might be utilized but higher up in the atmosphere, how would that get down there? I don’t know.
Michael: He makes mention of the fact that the methane is effectively can go into the membranes of the microbe and recovered in that particular way. And so it depends on where the bacteria actually are that is going to harvest the methane.
Michele: Yeah, I guess the question is how elevated is methane in the soil near a fracking operation? Is it or is it in the air mostly and dissipating? I don’t know.
Vincent: We don’t know.
Michael: Well, when DOE did the study of where greenhouse gases were and they put the satellite over South Carolina because we have so many pine trees, we have a tremendous amount of volatile organic carbon compounds that are being emitted into the atmosphere from the pine trees. And so it does go up and you can actually detect it via satellite. So there are concentrations in something like a tree is emitting a greenhouse gas in the terpenes that, because pine trees make turpentine after all.
Vincent: Yeah. Alright well we have one from Greg who writes,
I just discovered this in the web page for TWIM 133, which was an episode where Michael talked about a novel antibiotic from a commensal that grows in the human nose. Long time ago. Fantastic show, thank you so much for contributing to the enlightenment of the world community. I was trying to retell the story of this novel MRSA defense strategy using lugdunin which is essentially good bug vs bad bug and forgot the details told by Michael. So I will have to listen again, which is not a bad thing, as I often miss things when my mind wanders on the ramifications of a particular mechanism you bring to light and I don’t hit pause. My comment is I think it would increase the audience and usability of the material you present if it were also available as a transcript. Obviously this is a cost and it is preferable to have more content than less, but with notes available, perhaps your sponsors could pick up the tab and embed advertising in the transcript as it would surely increase your audience and exposure. So grateful for all I have learned and the insights you offer.
And this is interesting, because I got an email also the other day from someone who said I am deaf and I can’t listen, but if you had a transcript, I could read it. So that’s very interesting.
Michael: I think that technology is coming.
Vincent: It is coming.
Michele: Right.
Michael: It’s just not ready for prime time because we have been investigating it here at the university because we put up a lot of content on the web and a part of the American Disabilities Act is that we make our content available to the hearing impaired and of course transcripts are the best way of accomplishing that. So we have been investigating automatic transcription services, if you will. The technology is out there but you still need someone to review it and it struggles with scientific and medical words. It’s good for conversations but it really struggles with accents and technical terms.
Vincent: Also, if you have two or three people speaking, it cannot distinguish between you me and Michele. So you have to go through and annotate it, and that takes time as well.
Michele: Yeah. Point well taken.
Vincent: Yeah, I think it is a great idea, and I have looked into manual transcription services where you pay someone. It’s very expensive, it’s more than we can afford. So if we could pump the audio through a program that did most of it, and it will probably be in a couple years it will be ready right Michael?
Michael: I mean, look at your cellphone now. It’s effectively transcribing your voicemails to text and sending you a text message. It’s getting there, it’s not perfect.
Michele: My voicemail coming to my landline here in my office, it’s a mess of messages, they make no sense whatsoever. (laughs)
Vincent: Can you imagine an episode of TWIM, right? Alright. TWIM is on any smart device that you have that you listen to podcasts, your phone, your tablet. You have an app that plays them, you can just subscribe. So please do that so you get every episode. If you like what we do consider supporting us financially. You can give as little as a dollar a month, it would really help us. You can go to twim.tv/contribute for how you can do that. And send us your questions and comments, twim@mmicrobe.tvMichele Swanson is at the university of Michigan, thank you Michele.
Michele: Thank you, it was a pleasure.
Vincent: Michael Schmidt is at the medical university of South Carolina. Thank you, Michael.
Michael: Thank you.
Vincent: I’m Vincent Racaniello, you can find me at virology.ws. I would like to thank ASM for their support of TWIM and Ray Ortega for his technical help, and Ronald Jenkees for his music. You can find that at ronaldjenkees.com. Thanks for listening everyone, see you next time on This Week in Microbiology.
Content on This Week in Microbiology (microbe.tv/twim) is licensed under a Creative Commons Attribution 3.0 License.
Transcribed by Sarah Morgan.