So good afternoon and welcome to this hybrid seminar for you here at the LNB and for our online audience as part of Cambridge Biomedical Campus Tours. Before we begin, just a couple of technical details. Just at the end of the talk there'll be a chance for questions.
For our online audience, if you could write your questions in the Q&A and then I will read them out for you. So to start, a few words about the LNB. The MRC Laboratory of Molecular Biology is the institute where we are. and it's situated on Addenbrooke's campus. It's a world-leading research institute dedicated to understanding important biological processes at the levels of atoms, molecules, cells and organisms.
We aim to study difficult long-term problems and in doing so contribute knowledge needed to solve key issues in human health. Today I'm delighted to welcome Tanmay Bharat to give our talk today. Tanmay began his scientific career doing a BSc in Chemistry at the University of Delhi in India and a BA in Biological Sciences from the University of Oxford. In 2012 he achieved his PhD from the European Molecular Biology Laboratory in Heidelberg in Germany under John Briggs and then he came here to the LMBE to do some postdoctoral training with Jan Loewe. He then took up a position as a group leader at the University of Oxford.
before returning back here to the LMB as a program leader in 2022. In this talk, Tamer will share with us his research and how it is providing new insights into how bacteria can evade antibiotic treatment and how these findings of his team can be used to help find new ways to treat pathogenic disease. So thank you very much. Well, thank you very much for the Very kind introduction, and also thank you very much for coming, for the audience. So yeah, the title you've seen, we are surrounded in our world by the so-called microorganisms, which consist of bacteria and archaea.
We humans are eukaryotes, and we have cells that, we are made of cells, and these cells look like this, with a complex internal organization, with compartmentalization within the cells. which is not seen in our microbe counterparts. This is a schematic representation of a bacterial cell.
You can see on the inside there's very little compartmentalization. These cells are typically simpler compared to the eukaryotic counterparts, and eukaryotes are humans, plants, and other animals. So these cells have a characteristic shape, which allows them to navigate their environment. If you compare a microbial cell, so these cells of these microorganisms, they are extremely small compared to our cells. Therefore, they can only be perceived with the help of a microscope, and they are about 1 to 10 microns in size, which is 1 micrometer.
So this is just to give you an example. So this is the size of a human cell. 100 microns in size, prokaryotic cells are extremely small compared to them. Bigger than viruses, bigger than molecules, obviously bigger than us.
This is just to give you a scale of the things that we are looking at. So these microorganisms were the first organisms that appeared on Earth. So 5 billion years ago, approximately 5 billion years ago, when the Earth came about, shortly after that. these microorganisms appeared on Earth.
These microorganisms inhabited our planet, and then due to evolution, this led to the evolution of more complex beings, such as humans, and we really evolved comparatively not very long ago in the history of the evolution of life on Earth. So this is just giving you a perspective on how many organisms... arrived before the arrival of humans. These microorganisms are found everywhere on Earth. So it's estimated that there are over 1 trillion microbial species found on Earth, and there are over 10 to the power of 30 microbial cells on Earth.
So if we took all the plants, the weight of all the plants and all the animals on Earth, and we added it together, it would be less than the weight of all microorganisms found on Earth. These microorganisms can inhabit very diverse environments. So here.
These are hot springs, boiling hot springs, found in the Yellowstone National Park in the USA. It has this characteristic color, and this color is provided by pigments present in the microorganisms, and these pigments protect these microorganisms from sunlight and allowing them to proliferate in these harsh environments. Here, on the other end of the temperature spectrum, these microbes have also been found in very cold conditions like in Antarctica. This is a team of researchers from Chile who are isolating these microorganisms because they can eat pollutants and therefore are beneficial for humanity.
Here, this is the Dead Sea. So it is a sea found in the Middle East where the salt content of the sea is six times that of normal oceans. In this...
hyper saline environments, we can also find microbes that are found. So this sea is not actually dead. It's nearly dead.
So the take-home message is that these microbes can be found in many different parts of the earth. Just to explain that even further, you go from extremely cold conditions to extremely hot conditions, we can find these microbes. And even in very harsh environments created by humanity, for example, these nuclear contaminated sites, you can also find microbes. In our laboratory, we are working with one such microorganism that has been isolated at the Chernobyl nuclear reactor.
And this shows that these microbes find a way to survive in the harshest environments. So this is just a schematic of the kinds of habitats that are found on Earth. And in all these habitats... Microorganisms found find a way to survive and basically evolve ways to inhabit these.
Now when I say they can inhabit all sorts of habitat I really mean they can inhabit all sorts of habitats. So these microbes are also present on our body. So this is an estimate so there are over 100 trillion microbes in a single adult human.
and these microbes are found in our mouth, in the so-called oral microbiome. And microbiome is just a word for a particular geographical location inhabited by microorganisms. These microbes in our mouth help us digest food and they're quite beneficial for us. On our skin, there's also a group of microorganisms which protect us and they are generally beneficial.
And the same way we have microorganisms in our gut and also in our urogenital tract. So I don't want to alarm anyone. These microorganisms are normally very beneficial to us, and they don't cause any disease, and they help our body to function normally.
So there is intense debate in the literature, but all of the estimates suggest that there are at least as many microbial cells in our body as there are human cells. So there are a lot of microorganisms on our body. As I said, normally these microorganisms are beneficial, but sometimes some bad apples cause imbalance in our microbiome and can cause disease. And there are numerous bacteria that can cause different types of diseases in humans.
I'm just going to go through some of them. We can get meningitis, which is infection of the meninges. And bacteria such as Neisseria meningitis or Haemophilus influenzae, you might have seen in the news, they can cause this disease.
Whenever we get an ear infection, it's usually caused by bacteria. We can get nose infections or skin infections, which are caused by Staph aureus. And also sometimes we get lung infections, which are caused by Streptococcus pneumoniae.
So this is pneumonia. And also we have... other nasty diseases such as urinary tract infections caused by E. coli bacteria and other sexually transmitted diseases like Neisseria gonorrhoeae. These are all examples of diseases that are caused by bacteria that have either been acquired from the outside or is just one of these bacteria within a microbiome becoming an opportunistic pathogen.
just exploiting the opportunity to come and infect us and proliferate. Just to warn you, the next slide contains photos of infected patients, so if you are squeamish, please look away. So just like in nature, these microorganisms can infect...
really obscure parts of our body. So this is a wound victim which has been infected by Pseudomonas aeruginosa and this can cause a lot of problems in a hospital setting because these bacteria are difficult to eradicate. So this is an example of a bone infection.
This is actually my leg. So I had a bone infection in my leg and I've had all my life and it's no coincidence that I'm working on this problem. So in this case my bone had been infected by staph aureus, which didn't respond to treatment.
So the infected tissue had to be surgically removed and replaced by these antibiotic beads. And you can see this very painful operation, all these staples. So these microorganisms are a big problem medically. And the symptoms that the patient gets depends on the site of infection. For example, if the microorganism infects our blood, we usually get fevers, chills, muscle pain.
If we have a lung infection, it is usually accompanied by coughing or shortness of breath. If we get bone infections, we get reduced movements in the limbs and discharges in the limbs. So just to summarize, microbial infections give us fevers, chills, and fatigue. But depending on the site of infection, we can get swelling, pain.
organ dysfunction, and other indicators. So what can we do when we get infected by these microorganisms? So the common treatment, the good news is we have these drugs, which are called antibiotics, which were developed in the 20th century by a fortuitous discovery by Alexander Fleming in London, who discovered that a mold from a fungus could kill bacteria.
This led to the discovery of penicillin, which is a drug that allows us to treat microbial cells, leaving the human cells unharmed. Of course, this initial discovery was then taken up by other scientists like Ernst Chain and Howard Flory, which led to the starting of the Antibiotics. I was very fortunate to start my independent career at the Dunn School of Pathology, where all these developments took place.
This is a picture of Alexander Fleming. Flory and Jane receiving the Nobel Prize for their work and in the Dunn School in Oxford these were the kind of apparatus that were used for making large amounts of penicillin to treat bacterial infections. This is a picture of the first patient who was ever treated with an antibiotic it's a policeman called Albert Alexander and he had been infected by bacteria, he was successfully treated by penicillin, showing that this was something very useful for humanity.
I should also say that antibiotics have, or penicillin, has really changed the course of humanity, evidenced by the number of penicillin doses that, you know, when the Allies invaded Normandy, they took two million doses of penicillin, and this miracle drug changed the course of the Second World War, allowing the Allies to... basically not get these infections that the other side could get. So it's very important to have these kinds of drugs. Even at the LMB, there have been many studies that have been instrumental in the development of antibiotics. So here is the structure of the ribosome, solved by Venky Ramakrishnan.
The ribosome is one of the major targets for the modern-day antibiotics, and many antibiotics target bacterial ribosomes, leaving the human cells... unharmed. Knowing the atomic structure of this drug target allows researchers to solve the jigsaw of which small molecule can bind to this target, allowing drug development using these structural biology results. So what's the catch? The catch is that even though we have these antibiotics, these bacteria are extremely smart.
and they evolve ways to resist the antibiotic treatment. So this is a list published by the World Health Organization in 2017, which shows pathogenic bacteria which have evolved to a high degree of antibiotic resistance. So there are some really nasty bugs in here which resist antibiotics, like Pseudomonas aeruginosa, which my lab works on, Staph aureus. Maria Shigella. And yeah, so this is a big problem facing humanity that the antibiotics that were developed over the last few decades are now becoming...
Not very useful because the bacteria can evade them using various mechanisms. So one of the main mechanisms by which bacteria evade antibiotics is by forming multicellular aggregates called biofilm. Now biofilm formation is a process in which a bacterium can adhere to a surface, and this surface could be lung tissue of a human or any other tissue.
These adhered bacteria can then... grow, encase themselves in an extracellular matrix. So this is a protective layer where they encase themselves into, and this is known as a biofilm.
And this biofilm basically protects them against antibiotics. It's estimated that over 75% of human infections proceed with this biofilm formation, making it a very important problem that must be addressed if we want to successfully treat bacterial infections. One famous example of a biofilm is pseudomonas aeruginosa infections that patients with cystic fibrosis can get, particularly in hospital settings.
But just to also say that biofilms can form not only inside the human body, but they can also form in hospital devices like catheters, tubings. They can also form on hip implants, knee implants, so it's a major problem. So this is a gram stain smear of sputum from a cystic fibrosis patient.
And in this microscopy picture, you can see that we can see these bacterial cells, which have made this kind of a biofilm. And this biofilm is extremely difficult to treat with antibiotics, because it's a protective environment in which bacteria place themselves in. Now, what happens during biofilm formation? So, So after initial attachment, as I said, there's an extracellular matrix that is secreted by the bacterial cells.
This extracellular matrix, when it's established, leads to chronic infection. Now it's very difficult to clear these bacteria. On the molecular level, bacteria that are free swimming have various appendages that allow them to swim, to go into different places. But when they form a biofilm, they lose all these molecules.
And they secrete this extracellular matrix, so there's a dramatic change that occurs in the metabolism of these bacteria in going from a non-biofilm state. And it is this extracellular matrix which defines these biofilms, and this is what my laboratory is studying, because it's the hallmark of all biofilms, all different bacteria. So we try to understand the organizational principles within this biofilm. just give you two quick examples on the kind of. So just to say that we work on Pseudomonas aeruginosa, which is a major human pathogen.
It's one of the top five antibiotic resistant killers shown by this paper over the last decades, and it evades antibiotics by forming biofilms. And the title of my talk, so this would be the death star. So this is the thing that we have to kill.
And we use electron microscopy to study this problem. This is an electron microscopy picture of a pseudomonas biofilm. At this point, I would like to highlight that the LMB has played a pioneering role in the development of cryo-electron microscopy. And here is a picture of two former directors of the Institute, Aaron Klug and Richard Henderson, who played key roles in this. So now to some results from our laboratory.
So we are studying a master adhesive protein in Pseudomonas aeruginosa that allows bacteria to go into biofilm form. These are two tubes with approximately the same number of bacterial cells in them. In this tube, this protein is not expressed, meaning you get like a cloudy culture of bacteria. But when this protein is expressed, they go into this biofilm clumped form.
which you can see is evident by the different appearance of these tubes. Now, if you go look at these bacterial cells in the microscope and zoom into the cell surface, we see these appendages present on the surface. These are the appendages that allow bacterial cells to adhere to one another. These can also be seen in these images that we produce using the latest electron microscopy techniques that are present in the extracellular. Matrix how can we use this information for treatment?
So what we did is we purified this protein and we injected them into alpacas Alpacas have a very particular type of immune system, allowing them to produce these extremely small antibodies. One such antibody we showed using these mass spectrometry measurements to bind to this target in a 1 is to 1 ratio. And using this antibody or nanobody, you could efficiently kill bacteria inside these biofilms.
So now this is a killing assay. The live bacteria are blue-colored, and the dead bacteria are red-colored. So in presence of a sublethal dose of antibiotics, we saw that when we apply a nanobody, we can efficiently kill these bacteria.
And when we don't have the nanobody, the bacteria, basically, they don't care about the antibiotics. So using this approach of our imaging, we could develop a technique to kill the bacteria within the biofilms, allowing us to kill this death star, break through the defenses of the bacteria, a potential way to treat the infection. So how do these biofilms help bacteria tolerate large doses of antibiotics? This is another question in our laboratory.
To answer this question, we looked which are the proteins that are overexpressed within Pseudomonas aeruginosa biofilms. One such protein is a filamentous molecule called PF4. This molecule combines with other polymers that are secreted by the bacteria or sometimes present on the human airways.
assembles into these liquid crystalline droplets. These liquid crystalline droplets then encase bacterial cells, so basically they form a shield around the bacteria, protecting them from antibiotics. So this would be a death star with a force field.
So these bacteria have additionally put molecules that prevent the antibiotic from reaching them, thus protecting them from high doses of treatment. This is just an electron microscopy image showing how these filamentous molecules form this liquid crystal, and this liquid crystal surrounds the bacterial cell, forming this barrier, preventing the antibiotics to get through. This is another bacterial survival assay.
Dead bacteria are shown in blue, as you can see. So this is on antibiotic challenge. Bacteria that are not surrounded by this liquid crystalline sheath. killed by the antibiotic, whereas bacteria that are protected by the sheath, they aren't killed by the antibiotic. So it's demonstrating our hypothesis.
So what can we do? So they've made a shield around themselves. So how can we get past and kill these, kill the Death Star? So scientists are working very hard on this problem and one idea that is being floated around is to use viruses of bacteria.
So just like we have viruses that can infect and kill us, there are viruses that can kill bacteria and these viruses are called phages. They can bind to bacterial cells, go inside bacterial cells, make new copies of themselves and then come out of the bacteria killing the bacterial cell in the process. The good thing about these phages is that they can evolve.
So they... take part in this evolutionary arms race, sorry, with the bacteria. And yeah, so these phages can be used for therapy.
I apologize for the error, but the next slide contains it. So these phages have been used in the case of, this is a heart surgery, and during the surgery, there was an infection to the patient which didn't respond to antibiotics, and they used the phage. in addition to the antibiotics to successfully carry out this surgery.
They've also used phage therapy for this transplantation. So this was a toddler that had an infected liver, and only with the use of the bacteriophage together with the antibiotic, they could carry out the transplant successfully. In addition to the phages, new antibiotics are also being developed.
So this is the same idea as penicillin, using molecules that are found within nature. that can kill bacteria. These scientists, they took bacteria which could kill other bacteria, and from these experiments, isolated this compound called darobactin, which kills a lot of nasty pathogens like Pseudomonas aeruginosa, E. coli. Another idea is to go and sample the environment.
So this is a new antibiotic that has been developed against methicillin. So the scientists just went out into the environment, took bits of soil. and use this soil to see if there are compounds that could kill these pathogenic bacteria. And they found one compound, tyxobactin, and this is a new compound that helps in the fight against these pathogens.
Just to end here, while the work we are doing is quite fundamental in nature, the... The results that we get can be used for application to develop the next generation of drugs. Us fundamental researchers at the LMB must interact with clinicians, and we must also interact with the policymakers, the government, and when this whole pipeline is complete, only then we can have actual impact at the bedside and actually get rid of this death star. I just want to end. Oops, sorry.
With this statement from Sydney Brunner, who was at the LMB, new technologies, new discoveries, and new ideas, probably in that order. And with that, I would like to thank the people in my laboratory, some generous collaborators and funders, and also thank all of you for joining and listening. I'd just like to thank Tamo very much for a wonderful talk and open up for questions. Can all bacteria form biofilms? Yes, I think, yes, I would say so.
Obviously, all bacteria have not been tested. I think all bacteria will be able to form biofilms under some conditions. So, Shoyan asks, can we say that the biofilm's films serve purely as a physical barrier, or is there any biochemical or physiological mode of their action?
So, it's a physical barrier, but also there are chemical aspects to it. So, I should also add that bacteria have evolved other ways to evade antibiotics. which were not covered in this lecture.
Yeah, both. And another one, again from Sharon. Do you think that bioinformatics can help you with your research?
Yes, I think it's a good answer, the only answer I can give. So bioinformatics, for the audience, is an computational method to aid in our lab work. Another question from online. Asim asks, what is the composition of the biofilm with the envelope?
And is there a possible way to remove the components of the biofilm? Yes. So this is one of the open questions in the field.
So the biofilm is made of proteins and polysaccharides, sugars, proteins, and DNA. But the exact... arrangement and the exact composition is unknown, which is, I think, one of the major questions we are working on in our laboratory. So, predicting literature and... There's a question there, sorry.
That slide when you're talking about the human body and useful bacteria. Is that something which is gained after birth, or are you born with that bacteria? Gained after birth.
It's gained after birth. Yeah. If there are no more questions, I'd really like to thank Tamay again for an excellent seminar, and thank you all for coming.