Authors: Faherty C & Llanos-Chea A
In line with our July focus around enteric infections, we recently spoke to Christina Faherty from Massachusetts General Hospital (MA, USA) and Research Fellow from her lab, Alejandro Llanos-Chea, about the lab’s research on Shigella, Salmonella and E. coli and novel models for these in vitro.
They discussed organoid models for investigating bacterial pathogenesis, the impact of bile salts in infection and what the future might hold in terms of moving away from the current traditional laboratory methods – find out more in the interview below.
First, could you introduce yourselves and give a brief overview of the work you’re doing?
Christina: I’m Christina Faherty, an Assistant Professor at Massachusetts General Hospital with an academic affiliation at Harvard Medical School (MA, USA). Our research focuses on bacterial pathogenesis, host–pathogen interactions and virulence mechanisms, particularly for enteric pathogens like Shigella.
Alejandro: My name is Alejandro Llanos-Chea, I’m originally from Peru where I attended medical school before I trained in pediatrics here in the USA. I’m a research fellow in Dr. Faherty’s lab and I just completed my clinical fellowship in pediatric gastroenterology at Massachusetts General Hospital also with an academic affiliation at Harvard Medical School.
Much of your work focuses on Shigella flexneri – why do you think this is an area of unmet need?
Christina: Shigella causes staggering rates of global infections. There are four different species that cause millions of infections per year, mostly in children in the developing world under the age of 5 years. Hundreds of thousands of deaths can result.
In industrialized countries, approximately 500, 000 infections occur each year from foodborne, waterborne and day-care center outbreaks. Here in the USA, a recent example was the lead water crisis in Flint, Michigan (MI, USA) that actually resulted in a Shigella outbreak because many residents were scared to use the water and wash their hands. People ended up using baby wipes that were handed out by relief workers, which do not have the appropriate antibacterial components and thus facilitated the outbreak.
In addition to the number of cases there are two other significant problems – the first of which is antibiotic resistance is on the rise for many significant pathogens, and Shigella is no exception. The WHO recently included Shigella on a pathogen priority list in which they are encouraging new treatment options and ideas to combat antibiotic-resistant infections.
And most importantly, despite decades of research and attempts, there are no effective vaccines against Shigella. Shigella is a human-specific pathogen so reliable infection models and being able to incorporate the dynamic nature of the human gastrointestinal (GI) tract have been very difficult to develop and have hindered successful vaccine development.
Alejandro: As I said I’m from Peru and during my practice I took care of many pediatric patients with enteric infections including Shigella. Recurring infections from enteric pathogens can lead to chronic malnutrition in these patients and in the long run a consequence can be developmental delay.
Chronic malnourishment even affects developing countries at the macro level due to the socioeconomic burden of disease. More attention has recently been gained because of an understanding of the impact of these consequences, in addition to special attention specifically from organizations like the WHO or the Gates Foundation.
Could you outline the work you’ve done on bile salts and bacterial pathogens – what role do bile salts have in infection?
Christina: During my postdoc I started using bile salts. In the 1990s and 2000s, two different researchers demonstrated that bile salts exposure increased Shigella adherence and invasion of epithelial cells. Since we were interested in identifying how the bacteria make initial contact to the colonic epithelium, we applied the earlier studies to my research and utilize bile salts exposure. I was able to identify two small proteins – the OspE1 and OspE2 adhesins – that facilitate initial epithelial contact. Not only did bile salts increase the expression of the ospE1 and ospE2 genes, it also enabled the proteins stick to the outer membrane of the bacteria following secretion.
I was able to obtain a career development grant and move forward with this work. Bile is so important because the bacteria need to transit the entire length of the small intestine in order to cause infection in the colon. In our current research, our approach is to mimic exactly what the bacteria are encountering right before infection. We have been able to show that Shigella can resist the bactericidal nature of bile salts through the induction of efflux pumps, which are the same efflux pumps that help to facilitate antibiotic resistance. Moreover, the bacteria can form a biofilm in the presence of bile salts and this is important in their transit through the small intestine to the colon.
Finally, we’ve actually been able to mimic a bacterial dispersion event when the bacteria move from the ileum to the colon – since bile salts are mostly recycled back into the body at this point and do not enter the colon. We were able to demonstrate that following removal of the bile salts signal, the bacteria will disperse the biofilm and infect in a hyper-virulent state.
Thus, we saw that bile salt exposure really primed Shigella for infection – this is something you cannot see when you grow the bacteria in laboratory media. The model helped us to expand the infection paradigm for Shigella and to show how the bacteria exploit host factors to their benefit. Soon we are hoping to publish exciting updates to our adherence work and to keep moving forward with this important culture method to truly understand infection.
Why is understanding of molecular mechanisms of infection important?
Christina: I really think it is important for therapeutic and vaccine development for pathogens. We need to understand how bacteria are regulating virulence gene expression, how they are turning things on and off and how they are changing as they transition through the body.
Ideally, we want to get to the point where, when we put the bacteria in infection models, they feel like they are in the human host – then we can understand the gene expression profile, maybe identify new pathogen targets and help to advance new therapeutics. Again, this is something we cannot do with traditional laboratory media.
Focusing on methodology – what role do novel methods play in your research?
Christina: We have a two-pronged approach. First, we are using bile salts to mimic the pre-infection small intestinal transit; and second, we are using a very human-specific intestinal organoid infection model developed by our collaborators here at Massachusetts General Hospital, Dr. Stefania Senger and Dr. Alessio Fasano.
One of the reasons this model is exciting is because organoid spheroids cultivated from human intestinal stem cells can be opened and seeded onto transwells. It is difficult to work with spheroids from a bacterial pathogenesis perspective as you typically have to microinject bacteria and infection data can be limited. However, when the organoids are trypsinized to create the type of 2D system you would traditionally use for polarized T84 cells, this process allows you to examine factors such as the apical surface, the basolateral surface, cytokine secretion profiles and tight junction proteins.
Moreover, these models can be differentiated to not only have enterocytes but also mucus-producing goblet cells and specialized antigen-sampling M cells. So, we’ve been able to really get beyond the animal model limitations for Shigella. The appropriate cellular architecture is present for infection analysis. With tremendous effort from my postdoctoral fellow Kourtney Nickerson, we can demonstrate great infection patterns not only for Shigella but also for Salmonella and the pathovars of E. coli. We are really excited about this combined approach. We think it is the most human-specific analysis that we can get in the laboratory setting for Shigella and it has definitely given us some very interesting results.
Alejandro: Research often focuses on immortalized cell lines and animal models, but these models do not really duplicate what we see in the human GI tract. When I was a fellow, we used to look at pathological samples under the microscope. So, it is really fascinating to see that we can replicate that in vitro and see all the different cell populations reproduced from just stem cell crypts.
In addition, we can reproduce model lines from different parts of the GI tract. We have talked about Shigella, which is specific for the colon, but we have been able to obtain samples from the terminal ileum, the duodenum, and pediatric samples. Not only do we have the chance to assess how pathogens infect healthy tissue, but we can also obtain samples from patients with inflammatory bowel disease, or celiac disease, and we can even tweak the conditions to generate different situations where we can further assess how these pathogens interact with various human hosts.
Christina: One of the challenges with Shigella is that it invades at the basolateral side of the epithelial cells, whereas other pathogens like Salmonella invade at the apical surface. So with previous cell lines for Shigella, researchers had to flip the cells upside down and apply the bacteria directly to the basolateral side to see invasion. With this system, we can leave the cells in the proper orientation and we can detect Shigella transiting the M cells to gain access to the basolateral side as hypothesized in the traditional infection paradigm, which gives us much better infection rates. So, we are actually forcing the bacteria to mimic that natural infection process, which I think is going to be really important for us to understand the mechanisms of infection, especially early epithelial contact and how can we go in and intervene to try and prevent infection.
What further advances do you envisage with respect to new methodologies in the coming years?
Christina: We are hoping to include more colon specific conditions and expand the model to incorporate novel gene expression analyses, different aspects of host physiology, as well as different aspects of the innate immune response. Ideally this system will be able to essentially bypass animal models and allow us to advance therapeutic candidates into clinical trials without needing to do confirmation experiments with animals – especially since there are not any adequate animal models for Shigella infection.
Alejandro: I would say that in the coming years it would be good to make this process more efficient because the organoids do take care and dedication! It would be ideal to be able to reproduce them at larger scales, for larger surfaces and ensuring they are viable for longer periods. I think it is interesting to have multiple cell populations and there may even be potential to vascularize the organoids to have even more of an in vitro/in vivo hybrid type of model.
Christina: Just to add one more thing, there are a lot of good 3D models available, but they seem to be limited in certain aspects of infection analysis, especially cytokine secretions or tight junction integrity analysis. If there was a way to really combine the 2D and the 3D models to ensure we have the full structure but also the power to perform these analyses, the models could go really far, not just for pathogenesis researchers but for others too.
How do you hope this research might affect practice on the frontline of care?
Christina: I would definitely say we hope to influence and help design effective vaccines. There are other additional therapies that we would be interested in developing as well – novel inhibitors of infection that can be used potentially in conjunction with antibiotics. But more importantly, we hope our research shows that new antibiotic development needs to consider bile exposure for enteric pathogens since it is crucial to ensure that the pathogen’s bile-resistance strategies do not affect the drug efficacy. I think this consideration could really help for future clinical success.
Alejandro: Our research allows us to have a better and precise understanding of pathogenesis that can definitely be applied to vaccines and antibiotics. There is also a new interest on revamping other therapies, for example bacteriophage therapy.
We have recently submitted a proof-of-concept manuscript that analyzes phage therapy in the organoid model to see how the phage inhibits Shigella in a human-specific model.
Finally, where do you hope to see the field in the next 5–10 years?
Christina: For me, from a bacterial pathogenesis perspective, I really focus on how the pathogen survives to cause successful infection and try to incorporate the best growth conditions mimic what the bacteria encounter. What we are seeing with different types of human pathogens is that gene regulation is key. I think researchers need to make sure that the growth conditions are as specific as possible. Then, if we can get into these new and appropriate infection models, especially for human pathogens, I think we are really going to move beyond the limitations of traditional laboratory media and traditional animal models.
Alejandro: As you know Shigella infections continue to grow around the globe and pathogens continue to evolve. There is an interest in understanding these pathogens especially given concerns with antibiotic resistance. There is not only a role for microbiologists or immunologists, but also new approaches and further involvement of bioengineers. Bioengineering will help us ensure the success of these models – not only in developing the models but also to develop therapies. I think bioengineering will definitely influence the field over the next few years, and we even already see it happening.
Christina S. Faherty is an Assistant Professor at the Mucosal Immunology and Biology Research Center at Massachusetts General Hospital with an academic appointment at Harvard Medical School. Her research focuses on host-pathogen interactions in enteric bacteria. Specifically, she is interested in studying the effects of host signals on Shigella gene expression and virulence. With her laboratory and collaborators, Dr. Faherty has also optimized a novel human intestinal organoid-derived epithelial monolayer model for infection analysis with various pathogenic bacteria. Dr. Faherty received her Ph.D. in Infectious Diseases at the Uniformed Services University of the Health Sciences in Bethesda, Maryland in 2009. She did her postdoctoral work at the Center for Vaccine Development, University of Maryland School of Medicine in Baltimore, Maryland. Dr. Faherty became an Assistant Professor at MGH/HMS in 2013.
Alejandro Llanos-Chea, MD, FAAP is a Research Fellow at the Mucosal Immunology and Biology Research Center at Massachusetts General Hospital with an academic appointment at Harvard Medical School. His research interest focuses on host-pathogen interactions of enteric bacteria. He also has a special interest in environmental enteropathy, nutrition, and the subsequent effects on child health and gastrointestinal homeostasis. Dr. Llanos-Chea received his medical degree at the Universidad Peruana Cayetano Heredia in Lima, Peru in 2009. He completed his Pediatrics residency at John H. Stroger Jr. Hospital of Cook County in Chicago, Illinois in 2015; and his Pediatric Gastroenterology fellowship at Harvard Medical School program at the Massachusetts General Hospital in 2018.
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