Finding therapies
for Shigella infections
Christina Faherty
discusses her work with the pathogen Shigella and her efforts to develop
an effective solution for treating infections.
Please can you
introduce yourself and provide a brief overview of your research focuses?
I am an assistant
professor of pediatrics at the Mucosal Immunology and Biology Research Center
(MIBRC) at Massachusetts General Hospital and Harvard Medical School (both MA,
USA). My research focuses on bacterial pathogenesis, specifically on pathogens
that cause infectious diarrhea. I have worked with several different pathogens
such as pathogenic Escherichia coli and Salmonella, but most of
my research focuses on Shigella. Our goal is to identify novel targets to
develop improved therapeutics and new vaccines, to successfully treat
infections.
Why is Shigella infection
such a problem?
Shigella is a global infection problem and
every year throughout the globe it infects both children and adults in
developing and industrialized countries. Large epidemiological studies have
shown Shigella is the second leading cause of global diarrheal deaths
and the third leading cause of diarrheal death in children under 5 years of
age. There is also the problem of antibiotic resistance, to the extent that the
World Health Organization has listed Shigella as one of the priority
pathogens requiring new treatment therapies in
How are you
utilizing organoid models in your research?
Organoids are a great
infection model for us and provide a nice human-specific platform to
investigate host-pathogen interactions of the gastrointestinal tract,
especially for pathogens like Shigella that are human specific and
reliable animal models are lacking. Organoid models allow us to take tissue
culture technology and translate it into a more realistic representation of the
human cells that are encountered upon infection. We have collaborated with
researchers here at the MIBRC, Dr Stefania Senger and Dr Alessio Fasano, who
established this model for the center. We have been able to use the model and
apply it to our work with Shigella pathogenesis as well as to
investigate the other pathogens. We have even done some preclinical analyses to
evaluate potential therapeutic strategies that we are interested in trying to
further develop.
Based on your
research so far, what have you discovered about the effects of Shigella?
For Shigella
specifically, we have been able to validate much of the infection paradigm,
which is important since most of the previous work has been done using animal
models that typically do not produce the usual bloody diarrhea seen in humans.
The organoids are a little bit challenging to work with at times, especially
from the perspective of pathogenesis research. When organoids are grown in
culture, they form spheres and the apical surface of the epithelial cells that
the pathogens first encounter is actually on the inside of the sphere.
Therefore, a challenging approach to infect the spheroids is to use
microinjection systems. Our collaborators were able to help demonstrate that
the organoids can be trypsinized to break open the spheres to seed the cells
onto a Transwell system. This process helps to create a polarized, single
epithelial cell monolayer that has a top (apical) and a bottom (basolateral),
which represents the single layer of cells that lines your entire
gastrointestinal tract. Through differentiation reagents, we can make sure we
are producing mature cells such as enterocytes, mucus producing goblet cells
and antigen-sampling M cells that monitor the microenvironment and help
initiate the immune response if needed. Interestingly, Shigella exploits
M cells so that it can gain access to the basolateral side of the epithelium to
invade and enter the cells. With this model, we have been able to detect M cell
transit by Shigella, which is interesting and is something we are really
excited about. The data helped show that this infection model would be ideal to
examine early host-pathogen interactions and to understand infection mechanisms
to create successful therapeutics. Shigella invades epithelial cells,
tries to stay hidden as long as it can by attempting to regulate the immune
response, ultimately aiming to survive long enough to be transmitted to a new
host. So, understanding early infection is important.
Does Shigella
display specificity in where it imbeds in the gastrointestinal tract?
Shigella is specific to the colon and that
leads me to another part of my research: how does the bacteria survive host
transit and make it all the way to the colon following oral ingestion to
successfully establish infection? The organoids have been established from biopsy
samples throughout the intestinal tract. We have models from the duodenum, the
end of the small intestine called the terminal ileum, and the colon. We have
shown through our analyses that Shigella is specific for the colon. It
appears the bacteria have a tissue tropism and we are interested in figuring
out why. On a cellular level, the colon is pretty similar to the small
intestine, yet Shigella specifically targets the colon. We can see those
differences in the model, so we think we are going to be able to answer
questions as to why the colon is targeted.
You’ve mentioned
that the human specificity has been a challenge of studying Shigella,
what other challenges have you come across while you’ve been studying the
disease and how are you trying to overcome them?
The organoids really
help us get around the fact that there are no adequate animal models for Shigella.
Another important consideration is mimicking the host signals the bacteria
encounter in the human gastrointestinal tract. Laboratory media does not
adequately provide the bacteria with the appropriate host signals that Shigella
encounters during gastrointestinal transit. These signals are important for
both survival and for the activation of certain virulence factors. Shigella
is very efficient: it expresses certain genes at the right time and does not
like to waste energy expressing these factors unless needed. So, we’ve tried to
mimic what the bacteria encounter as they transition through the small
intestine by supplementing laboratory media with bile salts and glucose. Bile
is an interesting signal because it is mostly localized to the small intestine,
with only 5% entering the colon. For simple sugars like glucose, these signals
are available for bacterial utilization as our food is digested. So, it is a
nice combination of signals that we can use for these analyses.
All enteric pathogens
can resist bile, which typically involves the same mechanisms by which the
bacteria resist antibiotics. We found that Shigella, like other
pathogens, uses the AcrAB efflux pump to resist bile and that information helps
us to understand how Shigella survives host transit to cause infection
in the colon. It is intriguing to consider that the same mechanisms are used to
resist bile and antibiotics. So, the simple fact that pathogens are exposed to
bile essentially helps prime them for an antibiotic resistance phenotype, which
has important implications in our current age of antimicrobial resistance. We
also demonstrated that the combination of bile salts and glucose induces
biofilm formation in Shigella. Biofilms help bacteria to resist harsh
environments, so we hypothesize that the biofilm also facilitates Shigella
survival and transit through the small intestine. We even mimicked the loss of
the bile signal as the bacteria enter the colon and found that the bile signal
loss triggers Shigella to disperse the biofilm in a hypervirulent state
since infection is greatly enhanced upon this dispersion step. In fact, we are
close to publishing results that demonstrate Shigella expresses
adherence factors under these conditions. Many people in the field thought Shigella
cannot express adherence factors, especially since the structures are not
detected following growth in laboratory media. However, when we utilize the
media to mimic small intestinal transit, we can see the structures and are able
to characterize the genes. These conditions help us to understand how Shigella
is using the structures to make initial contact with the epithelial cell surface
and we are hoping we now have new therapeutic targets to pursue.
What methods are
you using in order to target Shigella infection?
Once we utilize the
supplemented media and the organoid infection model, we use several techniques
to study pathogenesis such as RNA sequencing or quantitative RT-PCR, and
protein expression and mutational analyses. We can evaluate changes in both
bacterial gene or protein expression and well as changes in the host cells.
Microscopy is also important, ranging from confocal microscopy to electron
microscopy, in order to get the detail of either visualizing adherence
structures or seeing how the bacterial surface changes, to examining the
bacterial-epithelial cell interaction at different time points of infection.
When it comes to
trying to tackle the disease, what kind of strategies and techniques are you
using to target the infection?
We are hoping we can
identify new virulence factors or antigens that can act as potential vaccine
targets to generate an antibody response – that would be the ultimate goal. The
field has tried for years to successfully develop a Shigella vaccine.
Candidates have routinely had problems or been unable to get through clinical
trials. We are hoping we can apply new information to develop novel vaccine
candidates and get the right formulation that is successful. I also think a
very important aspect of new therapeutic development is overcoming
antimicrobial resistance. Understanding that bile can trigger an antibiotic
resistance-like phenotype is, I think, important for trying to develop new
antimicrobials in the future. For Shigella, we need to target the
pathogen early enough to prevent the establishment of infection in the colon,
and so understanding all the changes that occur during small intestinal transit
is important to our future success.
Finally, what really
has developed in recent years, and is something that we have started to pursue
with collaborators at MIT (MA, USA), is the potential of phage therapy. Phage
therapy could give us something that is effective and bridges a gap as the
antibiotic resistance problem continues and we try to develop new and
successful vaccines.
Why has it been so
difficult to create a vaccine? What’s held that area of the research back?
For Shigella
specifically, it’s been challenging. A few antibodies have been considered the
correlates of protection. When anyone has tried to produce those antibodies
with a vaccine, either through injecting live attenuated strains of the
bacteria or using some sort of protein and/or lipopolysaccharide formulation to
generate an antibody response similar to infection, most of the vaccine
candidates have ended up failing. Even if some of the candidates have appeared
successful early on, especially in preclinical analyses or safe and well
tolerated in early clinical trials, the candidates end up failing during
challenge and efficacy studies. The candidates have been reactogenic,
especially working with attenuated strains; or the candidates do not provide
sufficient protection in challenge studies. In these challenge studies it is
difficult to detect differences between someone who has been challenged
with infection versus someone who first received a vaccine candidate and was
then challenged, so protection is not detected. Shigella infection does
not generate an adaptive T-cell response, which is a significant problem for
successful vaccine development. In fact, many researchers are trying to figure
out how Shigella subverts activation of the T-cell response; an
understanding that will certainly go a long way to eventually developing a
successful vaccine.
Phage therapy has
had a huge boost recently with a successful case, what does this latest
development mean for your research?
I think phage therapy
offers some great promise. We are at a point where we can see the potential of
phage therapy and start pursuing it, whether it is phage therapy by itself or
in combination with antibiotics. Phage therapy will provide specific approach
to killing the bacteria; or, if used in combinations with antibiotics, can
prevent the bacteria from quickly adapting to overcome either the antibiotic or
the phage. As I mentioned, we are collaborating with MIT and we are hoping to
push phage therapy into a new direction where we can use bioengineering, not
only to ensure its success, but also to ensure phages are specific to
pathogens. This concept is important for the gastrointestinal tract and the
ability to protect the microbiome, where current antibiotics would kill the
beneficial bacteria. We are trying to avoid the “total destruction” seen with
antibiotics and just specifically target the pathogen in question.
Bioengineering will also help to prevent the pathogens from quickly adapting to
resist the phage, which has been seen and is a possible challenge for phage
therapy. The potential of phage therapy is really important for controlling Shigella
infection since it mostly infects malnourished children under the age of five
years in the developing world. So, we need therapies that can prevent infection
in malnourished children and ensure that we are protecting their microbiome.
Malnourished children typically have recurrent diarrhea, which leads to growth
and developmental delays that significantly impact developing countries and is
very unfortunate. Phage therapy provides a new hope to help both malnourished
children and developing countries to truly enhance global health.
What do you think
needs to progress with phage therapy before it can become a fully-fledged
effective therapy that’s used in common practice?
From what I can see,
it seems like researchers are rapidly moving into phage therapies and looking
at the potential of phages, especially within the last 5 years. I think
efficacy and preclinical analyses will need to be performed to make sure we
have the targets right and we have hopefully reduced resistance forming in
pathogens. I think it is going to happen at a relatively quick pace, as fast as
it can be in science anyway. We can hopefully get phages into early clinical
trials and push them along the clinical trial trajectory to see it used as a
therapeutic. Researchers are quickly obtaining viable candidates from the lab,
so now it is a matter of getting them through clinical trials. There have been
several cases around the globe now where patients have been successfully
treated with phage, so I think regulatory agencies are very interested in
pursuing them as well. Many researchers in the field are at a ”let’s do it”
point, especially with the staggering increases in antibiotic resistance.
Shigella is mostly a pediatric disease, is
there a particular reason why it seems to be focused on children younger than
five?
Well, one of the ideas
is that the antibodies that are generated during infection can help to provide
protection in adults, at least somewhat partial protection. It could be a
scenario where we see mostly children under five because it is their first
exposure to Shigella, but that viewpoint might be changing a bit because
adults still get infected. It is a matter of whether adults are reporting the
infection or whether they are just dealing with the symptoms without going to
the doctor.
Interestingly, we have
also seen a resurgence of Shigella infection in adults in industrialized
nations, across Europe and in the United States. In certain populations,
especially in HIV populations of the homosexual community, there are
significant rates of transmission of Shigella. We are trying to
understand why that is occurring, and it seems to correlate with HIV infection.
Researchers in the field are trying to examine the epidemiology associated with
this type of Shigella infection. Also, in places like the U.S. and
Europe, there are many examples of water-borne or food-borne outbreaks. So, Shigella
is transmitting and causing infection. Usually the outbreak reports
are triggered when children or adults with additional health complications seek
medical treatment. When a child is sick and has watery diarrhea that suddenly
shifts to bloody diarrhea, parents quickly take notice and take their child to
the pediatrician. However, it will be interesting to see if we can
further investigate potential differences in disease patterns or microbiome
protection to truly understand why children are more susceptible to infection.
With the HIV
association, do you think it is because the HIV weakens the patient making them
more likely to feel the effects of the Shigella? Or is there an
implication that it might also have a sexually transmitted aspect to it?
There is a sexually
transmitted aspect to it, which has been documented. However, it is
interesting that it seems to be most the prevalent among patients with HIV, so
there could potentially be an important aspect to a weakened immune system that
helps Shigella establish infection. Tomado de envio de
biotechniques
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