Teamwork makes the dream work, but teamwork in E. coli may be more of a nightmare

Written by: Rebecca Tweedell

Original Article: Snoussi et al. eLife 2018.
The Gist of It
Most people are familiar with the phrase “Teamwork makes the dream work,” which is actually the title of a book about leadership by John Maxwell. What most people don’t know is that the full quote from the book is “Teamwork makes the dream work, but a vision becomes a nightmare when the leader has a big dream and a bad team.” This has never been more true than in a community of bacteria, where the “dream” is to grow and multiply and the “team” is full of bad pathogens that can make you sick. One of the body’s natural ways to stop pathogens from invading and multiplying is by producing antimicrobial peptides. These are similar to antibiotics and can stop the bacteria from growing and even kill them. Unfortunately, many bacteria have ways to block these antimicrobial peptides from doing their jobs. Dr. Snoussi and colleagues recently found that one bacterium, Escherichia coli, has a cooperative way of dealing with an important antimicrobial peptide called AMP LL37. The researchers found that when E. coli were exposed to AMP LL37, some of the bacteria would sacrifice themselves by absorbing lots and lots of AMP LL37 and dying. This decreased the amount of AMP LL37 floating around and allowed the rest of the bacteria to continue growing and thriving. This means that even more AMP LL37 would be needed to successfully kill all the bacteria. This is an important finding, as it sheds new light on how some pathogens may be able to escape our natural defenses. Finding ways to overcome this nightmare of bacterial “teamwork” will be essential for fighting these infections.

Teamwork in E. coli means living the dream and survival for them, but a nightmare for us. If one bacteria absorbs a lot of antimicrobial peptide (AMP) then it can save other bacteria by sacrificing itself.

The Nitty Gritty
To analyze the effects and minimal inhibitory concentration (MIC) of AMP LL37 on E. coli, researchers grew the bacteria in vitro in rich defined media. They found that the MIC for LL37 increased as a function of inoculum size, with the MIC increasing from 3.69 μM to 7.09 μM for inoculum sizes of 12.2 × 106 and 24.4 × 106 cells/mL, respectively. When E. coli were exposed to sub-MIC levels of LL37, the doubling time for the cells did not change, but the lag phase of growth was extended, suggesting that only a sub-set of the cells were being killed, leaving the rest of the population to grow and divide normally. Using a fluorescently labeled version of LL37, 5-FAM-LC-LL37, the researchers observed that in isogenic colonies (originating from a single cell), there was heterogeneous absorption of LL37. By tracking this fluorescent signal, they also found that LL37 was retained within the bacterium that originally absorbed it, keeping the peptide out of the supernatant and away from the neighboring cells. Furthermore, the researchers found an inverse correlation between the rate of LL37 uptake and the cell’s length, suggesting that smaller, growing cells were resistant to LL37 and that the larger, dividing cells took up the peptide. While the specific molecules within the bacteria responsible for this uptake remain unclear, the researchers used a minCDE mutant strain of E. coli to produce enucleated cells and found an increased rate of LL37 absorption in the neighboring mother cells with DNA content, suggesting the negative charge of DNA may be playing a role in absorption.
Original Research Article: Snoussi, et al. “Heterogeneous absorption of antimicrobial peptide LL37 in Escherichia coli cells enhances population survivability.eLife 7 (2018): e38174.

Intestinal parasites decrease inflammation after bone marrow transplant? Apparently!

Written By; Kaitlyn Sadtler

Original Article: Li et al. Journal of Immunology 2018
The Gist of It:
If you’ve watched ER or Grey’s Anatomy, you’ve probably heard of a patient “rejecting” their organ transplant. This happens when the patient’s body notices that the transplanted organ is not part of itself and starts to attack it because it is foreign. The part of our body that rejects a transplanted organ is our immune system, the same system that helps fight against infections like the flu. In certain kinds of transplants, like bone marrow transplants, immune cells are the “organ” transplanted into a patient, and sometimes things can go awry, leading to the transplanted immune cells attacking the patient because they see the whole patient’s body as foreign. This type of reaction is called graft versus host disease, or GVHD. This is caused by a type of inflammatory response that is similar to the response seen when fighting off an infection. Recently, researchers at the University of Iowa have discovered that the immune response to intestinal parasitic worms (which is different than the response during infections or GVHD) can actually decrease GVHD symptoms through a specific biologic pathway. The worm, Heligmosomoides polygyrus bakeri (we’ll just call it “worm”), makes the immune system secrete a certain protein called interleukin-4, or IL-4. This protein stimulates the growth of regulatory T cells – called Tregs. In general, Tregs help dampen the immune system and calm down inflammatory responses. In the case of having both a parasitic worm infection and GVHD from a bone marrow transplant, the worms stimulate these Tregs to calm down the inflammatory response of GVHD through a different protein called TGF-β. These studies could help us understand how to naturally decrease GVHD, a disease with minimal treatment options, and discover new therapies to help manage GVHD after bone marrow transplants.
(It is important to note that these proposed future therapies will require learning from our body’s response to parasitic worms, not the 1900’s-style order a tapeworm from a questionable source. If you know someone suffering from GVHD please consult a licensed physician for all treatments)

Ok, yes, intestinal parasites don’t look like mini dinosaurs but we got carried away with the glitter pens.

The Nitty Gritty:
Using a murine model of GVHD after bone marrow transplant with concurrent Heligmosomoides polygyrus bakeri infection, Li et al. showed that helminthic infection can reduce the symptoms of GVHD through induction of TGF-β and Treg expansion via IL-4 signaling that is dependent upon GATA3 activity. Though previous studies indicated invariable natural killer T cells (iNKTs) were a critical mediator of IL-4–dependent Treg induction by helminths, this study showed that in Ja8-/- mice, which lack iNKTs, helminth-induced Treg expansion was maintained, suggesting that a multitude of cells produce IL-4. Interestingly, production of TGF-β and expansion of Tregs were reliant upon the GATA3 transcription factor. These results could be used to identify new therapies; the current standard of care is immunosuppressive drugs, which can be minimally effective in GVHD and come with adverse side effects.
Original Research Article: Li, Yue, et al. “Helminth-Induced Production of TGF-β and Suppression of Graft-versus-Host Disease Is Dependent on IL-4 Production by Host Cells.” The Journal of Immunology 201.10 (2018): 2910-2922.

The devil in disguise: How contagious cancers have nearly wiped out Tasmanian devils

Written by: Padmini S. Pillai

Original Article: Caldwell et al, eLIFE 2018
The Gist of It:
Many of you may remember Taz, the beloved Tasmanian devil from The Looney Tunes Show, known for his voracious appetite and short temper. The real-life carnivorous marsupials that inspired this cartoon character are currently on the verge of extinction due to the rapid spread of a contagious cancer called Devil Facial Tumor Disease (DFTD). Recently, two different transmittable cancers have emerged in the devils, DFT1 in 1996 and DFT2 in 2014; these have resulted in the death of nearly 80% of the devil population. Tasmanian devils transmit the cancer through biting and then die from starvation as tumors grow uncontrollably on their face and mouth. Normally, cancers are not contagious because the immune system is able to distinguish foreign (non-self) cells from cells that belong within the body, using a molecule called major histocompatibility complex class I (MHC-I). T cells in the immune system recognize non-self MHC-I molecules on cells that don’t belong and destroy them. The process of identifying non-self MHC-I can protect us from bacteria and viruses, but it can also lead to organ transplant rejection. DFT1 cancer cells escape recognition by the Tasmanian devil immune system by simply not expressing MHC-I molecules on their surface. Without the non-self MHC-I, the body can’t identify that the cancer cells don’t belong. Until recently, it wasn’t clear whether the same was true for DFT2. A paper by Caldwell and colleagues used cells collected from tumors and cancer cells grown in the lab to explain how DFT2 spreads. Unlike DFT1, DFT2 cells still have MHC-I on their surface, but they express an MHC-I molecule that is genetically similar to those expressed in most Tasmanian devils. This allows the cancer cells to spread through the devil population without any T cells recognizing them as foreign. To make matters worse, analyses of tumors from many Tasmanian devils show that some DFT2 cancers are also beginning to lose their MHC-I, just like DFT1! This study gives us a close look at how contagious cancers can evolve rapidly, especially in endangered populations. Further research on how contagious cancers avoid recognition by our immune system could not only help save the Tasmanian devils, but could potentially shed light on ways to prevent transplant rejection.

DFT2 cancer cells escape elimination by immune cells by expressing an MHC-I molecule on their surface that is not recognized as foreign by the Tasmanian devil immune system.

The Nitty Gritty:
DFT2 cells were grown in culture and analyzed by flow cytometry to demonstrate expression of beta-2 microglobulin (β2M), a component of the MHC-I molecule. Quantification by RT-qPCR revealed that DFT2 cells expressed both classical and non-classical MHC-I heavy chain genes. Antibodies against classical and non-classical MHC-I heavy chains were generated and used to analyze expression of MHC-I in sections from primary DFT2 tumors. Staining revealed that surface expression of MHC-I in tumors is quite variable. Staining for CD3+ cells revealed that lymphocytic infiltration does occur in DFT2 tumors. Different DFT2 clones were found to express each of the five devil genomic MHC-I sequences, including some expressed by interferon gamma-simulated DFT1 tumor cells. Additionally, analysis of splenic mRNA samples from infected devils showed sequence similarity between MHC-I alleles expressed in three different hosts.
Original Research Article: Caldwell, Alison, et al. “The newly-arisen Devil facial tumour disease 2 (DFT2) reveals a mechanism for the emergence of a contagious cancer.” eLIFE 14;7 (2018): pii: e35314

ALS-on-a-chip: New ways to study Lou Gehrig’s Disease

Written By: Ritu Raman

Original Article: Osaki et al. Science Advances 2018
The Gist of It:
Treating a disease requires understanding it: What goes wrong? When does it go wrong? Does it get worse with time? With amyotrophic lateral sclerosis (ALS), more commonly known as Lou Gehrig’s disease, the cause of gradually and progressive death of the motor neurons (brain cells) that control skeletal muscle is generally unknown and, thus far, unsolved. A new way that scientists are trying to understand the onset and progression of diseases is through “organ-on-a-chip” technology. This is exactly what it sounds like – small volumes of engineered living tissues are grown inside a polymer device called a microfluidic chip. To study a disease like ALS requires an ALS-on-a-chip device that contains two types of tissues: motor neurons and skeletal muscle. In this paper, Osaki et al. develop an ALS-on-a-chip device using cells from a patient with sporadic ALS. They cultured neurons in one compartment of the device and muscle in another, and allowed them to form connections called “neuromuscular junctions” similar to how these tissues are connected in our bodies. In our bodies, when neurons are stimulated, the signal travels down through the neuromuscular junction to make the muscle contract. This group of scientists showed that, compared to healthy neurons, ALS neurons degrade and generate fewer contractions in the muscle. They were then able to test a few different drugs on their chip and showed that treatment with the drugs rapamycin and bosutinib in parallel could potentially help treat ALS. The platform Osaki et al. have developed allows us to study a debilitating and deadly disease and help test potential treatments in the lab before testing them in humans. This makes it more likely that a clinical trial conducted with these treatments will be effective. ALS-on-a-chip is one of many organ-on-a-chip systems being developed by engineers and scientists, and this technology could help us understand and treat many diseases that affect our lives and communities.

Organ-on-a-chip: Looking at the neuro-muscular junction

The Nitty Gritty
Osaki et al. manufactured microfluidic devices using poly (dimethylsiloxane) (PDMS). Within each device were distinct spatial regions for neurons and muscle, with a region in between for the formation of neuromuscular junctions. Both cell types were derived from human induced pluripotent stem cells (iPSCs). The ALS cells were harvested from a patient with sporadic ALS, and all neurons were genetically engineered to incorporate the light-sensitive ion channel, channelrhodopsin-2, ensuring that the neurons could be externally stimulated using blue light. Neurons and muscle cells were seeded separately and cultured until neurites extended to form neuromuscular junctions with muscle fiber bundles. Light stimulation of neurons was used to drive muscle contraction, and the force of muscle contraction was measured by tracking the deflection of flexible micro-pillars to which the muscle was attached inside the device. Immunostaining and fluorescent imaging of the tissue was used to confirm the spatial patterning and integration of the two cell types. The team then tested the effect of rapamycin and bosutinib on the morphology, viability, and functionality of the ALS-on-a-chip unit, demonstrating that this platform could be used to both understand disease progression and conduct high-throughput screening of therapeutic drugs with potential to treat ALS.
Original Research Article: Osaki, T., Uzel, S.G. and Kamm, R.D., 2018. Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neuronsScience advances4(10), p.eaat5847.


Let Sleeping Worms Lie: Sleep is important for survival and to prevent aging in larval C. elegans

Written By: Kathleen Cunningham

Original Article: Wu et al. Current Biology 2018
The Gist of It:
Anyone who has ever pulled an all-nighter has an intuitive understanding of the process of sleep. Despite our “I know it when I see it” understanding of sleep deprivation, there is still a lot that is not understood about why sleep is essential. Nearly every creature with a nervous system appears to sleep. In this study, Wu and colleagues investigated what environmental triggers in the nematode worm C. elegans cause sleep and what molecular pathways affect and are determined by sleep. Because C. elegans have such a simple nervous system, the researchers narrowed down the drive to sleep to a single neuron called the RIS neuron. The researchers found that during multiple life stages of the worm, prolonged starvation caused RIS neuron activation and made the worm sleep. When the researchers removed this neuron or prevented its activation, the worms were unable to sleep; when activation was allowed again, the worms fell back asleep and showed overall reduced brain activity, just like a sleep-deprived college student. When the worms were starved in early developmental stages, sleep deprivation caused early death. Further, sleep-deprived C. elegans juveniles showed signs of aging such as protein aggregation and mitochondrial defects in their muscles. Rather than providing generic rest, sleep in C. elegans appears to have specific roles at different stages, including preventing death and cellular aging.
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Sleepless in C. elegans.

The Nitty Gritty:
Wu et al. began this study by using a variety of harsh starvation conditions and GCaMP imaging in the RIS neurons, which control sleep, to determine how sleep occurs across various developmental stages. Consistent with previous literature, they found that short-term starvation increased activity and foraging behavior, while extended starvation stimulated sleep in adults, L1 larva, and dauer stages. Throughout all these sleep behaviors, the worms exhibited GCaMP activity in the RIS neuron. In mutants that lacked RIS neuropeptide or when apoptosis was induced in the RIS neuron in wild-type worms, starvation-induced sleep was dramatically reduced across all developmental stages. The researchers then behaviorally characterized L1 larval sleep using optogenetic or noxious stimuli, demonstrating that the strongly activating sensory neurons reversed quiescence and acutely inhibited the RIS neuron activity. In the sleeping larva, decreased movement also strongly tracked with decreased pan-neuronal activity. Since it is known that AMPK and FoxO signaling are required for starvation-induced sleep, the researchers tested whether knock-out mutants in the insulin receptor (DAF-2), FoxO (DAF-16), and/or AMPK (aak-1 and aak-2) genes demonstrated any sleep defects. Indeed, insulin receptor mutants demonstrated sleep in abnormal conditions which was dependent on FoxO; FoxO and AMPK double mutants had almost no sleep even under the strongest sleep-inducing conditions. The researchers also used tissue-specific rescue to demonstrate that FoxO signaling is required in muscle to induce sleep, while AMPK signaling in neurons, intestine, muscle, and multiple other tissues induced sleep. To understand why sleep may be required in response to starvation, the researchers observed the effects of ablating the RIS neuron on survival. In adults, RIS neuron ablation did not affect survival. However, during the L1 larval stage, sleep was required to survive starvation-induced arrest. Surprisingly, sleep was required in arrested worms even when they were provided with food, implying that the role of sleep goes beyond the conservation of energy or nutrients. Thus, the researchers used fluorescently labeled myosin, mitochondria, or an aggregation-prone polyglutamine to examine various markers of aging. Sleepless worms had early muscle fiber degradation, mitochondrial fission and fragmentation, and polyglutamine protein aggregation. Therefore, at least at certain stages, sleep is required in C. elegans to prevent aging and even death.
Original Research Article: Yin Wu, et al. “Sleep Counteracts Aging Phenotypes to Survive Starvation-Induced Developmental Arrest in C. elegans” Current Biology 28.22 (2018): 3610-3624.