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Building a Better Bacterial Trap for Sepsis

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NETs
Credit: Kandace Gollomp, MD, The Children’s Hospital of Philadelphia, PA

Spiders spin webs to catch insects for dinner. It turns out certain human immune cells, called neutrophils, do something similar to trap bacteria in people who develop sepsis, an uncontrolled, systemic infection that poses a major challenge in hospitals.

When activated to catch sepsis-causing bacteria or other pathogens, neutrophils rupture and spew sticky, spider-like webs made of DNA and antibacterial proteins. Here in red you see one of these so-called neutrophil extracellular traps (NETs) that’s ensnared Staphylococcus aureus (green), a type of bacteria known for causing a range of illnesses from skin infections to pneumonia.

Yet this image, which comes from Kandace Gollomp and Mortimer Poncz at The Children’s Hospital of Philadelphia, is much more than a fascinating picture. It demonstrates a potentially promising new way to treat sepsis.

The researchers’ strategy involves adding a protein called platelet factor 4 (PF4), which is released by clot-forming blood platelets, to the NETs. PF4 readily binds to NETs and enhances their capture of bacteria. A modified antibody (white), which is a little hard to see, coats the PF4-bound NET above. This antibody makes the NETs even better at catching and holding onto bacteria. Other immune cells then come in to engulf and clean up the mess.

Until recently, most discussions about NETs assumed they were causing trouble, and therefore revolved around how to prevent or get rid of them while treating sepsis. But such strategies faced a major obstacle. By the time most people are diagnosed with sepsis, large swaths of these NETs have already been spun. In fact, destroying them might do more harm than good by releasing entrapped bacteria and other toxins into the bloodstream.

In a recent study published in the journal Blood, Gollomp’s team proposed flipping the script [1]. Rather than prevent or destroy NETs, why not modify them to work even better to fight sepsis? Their idea: Make NETs even stickier to catch more bacteria. This would lower the number of bacteria and help people recover from sepsis.

Gollomp recalled something lab member Anna Kowalska had noted earlier in unrelated mouse studies. She’d observed that high levels of PF4 were protective in mice with sepsis. Gollomp and her colleagues wondered if the PF4 might also be used to reinforce NETs. Sure enough, Gollomp’s studies showed that PF4 will bind to NETs, causing them to condense and resist break down.

Subsequent studies in mice and with human NETs cast in a synthetic blood vessel suggest that this approach might work. Treatment with PF4 greatly increased the number of bacteria captured by NETs. It also kept NETs intact and holding tightly onto their toxic contents. As a result, mice with sepsis fared better.

Of course, mice are not humans. More study is needed to see if the same strategy can help people with sepsis. For example, it will be important to determine if modified NETs are difficult for the human body to clear. Also, Gollomp thinks this approach might be explored for treating other types of bacterial infections.

Still, the group’s initial findings come as encouraging news for hospital staff and administrators. If all goes well, a future treatment based on this intriguing strategy may one day help to reduce the 270,000 sepsis-related deaths in the U.S. and its estimated more than $24 billion annual price tag for our nation’s hospitals [2, 3].

References:

[1] Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis. Gollomp K, Sarkar A, Harikumar S, Seeholzer SH, Arepally GM, Hudock K, Rauova L, Kowalska MA, Poncz M. Blood. 2020 Mar 5;135(10):743-754.

[2] Sepsis, Data & Reports, Centers for Disease Control and Prevention, Feb. 14, 2020.

[3] National inpatient hospital costs: The most expensive conditions by payer, 2013: Statistical Brief #204. Torio CM, Moore BJ. Healthcare Cost and Utilization Project (HCUP) Statistical Briefs. Agency for Healthcare Research and Quality (US); 2016 May.

Links:

Sepsis (National Institute of General Medical Sciences/NIH)

Kandace Gollomp (The Children’s Hospital of Philadelphia, PA)

Mortimer Poncz (The Children’s Hospital of Philadelphia, PA)

NIH Support: National Heart, Lung, and Blood Institute


How Mucus Tames Microbes

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Scanning EM of mucus
Credit: Katharina Ribbeck, Massachusetts Institute of Technology, Cambridge

Most of us think of mucus as little more than slimy and somewhat yucky stuff that’s easily ignored until you come down with a cold like the one I just had. But, when it comes to our health, there’s much more to mucus than you might think.

Mucus covers the moist surfaces of the human body, including the eyes, nostrils, lungs, and gastrointestinal tract. In fact, the average person makes more than a liter of mucus each day! It houses trillions of microbes and serves as a first line of defense against the subset of those microorganisms that cause infections. For these reasons, NIH-funded researchers, led by Katharina Ribbeck, Massachusetts Institute of Technology, Cambridge, are out to gain a greater understanding of the biology of healthy mucus—and then possibly use that knowledge to develop new therapeutics.

Ribbeck’s team used a scanning electron microscope to take the image of mucus you see above. You’ll notice right away that mucus doesn’t look like simple slime at all. In fact, if you could zoom into this complex web, you’d discover it’s made up of mucin proteins and glycans, which are sugar molecules that resemble bottle brushes.

Ribbeck and her colleagues recently discovered that the glycans in healthy mucus play a long-overlooked role in “taming” bacteria that might make us ill [1]. This work builds on their previous findings that mucus interferes with bacterial behavior, preventing these bugs from attaching to surfaces and communicating with each other [2].

In their new study, published in Nature Microbiology, Ribbeck, lead author Kelsey Wheeler, and their colleagues studied mucus and its interactions with Pseudomonas aeruginosa. This bacterium is a common cause of serious lung infections in people with cystic fibrosis or compromised immune systems.

The researchers found that in the presence of glycans, P. aeruginosa was rendered less harmful and infectious. The bacteria also produced fewer toxins. The findings show that it isn’t just that microbes get trapped in a tangled web within mucus, but rather that glycans have a special ability to moderate the bugs’ behavior. The researchers also have evidence of similar interactions between mucus and other microorganisms, such as those responsible for yeast infections.

The new study highlights an intriguing strategy to tame, rather than kill, bacteria to manage infections. In fact, Ribbeck views mucus and its glycans as a therapeutic gold mine. She hopes to apply what she’s learned to develop artificial mucus as an anti-microbial therapeutic for use inside and outside the body. Not bad for a substance that you might have thought was nothing more than slimy stuff.

References:

[1] Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection. Wheeler KM, Cárcamo-Oyarce G, Turner BS, Dellos-Nolan S, Co JY, Lehoux S, Cummings RD, Wozniak DJ, Ribbeck K. Nat Microbiol. 2019 Oct 14.

[2] Mucins trigger dispersal of Pseudomonas aeruginosa biofilms. Co JY, Cárcamo-Oyarce, Billings N, Wheeler KM, Grindy SC, Holten-Andersen N, Ribbeck K. NPJ Biofilms Microbiomes. 2018 Oct 10;4:23.

Links:

Cystic Fibrosis (National Heart, Lung, and Blood Institute/NIH)

Video: Chemistry in Action—Katharina Ribbeck (YouTube)

Katharina Ribbeck (Massachusetts Institute of Technology, Cambridge)

NIH Support: National Institute of Biomedical Imaging and Bioengineering; National Institute of Environmental Health Sciences; National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases


Teaming Magnetic Bacteria with Nanoparticles for Better Drug Delivery

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Nanoparticles hold great promise for delivering next-generation therapeutics, including those based on CRISPR gene editing tools. The challenge is how to guide these tiny particles through the bloodstream and into the right target tissues. Now, scientists are enlisting some surprising partners in this quest: magnetic bacteria!

First a bit of background. Discovered in the 1960s during studies of bog sediments, “magnetotactic” bacteria contain magnetic, iron-rich particles that enable them to orient themselves to the Earth’s magnetic fields. To explore the potential of these microbes for targeted delivery of nanoparticles, the NIH-funded researchers devised the ingenious system you see in this fluorescence microscopy video. This system features a model blood vessel filled with a liquid that contains both fluorescently-tagged nanoparticles (red) and large swarms of a type of magnetic bacteria called Magnetospirillum magneticum (not visible).

At the touch of a button that rotates external magnetic fields, researchers can wirelessly control the direction in which the bacteria move through the liquid—up, down, left, right, and even “freestyle.” And—get this—the flow created by the synchronized swimming of all these bacteria pushes along any nearby nanoparticles in the same direction, even without any physical contact between the two. In fact, the researchers have found that this bacteria-guided system delivers nanoparticles into target model tissues three times faster than a similar system lacking such bacteria.

How did anyone ever dream this up? Most previous attempts to get nanoparticle-based therapies into diseased tissues have relied on simple diffusion or molecular targeting methods. Because those approaches are not always ideal, NIH-funded researchers Sangeeta Bhatia, Massachusetts Institute of Technology, Cambridge, MA, and Simone Schürle, formerly of MIT and now ETH Zurich, asked themselves: Could magnetic forces be used to propel nanoparticles through the bloodstream?

As a graduate student at ETH Zurich, Schürle had worked to develop and study tiny magnetic robots, each about the size of a cell. Those microbots, called artificial bacterial flagella (ABF), were designed to replicate the movements of bacteria, relying on miniature flagellum-like propellers to move them along in corkscrew-like fashion.

In a study published recently in Science Advances, the researchers found that the miniature robots worked as hoped in tests within a model blood vessel [1]. Using magnets to propel a single microbot, the researchers found that 200-nanometer-sized polystyrene balls penetrated twice as far into a model tissue as they did without the aid of the magnet-driven forces.

At the same time, others in the Bhatia lab were developing bacteria that could be used to deliver cancer-fighting drugs. Schürle and Bhatia wished they could direct those microbial swarms using magnets as they could with the microbots. That’s when they learned about the potential of M. magneticum and developed the experimental system demonstrated in the video above.

The researchers’ next step will be to test their magnetic approach to drug delivery in a mouse model. Ultimately, they think their innovative strategy holds promise for delivering nanoparticles carrying a wide range of therapeutic payloads right to a tumor, infection, or other diseased tissue. It’s yet another example of how basic research combined with outside-the-box thinking can lead to surprisingly creative solutions with real potential to improve human health.

References:

[1] Synthetic and living micropropellers for convection-enhanced nanoparticle transport. Schürle S, Soleimany AP, Yeh T, Anand GM, Häberli M, Fleming HE, Mirkhani N, Qiu F, Hauert S, Wang X, Nelson BJ, Bhatia SN. Sci Adv. 2019 Apr 26;5(4):eaav4803.

Links:

VIDEO: Synthetic and Living Micropropellers Stir Up Nanoparticles for Enhanced Drug Transport Powered by Magnetism

Nanotechnology (NIH)

What are genome editing and CRISPR-Cas9? (National Library of Medicine/NIH)

Sangeeta Bhatia (Massachusetts Institute of Technology, Cambridge, MA)

Simone Schürle-Finke (ETH Zurich, Switzerland)

NIH Support: National Cancer Institute; National Institute of General Medical Sciences


Targeting the Microbiome to Treat Malnutrition

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Caption: A Bangladeshi mother and child in the Nutritional Rehabilitation Unit.
Credit: International Centre for Diarrhoeal Disease Research, Bangladesh

A few years ago, researchers discovered that abnormalities in microbial communities, or microbiomes, in the intestine appear to contribute to childhood malnutrition. Now comes word that this discovery is being translated into action, with a new study showing that foods formulated to repair the “gut microbiome” may help malnourished kids rebuild their health [1].

In a month-long clinical trial in Bangladesh, 63 children received either regular foods to treat malnutrition or alternative formulations for needed calories and nutrition that also encouraged growth of beneficial microbes in the intestines. The kids who ate the microbiome-friendly diets showed improvements in their microbiome, which helps to extract and metabolize nutrients in our food to help the body grow. They also had significant improvements in key blood proteins associated with bone growth, brain development, immunity, and metabolism; those who ate standard therapeutic food did not experience the same benefit.

Globally, malnutrition affects an estimated 238 million children under the age 5, stunting their normal growth, compromising their health, and limiting their mental development [2]. Malnutrition can arise not only from a shortage of food but from dietary imbalances that don’t satisfy the body’s need for essential nutrients. Far too often, especially in impoverished areas, the condition can turn extremely severe and deadly. And the long term effects on intellectual development can limit the ability of a country’s citizens to lift themselves out of poverty.

Jeffrey Gordon, Washington University School of Medicine in St. Louis, and his NIH-supported research team have spent decades studying what constitutes a normal microbiome and how changes can affect health and disease. Their seminal studies have revealed that severely malnourished kids have “immature” microbiomes that don’t develop in the intestine like the microbial communities seen in well nourished, healthy children of the same age.

Gordon and team have also found that this microbial immaturity doesn’t resolve when kids consume the usual supplemental foods [3]. In another study, they turned to mice raised under sterile conditions and with no microbes of their own to demonstrate this cause and effect. The researchers colonized the intestines of the germ-free mice with microbes from malnourished children, and the rodents developed similar abnormalities in weight gain, bone growth, and metabolism [4].

All of this evidence raised a vital question: Could the right combination of foods “mature” the microbiome and help to steer malnourished children toward a healthier state?

To get the answer, Gordon and his colleagues at the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, led by Tahmeed Ahmed, first had to formulate the right, microbiome-friendly food supplements, and that led to some interesting science. They carefully characterized over time the immature microbiomes found in Bangladeshi children treated for severe malnutrition. This allowed them to test their new method for analyzing how individual microbial species fluctuate over time and in relationship to one another in the intestine [5]. The team then paired up these data with measurements of a set of more than 1,300 blood proteins from the children that provide “readouts” of their biological state.

Their investigation identified a network of 15 bacterial species that consistently interact in the gut microbiomes of Bangladeshi children. This network became their means to characterize sensitively and accurately the development of a child’s microbiome and/or its relative state of repair.

Next, they turned to mice colonized with the same collections of microbes found in the intestines of the Bangladeshi children. Gordon’s team then tinkered with the animals’ diets in search of ingredients commonly consumed by young children in Bangladesh that also appeared to encourage a healthier, more mature microbiome. They did similar studies in young pigs, whose digestive and immune systems more closely resemble humans.

The Gordon team settled on three candidate microbiome-friendly formulations. Two included chickpea flour, soy flour, peanut flour, and banana at different concentrations; one of these two also included milk powder. The third combined chickpea flour and soy flour. All three contained similar amounts of protein, fat, and calories.

The researchers then launched a randomized, controlled clinical trial with children from a year to 18 months old with moderate acute malnutrition. These young children were enrolled into one of four treatment groups, each including 14 to 17 kids. Three groups received one of the newly formulated foods. The fourth group received standard rice-and-lentil-based meals.

The children received these supplemental meals twice a day for four weeks at the International Centre for Diarrhoeal Disease Research followed by two-weeks of observation. Mothers were encouraged throughout the study to continue breastfeeding their children.

The formulation containing chickpea, soy, peanut, and banana, but no milk powder, stood out above the rest in the study. Children taking this supplement showed a dramatic shift toward a healthier state as measured by those more than 1,300 blood proteins. Their gut microbiomes also resembled those of healthy children their age.

Their new findings published in the journal Science offer the first evidence that a therapeutic food, developed to support the growth and development of a healthy microbiome, might come with added benefits for children suffering from malnutrition. Importantly, the researchers took great care to design the supplements with foods that are readily available, affordable, culturally acceptable, and palatable for young children in Bangladesh.

A month isn’t nearly long enough to see how the new foods would help children grow and recover over time. So, the researchers are now conducting a much larger study of their leading supplement in children with histories of malnutrition, to explore its longer-term health effects for them and their microbiomes. The hope is that these new foods and others adapted for use around the world soon will help many more kids grow up to be healthy adults.

References:

[1] Effects of microbiota-directed foods in gnotobiotic animals and undernourished children. Gehrig JL, Venkatesh S, Chang HW, Hibberd MC, Kung VL, Cheng J, Chen RY, Subramanian S, Cowardin CA, Meier MF, O’Donnell D, Talcott M, Spears LD, Semenkovich CF, Henrissat B, Giannone RJ, Hettich RL, Ilkayeva O, Muehlbauer M, Newgard CB, Sawyer C, Head RD, Rodionov DA, Arzamasov AA, Leyn SA, Osterman AL, Hossain MI, Islam M, Choudhury N, Sarker SA, Huq S, Mahmud I, Mostafa I, Mahfuz M, Barratt MJ, Ahmed T, Gordon JI. Science. 2019 Jul 12;365(6449).

[2] Childhood Malnutrition. World Health Organization

[3] Persistent gut microbiota immaturity in malnourished Bangladeshi children. Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M, Alam MA, Benezra A, DeStefano J, Meier MF, Muegge BD, Barratt MJ, VanArendonk LG, Zhang Q, Province MA, Petri WA Jr, Ahmed T, Gordon JI. Nature. 2014 Jun 19;510(7505):417-21.

[4] Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. Blanton LV, Charbonneau MR, Salih T, Barratt MJ, Venkatesh S, Ilkaveya O, Subramanian S, Manary MJ, Trehan I, Jorgensen JM, Fan YM, Henrissat B, Leyn SA, Rodionov DA, Osterman AL, Maleta KM, Newgard CB, Ashorn P, Dewey KG, Gordon JI. Science. 2016 Feb 19;351(6275).

[5] A sparse covarying unit that describes healthy and impaired human gut microbiota development. Raman AS, Gehrig JL, Venkatesh S, Chang HW, Hibberd MC, Subramanian S, Kang G, Bessong PO, Lima AAM, Kosek MN, Petri WA Jr, Rodionov DA, Arzamasov AA, Leyn SA, Osterman AL, Huq S, Mostafa I, Islam M, Mahfuz M, Haque R, Ahmed T, Barratt MJ, Gordon JI. Science. 2019 Jul 12;365(6449).

Links:

Childhood Nutrition Facts (Centers for Disease Control and Prevention)

Gordon Lab (Washington University School of Medicine in St. Louis)

NIH Human Microbiome Project

International Centre for Diarrhoeal Disease Research (Dhaka, Bangladesh)

NIH Support: National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of General Medical Sciences; National Institute of Arthritis and Musculoskeletal and Skin Diseases; National Center for Advancing Translational Sciences; National Cancer Institute


Electricity-Conducting Bacteria May Inspire Next-Gen Medical Devices

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Nanowires
Credit: Edward H. Egelman

Technological advances with potential for improving human health sometimes come from the most unexpected places. An intriguing example is an electricity-conducting biological nanowire that holds promise for powering miniaturized pacemakers and other implantable electronic devices.

The nanowires come from a bacterium called Geobacter sulfurreducens, shown in the electron micrograph above. This rod-shaped microbe (white) was discovered two decades ago in soil collected from an unlikely place: a ditch outside of Norman, Oklahoma. The bug can conduct electricity along its arm-like appendages, and, in the hydrocarbon-contaminated, oxygen-depleted soil in which it lives, such electrical inputs and outputs are essentially the equivalent of breathing.

Scientists fascinated with G. sulfurreducens thought that its electricity had to be flowing through well-studied microbial appendages called pili. But, as the atomic structure of these nanowires (multi-colors, foreground) now reveals, these nanowires aren’t pili at all! Instead, the bacteria have manufactured unique submicroscopic arm-like structures. These arms consist of long, repetitive chains of a unique protein, each surrounding a core of iron-containing molecules.

The surprising discovery, published in the journal Cell, was made by an NIH-funded team involving Edward Egelman, University of Virginia Health System, Charlottesville. Egelman’s lab has had a long interest in what’s called a type 4 pili. These strong, adhering appendages help certain infectious bacteria enter tissues and make people sick. In fact, they enable bugs like Neisseria meningitidis to cross the blood-brain barrier and cause potentially deadly bacterial meningitis. While other researchers had proposed that those same type 4 pili allowed G. sulfurreducens to conduct electricity, Egelman wasn’t so sure.

So, he took advantage of recent advances in cryo-electron microscopy, which involves flash-freezing molecules at extremely low temperatures before bombarding them with electrons to capture their images with a special camera. The cryo-EM images allowed his team to nail down the atomic structure of the nanowires, now called OmcS filaments.

Using those images and sophisticated bioinformatics, Egelman and team determined that OmcS proteins uniquely fit into the nanowires’ long repetitive chains, spacing their iron-bearing cores at regular intervals to transfer electrons and convey electricity. In fact, bacteria unable to produce OmcS proteins make filaments that conduct electricity 100 times less efficiently.

With these cryo-EM structures in hand, Egelman says his team will continue to explore their conductive properties. Such knowledge might someday be used to build biologically-inspired nanowires, measuring 1/100,000th the width of a human hair, to connect miniature electronic devices directly to living tissues. This is one more example of how nature’s ability to invent is pretty breathtaking—surely one wouldn’t have predicted the discovery of nanowires in a bacterium that lives in contaminated ditches.

Reference:

[1] Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Wang F, Gu Y, O’Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. Cell. 2019 Apr 4;177(2):361-369.

Links:

Electroactive microorganisms in bioelectrochemical systems. Logan BE, Rossi R, Ragab A, Saikaly PE. Nat Rev Microbiol. 2019 May;17(5):307-319.

High Resolution Electron Microscopy (National Cancer Institute/NIH)

Egelman Lab (University of Virginia, Charlottesville)

NIH Support: National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases; Common Fund


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