Tag Archives: Vanderbilt University Medical Center

Vanderbilt University Medical Center Scientists Demonstrated C. diff. Exposed To Heme Increases Expression of a Protein System Not Previously Studied

 

Vanderbilt University Medical Center scientists have identified a C. diff protein system that senses and captures heme (part of hemoglobin) to build a protective shield that fends off threats from our immune system and antibiotics. The findings, reported in the journal Cell Host & Microbe, reveal a unique mechanism for C. diff survival in the human gut and suggest novel strategies for weakening its defenses.

In a cruel twist, the bacterium Clostridioides difficile (C. diff) makes us bleed and then uses our blood to defend itself against us.

C. diff the most common cause of health care-associated infections (HAI’s) in the United States causes diarrhea and inflammation of the colon (colitis). Individuals taking antibiotics, which disturb the protective gut microbiota, have increased risk for acquiring a C. diff infection, and 20% of patients suffer recurrent C. diff infections despite treatment.

When C. diff colonizes the gut, it produces toxins that cause tissue damage and inflammation. Blood cells burst, releasing heme, the part of hemoglobin that binds iron and oxygen.

Eric Skaar, Ph.D., MPH, Ernest W. Goodpasture Professor of Pathology, Microbiology and Immunology, and colleagues have studied how bacteria respond to heme, which is both a source of the nutrient iron and a reactive, toxic compound.

“Organisms that experience large amounts of heme have to have ways to deal with heme toxicity,” said Skaar, director of the Vanderbilt Institute for Infection, Immunology and Inflammation (VI4). “We wanted to understand how C. diff deals with heme exposure.”

The investigators demonstrated that C. diff exposed to heme increases expression of a protein system that had not been previously studied. They named the system HsmRA (heme sensing membrane proteins R and A) and showed that HsmR senses heme and deploys HsmA to capture it. They also found that the HsmRA system is genetically conserved in many bacterial species.

The binding of heme in the bacterial membrane by HsmA serves a protective purpose first by simply reducing the concentration of free heme, Skaar explained. The researchers also discovered that HsmA uses heme binding to protect C. diff from oxidative stress, including that produced by neutrophils and macrophages from our immune system to kill bacteria.

“C. diff is using cofactors from our own cells as a shield to protect against our innate immune response,” Skaar said.

Oxidative stress also plays a role in antibiotic action.

“Antibiotics have different molecular targets—they may prevent cell wall synthesis; they may prevent protein translation—but the net result of that stress on the cell is often the massive accumulation of oxidative stress that many believe to be a major contributor to why antibiotics kill bacteria,” Skaar said.

The investigators studied whether the HsmRA system protected C. diff against antibiotics.

“We found a really impressive phenotype with vancomycin and metronidazole, two of the front-line antibiotics used to treat C. diff,” Skaar said. “C. diff that expresses HsmA, when HsmA is bound to heme, is much more resistant to vancomycin and metronidazole.”

They also showed that C. diff strains with inactivated HsmR or HsmA had reduced colonization in a mouse model of relapse C. diff infection.

Skaar said it has not been clear why C. diff produces toxins that cause so much tissue damage.

“It’s interesting to speculate that a benefit of toxin-related damage is that C. diff can capture liberated heme and use it as a shield to protect itself against various insults that cause oxidative stress—that would be immune cells, antibiotics and potentially other bacteria.”

The findings suggest that targeting the HsmA-heme shield might increase the sensitivity of C. diff to antibiotics such as vancomycin and metronidazole. It’s not clear that HsmA, a membrane protein, will be a druggable target, Skaar said.

It might be possible, however, to deprive C. diff of heme building blocks by reducing tissue damage or by administering proteins that bind heme, he said. The researchers will explore whether they can increase the sensitivity of C. diff to antibiotics by co-administering a heme-binding protein during infection in an animal model.

“We’re excited about this as a potentially powerful strategy for treating C. diff,” Skaar said.

In other studies, the researchers will explore if the HsmRA system that is genetically conserved in many different organisms has the same functional role to protect against reactive oxygen species. They are also trying to understand the exact mechanism that HsmA-heme uses to detoxify oxidative stress.

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https://phys.org/news/2020-06-clostridioides-difficile-captures-blood-cell.html

Moving the Dial A Little Closer To Better Treatment and Prevention Of C. diff. Infections

 

 

 

Researchers at Vanderbilt University Medical Center have obtained the crystal structure of a toxin from the bacterium Clostridium difficile (C. diff)   a leading hospital-acquired infection  in the United States.

“This is basic science. I think it gives a framework for understanding how, once you do have an infection, the toxins are causing the disease,” says senior author D. Borden Lacy, PhD, associate professor of Pathology, Microbiology and Immunology and of Biochemistry.

Like anthrax, diphtheria and botulism, C. diff infection is a toxin-mediated disease. The bacterium actually produces two similar toxins, toxin A and toxin B. But unlike the other infections, there is as yet no vaccine or other treatment that can effectively block C. diff toxins.

Meanwhile, C. diff has become a major public health menace. In 2011, the bacterium caused nearly half a million infections in the United States, and approximately 29,000 people died from intestinal complications, including a form of colitis, within a month of the initial diagnosis.

In 2012 and 2013, Lacy and her colleagues reported the mechanism by which toxin B kills cells. Earlier this year, they reported the identification of the cellular receptor that binds the toxin.

After binding to their receptors, the toxins are enveloped by an endosome, or tiny vesicle. Through a pore it drills into the cell membrane, each toxin then sends pieces of itself with two enzymatic activities into the cell. The enzymes modify the activity of cellular proteins, ultimately killing the cell.

In the current study, the researchers, led by Lacy’s research assistant, Stacey Rutherford, generated the crystal structure of C. diff toxin A. Benjamin Spiller, PhD, associate professor of pharmacology and of pathology, microbiology and immunology, also contributed to the crystallography.

At Argonne National Laboratory outside Chicago, the researchers bounced a highly focused X-ray of a specific wavelength off the crystal. The resulting diffraction pattern was then converted using computational methods into a model of the toxin.

They found that one small section of the toxin is “highly conserved,” meaning that its sequence of amino acids is identical to the same sequence in other Clostridium species.

This “suggests that antibodies specific for this conserved region could provide protection against multiple toxin-mediated clostridium infections and points to a generalizable strategy for generating safe vaccine antigens for this class of toxins,” they conclude.

In addition, Nicole Chumbler, a graduate student in the Lacy lab who is now a postdoctoral fellow at Harvard Medical School, found that zinc is bound to the toxin and is required for its activity. Small molecules targeting the zinc-binding enzyme could block the toxin’s effects.

Much remains to be discovered, Lacy says, but each study moves the dial a little closer to better treatment and prevention of C. diff infections.

 

The research was supported in part by National Institutes of Health grants AI095755 and GM042569.

 

Source: Vanderbilt University Medical Center

 

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http://www.infectioncontroltoday.com/news/2016/01/researchers-closer-to-a-better-treatment-for-clostridium-difficile.aspx