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11.10.2015

Dr. Eckhard Podack and Research Team Find Perforin-2 to be Potent Weapon against Bacterial Infection

The late eminent Miller School of Medicine researcher Eckhard Podack, M.D., Ph.D., and a multi-disciplinary team of investigators have discovered that the protein Perforin-2 is a highly potent mechanism of the immune system for fighting bacterial infection. Their findings may provide an answer to the looming medical threat of antibiotic-resistant bacteria.

Perforin-2 is an ancient protein found in the cells of a wide range of organisms, from sea sponges to humans. It has been known for some time that this protein has a role as an antimicrobial agent, but the exact function Perforin-2 has in the innate immune system and the mechanisms it uses to combat bacterial infections have remained elusive.

Now, the researchers have learned that Perforin-2 is a vital component of our immune system and our body’s first line of defense against both intracellular and extracellular bacteria. Their findings describe how Perforin-2 is deployed by the immune system, and the means that bacteria have to combat it. The results may lead to the development of therapies that overcome antibiotic resistance.

“These findings open up previously unimagined avenues for the development of novel therapeutics that either prevent pathogens from interfering with Perforin-2 or jump start its activity,” said George P. Munson, Ph.D., associate professor of microbiology and immunology and one of the corresponding authors of the study. “These novel drugs would restore the potent bactericidal activity of Perforin-2 and allow our own innate immune system to successfully clear the infection.”

The new findings contribute to the legacy of Podack, a world-renowned cancer researcher and immunologist who was the Sylvester Distinguished Professor at Sylvester Comprehensive Cancer Center and chaired the Department of Microbiology and Immunology for 21 years. His pioneering work with the innate immune system led to the discovery of two other pore-forming killer molecules — poly-C9, which kills extracellular bacteria, and Perforin-1, which kills virus-infected cells and cancer cells.

“His seminal discoveries laid the groundwork for the current studies, for which he was a principal investigator, corresponding author, guiding intellect, and enthusiastic supporter,” Munson said.

The current findings are published in two breakthrough studies by the journal eLIFE. Building upon Podack’s previous work with other effectors of the innate immune system, the team showed that Perforin-2 forms lethal pores in the membranes of target cells. Unlike other pore-forming proteins that are found only in specific cells, every cell within the human body uses Perforin-2 to defend itself.

“Perforin-2 is also unique among pore-forming proteins in that it is able to kill a vast variety of human pathogens including MRSA, Salmonella and bacteria that cause tuberculosis,” said Ryan M. McCormack, an M.D./Ph.D. student in Podack’s laboratory and first author of both studies. “Even bacteria that are resistant to antibiotics are rapidly destroyed by Perforin-2.”

The team also found that when mammalian cells detect a bacterial infection, they rapidly mobilize Perforin-2 and deliver it to the site of infection. This is accomplished by a CRL (Cullin-RING ubiquitin ligase), which is a protein complex that adds a tag to Perforin-2 upon detection of bacteria. This labeling process is critical to Perforin-2 antibacterial function.

“The tag functions as a sorting signal — the cellular equivalent of a package’s shipping label that tells the cellular machinery to mobilize Perforin-2 from its storage sites within the cell, and deliver it to the site of infection so that it can destroy the pathogen,” Munson said.

Surprisingly, the activity of other bactericidal molecules was found to require Perforin-2. Nonetheless, how infections occur in spite of the efficient killing mechanism of Perforin-2 remained unknown. One possibility is that the evolutionary arms race between host and pathogens has produced bacteria that can overcome the antibacterial action of Perforin-2.

To test this hypothesis the team used pathogenic strains of E. coli and Yersinia pseudotuberculosis in one of the studies. They found that these pathogens insert a protein, called Cif (cell cycle inhibiting factor), into healthy host cells. This action inhibits the CRL complex by inactivating a protein called NEDD8, which is necessary for tagging Perforin-2, leaving the host vulnerable to infection, even in the presence of other bactericidal molecules.

The study revealed the specific mechanism by which two human pathogens circumvent the bactericidal activity of Perforin-2. It is likely that most pathogens have also evolved strategies to interfere with the expression, activation, and/or mobilization of Perforin-2. Hence the findings have far-reaching implications. They can inform the development of treatments that restore or supplement the antibacterial effect of this protein.

“Our studies suggest that if microbial invaders were unable to disarm Perforin-2 our own bodies would be able to successfully combat them,” Munson said. “This strategy would also be an answer to our society’s greatest looming medical threat — which some suggest is already upon us — which is the prevalence and spread of bacterial pathogens that have developed resistance to antibiotics.”

“The untimely death of Dr. Podack is a tragic loss for our team, his department, the university and the worldwide community of researchers,” McCormack said. “Nevertheless, the entire team is committed to the further development and advancement of the groundbreaking research that he initiated.”

Currently, the multi-disciplinary team is exploring the role of Perforin-2 from both sides of the arms race. On the host side they are investigating the cellular mechanisms of Perforin-2 activation and mobilization. They are also characterizing several unique mechanisms that pathogens use to defeat Perforin-2.

The studies are titled “Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria,” available online here, and “Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2,” available online here. The latter article has been recommended in the scientific publishing platform F1000Prime, as being of special significance in its field. Word of the articles’ acceptance for publication was received by Podack just prior to his death.

Faculty co-authors in the studies were Mathias Lichtenheld, M.D., associate professor of microbiology and immunology, Natasa Strbo, M.D., Ph.D., research assistant professor of microbiology and immunology, Olivera Stojadinovic, M.D., research assistant professor of dermatology and cutaneous surgery, Gregory Plano, Ph.D., professor of microbiology and immunology, Marjana Tomic-Canic, Ph.D., professor of dermatology and cutaneous surgery, and Director of the Wound Healing and Regenerative Medicine Research Program in the Department of Dermatology and Cutaneous Surgery, and Robert S. Kirsner, M.D., Ph.D., Harvey Blank Professor and interim Chair of the Department of Dermatology and Cutaneous Surgery, and professor of public health sciences.

Current and former researchers and graduate students from the Department of Microbiology and Immunology who were co-authors were Lesley R. de Armas, Ph.D., Motoaki Shiratsuchi, Ph.D., Desiree G. Fiorentino, M.D., Melissa L Olsson, Alejo Morales, Ph.D., Kirill Lyapichev, M.D., and Louis E. Gonzalez, Ph.D.

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