Scientists in the U.S. and Italy have discovered that fragments of the stomach enzyme pepsinogen, which when activated normally help to digest proteins in our food, also have antibiotic activity that can kill food-borne and lung pathogens. The researchers, at Massachusetts Institute of Technology (MIT), and the University of Naples Federico II, used a computational-experimental framework to look for peptide and protein sequences that are similar to those of the body’s own antimicrobial peptides (AMPs), which help the immune system to fight infection. The pepsinogen fragments identified were found to have antibacterial activity against pathogens including Salmonella, Escherichia coli, and Pseudomonas aeruginosa, both in vitro and in an animal model.
The researchers hope to further modify the peptides with a view to generating a new class of synthetic peptide antibiotics that could help to fight drug-resistant infections. The team is also using the same computational technique to look for additional promising antimicrobial peptides in humans and in other organisms. “We now have an atlas of these molecules, and the next step is to demonstrate whether each of them actually has antimicrobial properties and whether each of them could be developed as a new antimicrobial,” comments MIT's Cesar de la Fuente-Nunez, Ph.D., who is also an Areces Foundation Fellow, and one of the senior authors of the team’s paper, published in ACS Synthetic Biology. “These peptides really constitute a great template for engineering. The idea now is to use synthetic biology to modify them further and make them more potent.”
The researchers' studies are reported in a paper titled, “Identification of Novel Cryptic Multifunctional Antimicrobial Peptides from the Human Stomach Enabled by a Computational−Experimental Platform.”
Drug-resistant organisms kill an estimated 23,000 people in the U.S. every year, and it is predicted that by 2050 antimicrobial drug resistance will lead to more than 10 million deaths worldwide annually, the researchers write. “… new treatment options to combat antibiotic resistance are urgently needed.” In the hunt for new antibiotic classes, antimicrobial peptides, also known as host defense peptides (HDP), represent what the authors describe as “promising alternative” to conventional antibiotics.
AMPs are key components of the body’s innate immune system. Working in partnership with immune system components, they represent a first line of defense against invading bacteria, fungi, parasites, and viruses. In addition to well-recognized HDPs such as defensins, other HDPs in the human body are known as “cryptic HDPs/AMPs.” These are produced when larger proteins – including those that are not involved in host defenses – are split into smaller peptides
The team developed a novel computational tool for detecting cryptic AMPs, which they used to analyze databases of human protein sequences in the search for peptides that might be similar to known AMPs. “It's a data-mining approach to very easily find peptides that were previously unexplored,” Dr. de la Fuente-Nunez says. “We have patterns that we know are associated with classical antimicrobial peptides, and the search engine goes through the database and finds patterns that look similar to what we know makes up a peptide that kills bacteria.”
The team's screen of some 2,000 human proteins identified about 800 molecules with potential antimicrobial activity. The work reported in the ACS Synthetic Biology paper focused on the protease pepsinogen, a peptide that is secreted into the stomach. Pepsinogen itself is inactive, but in the acidic environment of the stomach the peptide split into the active proteolytic enzyme pepsin A, and a number of smaller fragments.
It is these fragments that were highlighted as candidates in the team’s screen. Initial in vitro tests showed that the three peptides were active against a wide range of Gram-negative and Gram-positive bacteria, including gut pathogens and clinical multidrug-resistant strains. The peptides were active neutral pH values, as well as at the much low pH that would be found in the stomach. “The human stomach is attacked by many pathogenic bacteria, so it makes sense that we would have a host defense mechanism to defend ourselves from such attacks,” notes Dr. de la Fuente-Nunez.
Subsequent in vivo experiments demonstrated that one of the fragments, (P)PAP-A3, was capable of reducing P. aeruginosa bacterial load fourfold in a mouse model of skin infection. The other two fragments, though less potent, were still able to reduce bacterial load by about two orders of magnitude. “(P)PAP-A3 thus represents a novel peptide antibiotic that may be exploited for the treatment of bacterial infections,” the authors write. Encouragingly, none of the fragments showed any toxicity to human cells. The team's studies also indicate that other pepsinogens may have similar AMP activity. “Our in silico analysis also suggests that several other, even if not all, mammalian pepsinogens A may have antimicrobial properties,” they write.
In the stomach, low pH and the proteolytic activity of pepsin A and other proteases act as a natural barrier to infection. The reported studies hint that the three pepsinogen-derived fragments – the function of which wasn't previously known – may represent another cryptic AMP tier to the gut’s natural defenses. The authors say the findings could explain the observation that a small amount of pepsinogen is secreted in the bloodstream, where it circulates and is eventually filtered, unchanged, by the kidneys. It’s possible that this uropepsinogen could represent another AMP precursor.
“On the whole, our findings suggest the feasibility of developing topical antimicrobial agents based on PAP-A3 and its fragments and demonstrate that the computational−experimental platform we have developed could lead to the discovery and exploitation of bioactive peptides from previously unexplored sources,” the authors conclude.