Necrotizing fasciitis, or flesh-eating disease, occurs when certain types of streptococcal bacteria start visiting harm that is more than skin deep. Instead of limiting themselves to the epithelium and subepithelium, these strep bacteria keep burrowing, invading the soft tissues beneath and establishing life-threatening conditions. These bacteria presumably carry genes that distinguish them from their self-limiting brethren. Yet the genes that turn strep bacteria into voracious “flesh eaters” remain obscure, even though strep bacteria genomes have been sequenced.

Just because strep bacteria genes have been identified doesn’t mean that that we know which genes account for the flesh-eating phenotype. To find the genes responsible for the phenotype, and possibly identify therapeutic targets, a team of scientists based at the University of Maryland devised a transposon-sequencing (Tn-seq) screen. This screen, the scientists report, found that two previously uncharacterized genes contribute to certain strep bacteria’s ability to spread beneath the surface of the skin or throat and invade the underlying soft tissue.

Details of the scientists’ work appeared August 23 in the journal PLOS Pathogens, in an article entitled “Genome-Wide Discovery of Novel M1T1 Group A Streptococcal Determinants Important for Fitness and Virulence during Soft-Tissue Infection.” The article describes how the scientists used a clinically relevant M1T1 group A Streptococcus strain, GAS 5448, to perform the first in vivo Tn-seq screen to characterize, on a whole-genome level, those GAS genetic determinants (subcutaneous fitness genes, scf) functionally required within the subepithelial niche, as well as mutations that could potentially confer a selective advantage during the infection process.

Historically, GAS isolates from the globally distributed M1T1 serotype have been commonly used to investigate GAS virulence potential in vivo, the authors noted. Among these, GAS strain 5448 is a clinical isolate representative of the M1T1 serotype that has been successfully employed in different mouse models of skin and tissue infections as well as necrotizing fasciitis.

The genes found with the Tn-seq screen are called subcutaneous fitness genes A (scfA) and B (scfB). They may prove to be promising clinical targets in the fight against these infections, as there are no vaccines against GAS or effective treatments for invasive infections.

“Although many were annotated with specific roles, we found that a substantial portion of the identified scf genes were of unknown function,” wrote the authors of the PLOS Pathogens article. “Of these, we selected two undetermined genes, scfA and scfB, present in the GAS core genome and confirmed their role in vivo during soft tissue infection and GAS dissemination into the bloodstream.”

“Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream,” the authors continued. “We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.”

Essentially, the scientists zeroed in on scfA and scfB by performing transposon sequencing on the entire GAS genome. Transposons, also known as jumping genes, are short sequences of DNA that physically move within a genome, mutating genes by inserting into them. If the mutation causes an interesting effect, scientists can identify the mutated gene by locating the transposon, sequencing the DNA surrounding the transposon, and mapping its location in the genome.

“Invasion under the skin, or subcutaneously, is not the norm for GAS bacteria; it's actually very rare,” explained Kevin McIver, Ph.D., senior author of the current study and professor of cell biology and molecular genetics at the University of Maryland, College Park. “We hypothesized that there must be genes in the bacteria important for invading soft tissues and surviving under the skin. And we tested that theory by using transposons to make thousands of different individual mutants that we used to infect a subcutaneous environment in mice.”

McIver and his colleagues used a transposon called Krmit—which they created in a previous study—to generate a collection of approximately 85,000 unique mutants in a GAS strain. They injected the mutant strains into mice, which resulted in humanlike infections. The transposon was named for the Muppets character Kermit the frog, whose creator Jim Henson, a 1960 College Park alumnus, died of toxic shock syndrome following GAS pneumonia.

“We were particularly interested in the mutations that didn't come out the other end—the ones not found in the surviving bacteria from the infected tissue,” noted McIver. “These genes would be good targets for a vaccine or treatment because the bacteria missing these genes did not flourish in the infection site.”

The researchers identified 273 scf genes as potentially involved in establishing infection under the skin, but two genes stood out: scfA and scfB. Based on patterns in their DNA sequences, these genes likely encode proteins in the bacterial membrane. This is a prime location for gene products involved in infection because many dangerous bacteria secrete toxins or proteins through the membrane to attack the host. Additional experiments showed that bacteria lacking scfA or scfB had difficulty spreading from under the skin to the bloodstream and other organs.

“The next steps will be to expand the study to include multiple animal models, and these experiments are already underway,” said Mark Shirtliff, Ph.D., a co-author of the study and a professor in the department of microbiology and immunology at the University of Maryland School of Medicine and the department of microbial pathogenesis at the University of Maryland School of Dentistry. “We can also begin to formulate improved therapies and vaccines against GAS infections and their complications, such as rheumatic heart disease, pneumonia, and necrotizing fasciitis.”

McIver also looks forward to using transposon sequencing to study other ways bacteria attack humans.

“Transposon sequencing can be used to probe how bacteria infect humans in any environment you can think of,” stated McIver. “Like GAS, many pathogenic bacteria have completely sequenced genomes, but we don't know what most of the genes are doing. We're excited to have a method to interrogate all that unknown genetic material to better understand human infections.”

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