Graf Lab highlighted by the Daily Campus:

Inside the Lab: Medicinal leech study explores life of bacteria

On the fourth floor of the biophysics building, assistant professor Joerg Graf’s lab is alive with activity. Jeremiah Marden, a post-doctorate, guides fifth-semester molecular and cell biology major Spoorthi Sampath through the process of heating a beaker under a fume hood.

“I’m making a gel to run out so we can see if the cells we have transformed have actually changed,” Sampath said.

Sampath is working on a project to use the system of Aeromonas veronii, the bacteria that lives inside Hirudo medicinalis, a medicinal leech, to learn more about the genes that allow bacteria to live inside their hosts.

“There’s a whole range of benefits associated with these microorganisms,” Graf said. “We want to understand the physiology of the microbial community and how it changes over time.”

Graf’s lab includes six undergraduates, four Ph.D. students, two postdoctoral workers, a master’s student and a lab technician.

Graf said every human being has a different microbial community living inside, but these communities are often too complex to study. He said that the lab focuses on microbial communities in medicinal leeches because they are simpler than communities found in humans.

Humans tolerate bacteria because of a symbiotic relationship in which both humans and bacteria benefit. These bacteria live in sterile environments such as the digestive tract, but when pathogenic bacteria enter that environment, it is no longer sterile and causes an infection, Graf said.

In collaboration with doctors and diagnosticians from different countries, leech therapy, which involves the use of leeches on wound sites, is being used to understand beneficial bacteria, pathogens and antibiotic resistance, Graf said. Antibiotic resistance genes can quickly spread in a bacterial colony as antibiotics kill off non-resistant bacteria, leaving the resistant bacteria to flourish.

The lab works on understanding what makes bacteria pathogenic through a variety of procedures that include growing bacteria, isolating DNA and building gene libraries.

Marden, one of the two post-doctorates in the lab, said bacteria must have at least three secretion systems to inject toxins that function in hundreds of different ways within the host. By deleting the genes that code for the secretion systems, Marden makes mutants, which he then inserts into hosts. The hosts are compared in order to understand the effects of the gene deletion.

“We take the gene that codes for something like a virulence factor, we put in into yeast, which expresses the factor and then you can see if it kills off the yeast,” Marden said.

This research is associated with the United States Department of Agriculture, Marden said, which wants to see how Aeromonas veronii affects fish in aquaculture settings and what strains of the bacteria should be used to make vaccines for the fish.

To understand the genes of microorganisms living symbiotically within organisms, DNA must be sequenced to determine species.

Susan Janton, a research assistant, sequences a variable part of the bacterial genome within the 16S ribosomal RNA, or ribonucleic acid, gene. That gene is responsible for coding the RNA molecules that make up ribosomes where amino acids are strung together to make proteins essential for life.

Because the region is variable, it helps Janton identify specific species found in microbial communities of interest. In addition to the 16S gene, Janton sequences entire genomes of bacteria for comparisons between species.

Using next generation sequencing, Graf said, the lab is able to sequence genes and genomes quickly and efficiently.

In a separate room down the hall, a black box that resembles a large printer is sequencing samples of DNA. The MiSeq sequencer uses a flow cell, which can have 25 to 30 million pieces of DNA stuck to it, Graf said.

Back in the lab, Marden and Sampath have just poured a hot mixture into a container. The mixture will need about a half hour to solidify before samples can be pipetted into a neat row of chambers and pushed through the gel by electricity for visualization later.

“It really is just basic science,” Marden said.