Sarah Pyne-Gehen and Dr. David Erickson, Department of Microbiology and Molecular Biology
Yersinia pestis, the causative agent of plague, is transmitted by fleas such as the Oriental rat flea Xenopsylla cheopis. Very little is known about the Y. pestis factors that are important in establishing a transmissible infection in fleas. The Yersinia Murine Toxin (Ymt) is an intracellular phospholipase enzyme that is required for Y. pestis to survive inside the midgut after fleas take an infectious blood meal (1). The basis for the Ymt requirement is unknown.
Since ymt mutants of Y. pestis can survive in fleas that feed on washed red blood cells without serum, yet ymt mutants grow in serum in vitro, it is likely that a component of serum digestion kills Y. pestis (1). Serum contains thiocyanate (SCN-) ions and previous research performed in the Erickson lab show that flea midguts contain high levels of hydrogen peroxide (H2O2). SCN- and H2O2 can be converted by a peroxidase, such as lactoperoxidase (LPO) into the potent antimicrobial hypothiocyanate (OSCN-).
My research specifically focused on the interaction of Ymt and OSCN-. I hypothesized that Ymt prevents killing of Y. pestis via OSCN- inside the flea midgut. I tested the in vitro susceptibility of Y. pestis wild type and ymt mutant strains to OSCN-. To do this, cells were challenged with OSCN-. Both wild type and ∆ymt strains of Y. pestis was grown in liquid cultures of LB broth and diluted in PBS. Control groups contained cells and LPO or cells, LPO, and H2O2. OSCN- was made in test groups by combining cells with LPO, H2O2 and SCN-. H2O2 was added in 1ul increments every 5min for 30min. After incubating for 2hr, samples were plated on LB plates and colonies were later counted. I found that both wild type and ymt mutant Y. pestis were equally susceptible to OSCN-.
To test my hypothesis in vivo, I fed fleas on Y. pestis ∆ymt infected blood with serum removed, with serum removed +SCN-, or whole blood. Fleas were allowed to live for 3 days and then crushed and plated on selective media. Colonies were later counted and microbial growth was compared between the three groups. I found that adding SCN- to washed red blood cells did not affect survival of Y. pestis ∆ymt in flea infections.
While OSCN- does not appear to be the toxic component in fleas, Ymt is playing a pivotal part in the survival of Y. pesitis inside the flea. One of my fellow lab partners is currently investigating the hypothesis that a digestion product of serum triggers a lytic pathway in Y. pestis and that Ymt redirects or blocks this pathway. To identify Y. pestis gene products that may be altered by Ymt during infection, she subjected Y. pestis ∆ymt to random transposon mutagenesis to identify suppressor mutations. Fleas were infected with pools of transposon mutants and identified one mutant that was able to survive for one week in flea guts in the absence of Ymt.
The transposon insertion site was mapped to the y3762 gene, with homology to ligases (WaaL) that attach surface carbohydrate polymers to the lipid A core. These results suggest that the WaaL modulates the outer structure of Y. pestis, enhancing susceptibility towards toxic product(s) of serum digestion. We hypothesize that Ymt either acts on the toxic product(s) directly or inhibits the function of WaaL so that the toxic product(s) cannot enter the cell.
In the end, my results indicated that Hypothiocyanate alone is likely not the toxic agent of serum digestion. However, our laboratory has recently discovered that WaaL, a proton similar to a ligase, is somehow involved in the killing mechanism of Y. pestis. When both WaaL and Ymt are absent, Y. pestis can survive within the flea. We predict that Waal modifies the cell membrane of Y. pestis and thus allows toxin produced from serum digestion or a lytic pathway signal to enter the cell. Ymt counteracts this by blocking the toxin or lytic factor.
References
- Hinnebuschi B., Rudolph J., Cherepanov A., Dixon P., Schwan J., Forsberg T., 2002. Role of Yersinia Muring Toxin in Survival of Yersinia pestis in Midgut of the Flea Vector. Science. 296: 733-736.