Our Work

 


Regulation of the Type III Secretion System

 

Target Cell Selection:

Bacteria are faced with a wide array of cells when entering a mammalian host; however most bacteria will only colonize specific tissues. Plague bacteria and other Yersinia species share a common tropism for lymphoid tissues (liver, spleen, lymph nodes). In vitro, however, many cell types including epithelial, hepatic, and macrophage cell lines can all be targets for injection by the type III secretion system. How then is tissue tropism determined? To identify the cells targeted by yersiniae, a fluorescent reporter assay was developed to visualized injected cells. In this assay, a Yop protein is fused to beta-lactamase to generate a hybrid protein which can be injected by the type III pathway. Host cells that have been injected can be distinguished from uninjected cells based on a shift from green to blue fluorescence upon addition of a fluorescent beta-lactamase substrate.

Schematic of B-lactamase reporter assay to measure injection of Yops into host cells. Eukaryotic cells are stained with a fluorescent B-lactamase substrate called CCF2, which is green when intact or blue when cleaved. B-lactamase can be fused to a Yop. When Y. pestis expressing the Yop-Bla reporter is used to infected HeLa cells, the Yop-Bla reporter will be injected and will cause the cell to turn blue. This has been used successfully on several different Yops (Marketon et al. Science, 2005 and unpublished data).

With this technology, we showed that plague bacilli are able to specifically target cells of the innate immune system (neutrophils, macrophages, dendritic cells) in the spleens of infected mice. While this helps to explain tissue tropism, it also opens the door to many other questions. Ongoing work in our lab strives to shed light on the mechanisms controlling the type III secretion system and its interaction with host immune cells.

Y. pestis expressing the YopM-Bla reporter were used to infect mice to determine which cell types were targeted during infection. The results show that Y. pestis specifically targets innate immune cells (Marketon et al. Science, 2005).

 

Regulatory Mechanism of YopK:

HeLa cells were infected with Y. pestis expressing the YopK-Bla reporter and stained with the CCF2-AM substrate. The results show that YopK is injected into host cells by the TTSS (Marketon, unpublished data).


YopK, also known as YopQ, is a substrate of the type III secretion system and has long been a mystery. While yopK mutants inject greater quantities of other Yops into tissue culture cells, they display a severe virulence defect in mice. Our recent work showed that once YopK is delivered to host cells via the type III secretion system, it functions within the host cells to down-regulate injection of Yops. While YopK is not the only Yop to regulate the process of injection from within host cells, our data suggests that its activity is separate and independent of the other Yops. We are currently investigating the mechanism behind this intriguing regulator. Based on our preliminary data we propose a model for YopK regulation of Yop injection: Yops, including YopK are injected into host cells. YopK traffics to the surface of the host cells where it interacts somehow with the secretion machinery to provide negative feedback. This feedback provides a detachment signal that allows the bacterium to find another target cell. In the absence of YopK, there is no detachment, which leads to continual injection of effector Yops and also prevents further spread and colonization of host tissues.

This is our working model to explain the regulatory role of YopK. Based on preliminary data, YopK is injected into host cells in relatively low amounts compared to other Yops. Once inside, YopK interacts with the TTSS to send a detachment signal to the bacterium. In the absence of YopK, this negative feedback is not achieved, so the bacterium remains docked on the host cell and continues to inject Yop effectors.

 

Our Newest Project: Investigating the Flea Niche

Though mammalian plague infections receive much of the attention, the flea vector represents another important aspect of the Y. pestis life cycle. Flea bites are the primary source of infection in rodent reservoirs but little is known about the factors required for bacterial survival within the flea vector. To study this interaction, we have developed a model system using the fruit fly Drosophila melanogaster. We are currently characterizing the interaction between Y. pestis and the insect gut and we plan to take advantage of both bacterial and fly genetics to identify factors that influence Y. pestis colonization of insect vectors.

 

 

 

Relevant Pubs:

  1. Dewoody, R., and M.M. Marketon. 2012. Regulation of the type III secretion system injectisome: Hall monitors for translocation. Submitted to Frontiers in Cellular and Infection Microbiology.
  2. Dewoody, R., P.M. Merritt, and M.M. Marketon. 2012. YopK control both rate and fidelity of Yop translocation. Submitted to Molecular Microbiology.
  3. Houppert, A.S., L. Bohman, A.J. Caulfield, W.W. Lathem, and M.M.Marketon. 2012. RfaL is required for Yersinia pestis type III secretion and virulence. Submitted to Infection and Immunity.
  4. Houppert, A. S., E. Kwiatkowski, E. M. Glass, K. L. DeBord, P. M. Merritt, O. Schneewind, and M. M. Marketon. 2012. Identification of Chromosomal Genes in Yersinia pestis that Influence Type III Secretion and Delivery of Yops into Target Cells. PLoS ONE 7(3): e34039. doi:10.1371/journal.pone.0034039. PMCID: PMC3316589.
  5. Dewoody, R., P.M. Merritt, A.S. Houppert, and M.M. Marketon.  2011. YopK Regulates the Yersinia pestis Type III Secretion System from Within Host Cells. Mol Microbiol. 79:1445-1461. PMCID: PMC3210821.
  6. DeBord, K.L., D.M. Anderson, M.M. Marketon, K.A. Overheim, R.W. DePaolo, N. Ciletti, B. Jabri, and O. Schneewind. 2006. Immunogenicity and protective immunity against bubonic plague and pneumonic plague by immunization of mice with the recombinant V10 antigen, a variant of LcrV. Infect Immun. 74: 4910-4914. PMCID: PMC1539636.
  7. DeBord, K.L., D.M. Anderson, M.M. Marketon, K.A. Overheim, R.W. DePaolo, N. Ciletti, B. Jabri, and O. Schneewind. 2006. Immunogenicity and protective immunity against bubonic plague and pneumonic plague by immunization of mice with the recombinant V10 antigen, a variant of LcrV. Infect Immun. 74: 4910-4914. PMCID: PMC1539636.
  8. Sorg, J.A, N.C. Miller, M.M. Marketon, and O. Schneewind. 2005. Rejection of Impassable Substrates by Yersinia Type III Secretion Machines. J. Bacteriol. 187: 7090-7102. PMCID: PMC1251613.
  9. Marketon, M.M., R.W. DePaolo, K.L. DeBord, B. Jabri, and O. Schneewind. 2005. Plague Bacteria Target Immune Cells during Infection. Science 309: 1739-1741. PMCID: PMC3210820.

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