Our Work

 


Regulation of the Type III Secretion System:

A schematic of the stepwise assembly of the TTSS. Image courtesy of Olaf Schneewind.

 

Y. pestis encounters both mammalian hosts and insect vectors during its life cycle. Each host presents a different set of environmental conditions, and the expression of a large number of genes having basic metabolic as well as pathogenic functions is regulated accordingly. Signals such as temperature, calcium and serum protein and amino acids serve to regulate Yop delivery until contact with an appropriate target cell has been made. Several regulatory proteins that respond to these signals have already been identified (such as LcrF, YopN, LcrV, LcrG, LcrQ, YopB, and YopD, and there respective chaperones). However the gene products responsible for sensing the environmental cues and then relaying that information to the known regulatory components of the secretion machinery remain a mystery. We therefore hope to identify the sensors and signal transduction pathways involved in controlling type III secretion. Our work has revealed a two component system, which in other organisms such as Salmonella, Pseudomonas, and E. coli, functions as part of an elaborate signal transduction network. Several uncharacterized genes have also been linked to the type III pathway. Future studies will address the mechanism by which these gene products contribute to the processing of environmental cues and regulation of type III secretion.

Y. pestis cells are stained with a fluorescent maleimide reagent that reacts with the TTSS needles. The needles can be seen as punctate dots on the surface of the cell. Image taken by Warren Kilmer.

 

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 other questions: 1) Are the same type of cells targeted in different organs? 2) Do different routes of infection influence the selection process? 3) Are all Yop proteins delivered with the same efficiency into all targeted cell types? 4) How is the selection process mediated; i.e., what are the bacterial and host genes involved in target cell identification?

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. Furthermore, the location of YopK during infection was unknown. Using the beta-lactamase reporter assay described above, we found YopK injected into host cells. Bioinformatic analysis reveals only a putative CaaX box, which in eukaryotes is a signal for prenyl modification. We will determine whether this CaaX box is functional and if host prenylation contributes to YopK function. Various biochemical approaches will also be employed to investigate the subcellular location of YopK and to identify interacting proteins. 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.