Final Report Summary - IPAH FUNCTIONS (Role and natural host targets of Shigella IpaH during infection)
Executive Summary:
Objective 1:
Determine the Spatio-temporal resolution of the expression, injection and host cell subcellular localization of the IpaH
To determine the spatio-temporal expression of the IpaH we wanted to develop transcriptional reporters based on the expression of ultrafast maturing GFP from IpaH promoters. Importantly, all IpaH promoters are regulated by the transcription actor MxiE, which is directly regulated by the secretion activity of the type three secretion apparatus (T3SA). Therefore, we also used our newly designed tools to address the dynamic of the T3SA inside infected cells. In order to determine the host cell sub-cellular localization of the IpaH upon secretion we used epitope tagged version of the IpaH under the control of their native promoter.
Objective 2:
Determine the Polyubiquitinome Remodelling induced by Infection (PRI): identification of the substrate(s) of the IpaH proteins
Our initial objective was to develop a large scale approach to biochemically purify the total cellular polyubiquitinated protein, which we dubbed the polyubiquitinome, of cells infected with the WT bacteria and to compare it to an IpaH defective mutant by quantitative mass spectrometry to identify the IpaH substrates. During the development phase of the project we decided to adopt a more targeted approach to do so relying on the biochemical purification of epitope tagged catalytically inactive IpaH proteins, on the basis of the constructs described under the previous objective.
Results:
A considerable amount of efforts during the project has been dedicated to develop the most sensitive and dynamic transcriptional reporters of IpaH expression. After initial results, we rapidly realized that the key characteristic of the ideal fluorescent protein (FP) to design the reporter was maturation rate of the chromophore. Relying on the literature, we selected GFPmut2 variant is clearly faster at maturing than other variants. To improve its behavior in bacteria, including faster folding of the polypeptide chain and minimize aggregation in the cytoplasm, we introduced the GFPmut2 mutations in the GFPsf background. After making the proof of principle that the IpaH7.8 promoter has the better signal-to-noise ratio, we improved the general level of fluorescence by optimizing translation initiation. Last but not least, to improve the dynamic of the reporter, we have fused the ssrA-encoded peptide to the C-terminus of the FP, hence reducing its half-life from 8 hours originally to approximately 40 minutes.
Using this reporter, we have been able to determine the cellular context in which the IpaH are expressed and used it to measure the successive activation and inactivation of the T3SA inside infected cells. Our results clearly indicate that after initial entry the T3SA is inactivated and is only reactivated upon transferring to neighboring cells. Cell-to-cell spread absolutely necessitates the motile phenotype conferred by the pathogen protein IcsA, which induces microfilaments remodelling to propel the bacteria. Finally we have shown that entry into plasma membrane protrusions and vacuole is required, but sufficient to allow for activation of the T3SA. This manuscript was recently accepted for publication in Cell Host and Microbe.
In addition, we have generated reporter of the secretion of the four IpaH genes encoded by the virulence plasmid by inserting an epitope tag to their C-terminus and keeping them under the control of their endogenous promoter. Our characterization of the cellular localization of injected IpaH7.8 and 9.8 have shown that they are associated with different and so far unknown structures in infected cells that are not observed upon mere transfection of the genes inside mammalian cells. Using epitope tagged catalytically inactive variant of IpaH7.8 and 9.8 we have initially identified by mass spectrometry a dozen of their potential substrates. In the last months this strategy has been extended to the other virulence plasmids IpaH1.4 and 4.5.
Conclusion
The delineation of the context of activation of the T3SA has indicated that the secretion of effectors is highly constrained both in time and in space. This observation emphasizes the importance of developing approaches to study the function of effectors in the context of infection and, more specifically, support the approaches that we have taken to identify the IpaH substrates. Within the funding period, I have been able to put in place the experimental strategy to identify these substrates by mass-spectrometry. In the coming months, we will perform the replicates of this mass-spectrometry study and validate some of those substrates using complementary approaches. Our methodology and findings could have broad applications for the study of secretion and for determining the functions of other bacterial E3 ligases found in several pathogenic bacteria.