Identification and molecular characterization of secretion signals in the virulence proteins secreted by the bacterial type 2 secretion system – SecPath

An innovative aspect of our project is the use of a highly integrative approach, which will give insight from the atomic to the cellular level. To perform this work, we will take advantage of our complementary expertises in a wide range of methods from proteomics, genetics, biochemistry, microbiology, to in silico modelling and X-ray crystallography. Another originality of the project is the use of two bacterial models (a human pathogen, K. oxytoca, and a plant pathogen, E. chrysanthemi) and an array of secreted proteins.

To elucidate how the T2SS, a highly dynamic multiprotein machinery, recognizes and translocates folded proteins across the bacterial outer membrane, we study the two sides of this process: 1) identification and characterization of the structural elements of secreted exoproteins that constitute the secretion signals: structure-function analyses of PelI, Pel3 and PulA (structure determination, systematic site-directed mutagenesis, domain swapping); in silico analyses and development of software for comparison of 3D structure of secreted proteins; 2) identification of T2SS components involved in substrate recognition and molecular characterization of their interactions: a systematic approach (site-directed in vivo cross-linking, supressors) and a targeted approach (bacterial two-hybrid, pull-down, NMR spectroscopy).

Understanding the mechanism of T2S and determining the molecular basis of secretion specificity will not only improve our fundamental knowledge but may also open the way to new anti-bacterial agents that function by blocking secretion. The development of small molecules that, by interacting with secretion signal or with the components of the T2SS, could block the secretion of the virulence factors is a possible alternative to antibiotics to fight pathogenic bacteria.

1. Gu S., Rehman S., Wang X., Shevchik V.E., Pickersgill R.W. (2012) Structural and functional insights into the pilotin-secretin complex of the type II secretion system. PLoS Pathog. 8(2): e1002531. doi:10.1371/journal.ppat.1002531.
2. Gu S., Kelly G., Wang X., Frenkiel T., Shevchik V.E., Pickersgill R.W. (2012) Solution structure of homology region (HR) domain of type II secretion system. J. Biol. Chem. 287: 9072-9080.
3. Wang X., Pineau C., Gu S., Guschinskaya N, Pickersgill R.W., Shevchik V.E. (2012) Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the T2SS. J. Biol. Chem. 287: 19082-19093.

Many pathogenic Gram-negative bacteria use common invasion strategies and share the same macromolecular machineries to invade plants and animals. The type II secretion system (T2SS) is widely exploited by animal and plant pathogens (Vibrio, Klebsiella, Pseudomonas, Yersinia, Erwinia, Xanthomonas etc.) to secrete toxins and lytic enzymes. These secreted virulence factors are directly involved in destruction of host tissues, facilitating invasion and infection. The pathogenic bacteria carrying T2SS cause serious diseases in human and animals and provoke important crop losses. The T2S is a two-step process. The exoproteins are synthesized with an N-terminal signal sequence and cross the cytoplasmic membrane either by the Sec or by the Tat system. Once in the periplasm the exoproteins are folded and then translocated across the outer membrane by the T2S machinery composed of 12 to 15 proteins. Although it is studied in diverse bacteria, the architecture and mode of action of this multiprotein nano-machinery is not yet understood. The proteins secreted by the T2SS have no obvious linear secretion sequence and despite numerous attempts to elucidate the mechanisms of exoprotein recognition, the secretion signal has remained elusive. The major aim of this proposal is to elucidate how the T2SS, a highly organized multiprotein machinery, recognizes and translocates folded proteins across the bacterial outer membrane. This proposal therefore addresses the problem of exoprotein recognition by the T2SS: what is the secretion signal and how recognition and secretion of the exoproteins are achieved.
An innovative aspect of our project is the use of a highly integrative approach, which will give insight from the cellular to the atomic level. To perform this work, we will take advantage of our complementary expertises in a wide spectrum of methods from proteomics, genetics, biochemistry, microbiology, to in silico modelling and X-ray crystallography. Indeed, molecular modelling and structural analysis of the proteins secreted by the T2SS is a very powerful tool to search for putative secretion signals, but alone it is not sufficient to provide valuable functional information. Conversely, identification and characterization of secretion signals by biochemical and genetic approaches requires a dedicated structural expertise. This interdisciplinary approach makes our research strategy original and efficient.
Another originality of our project is the use of two bacterial models (E. chrysanthemi and K. oxytoca) and an array of secreted proteins. A dozen soluble proteins are secreted by the Out system of E. chrysanthemi, while the Pul substrate is an inner membrane lipoprotein. Complementary technical strategies used by each partner and applied to different models will allow to extract valuable data common to all the T2SS.
Understanding the mechanism of T2S and determining the molecular basis of secretion specificity will not only improve our fundamental knowledge but may also open the way to new anti-bacterial agents that function by blocking secretion. The development of small molecules that, by interacting with secretion signal or with the components of the T2SS, could block the secretion of the virulence factors is a possible alternative to antibiotics to fight pathogenic bacteria. This finding could also be exploited to direct secretion of heterologue proteins by the T2SS. It has long been thought that T2SS could be engineered to become a “universal” secretion machinery, a goal that could never be reached. A more efficient way could be to modify the surface of proteins without affecting their biologic activity to enable their secretion by T2SS. A similar strategy has been apparently used by bacteria during evolution, which were able to adapt multiple exoproteins and target them to the same secretion system. Identification of secretion signals will allow to apply a rational strategy for these applications.