Type I secretion in E. coli - molecular mechanisms of the toxicity of haemolysin A
Abstract
A Type I secretion apparatus is a rather ‘simple' molecular machine, composed only of two inner membrane proteins, a so called membrane-fusion-protein (MFP), an ATP Binding Cassette (ABC) transporter, and one outer membrane protein extending into the periplasm. These three components form a continuous tunnel to the exterior and the substrate is secreted in one step accross two cellular membranes. In the model organism E. coli the paradigm for Type I secretion is the haemolysin system which was discovered in the early 1980s with the corresponding proteins being HlyD (MFP), HlyB (ABC transporter) and TolC in the outer membrane and secretes the best characterized allocrite for a Type I secretion machinery, the 110 kDa haemolytic toxin, haemolysin A (HlyA). Cross-linking procedures and subsequent affinity purification have shown that all three transport components for HlyA secretion can be assembled during the transport process, forming a channel-tunnel direct to the exterior from the cytoplasm. How transcription, translation and secretion of Type I proteins are triggered is of great importance, since this system has considerably potential in vaccination procedures for antigen delivery from attenuated bacteria in the gut. This potential application requires that the regulation of secretion is properly understood and can therefore be manipulated for optimal efficiency. Almost all substrates of Type I systems have crucial functions in attacking host cells either directly as exemplified by HlyA or by being essential, for example, for host colonization. These transport substrates can be divided in several subgroups: RTX toxins, which can be further subdivided in pore-forming cytotoxins, metalloproteases, lipases and S-layer proteins, and non-RTX proteins like hemophores and bacteriocins. They all differ in size and function but all contain a secretion signal located at the C-terminal end of the secreted protein. The location of this signal sequence is therefore different from the classical Sec-dependent secretion pathway but also in distinction to the Sec-pathway, the signal sequence is not cleaved off during or after transport. The diagnostic structural feature for this family is the presence of a characteristic nonapeptide glycine rich repeat (GGXGXDXXX, where X can be any amino acid) that specifically binds calcium ions, giving rise to the name RTX toxins (Repeats in ToXins).
However, it is currently completely unclear how the secretion process is initiated and how substrates cross the funnel-channel of a Type I secretion machinery. We have analyzed the secretion capability of several MalE-HlyA fusion proteins that differ in their folding but not unfolding rate. Based on the results obtained, we propose a kinetic partitioning model. Here, slowly folding substrates are secretion competent while those substrates that fold faster than a certain threshold will not be secreted through a Type I secretion machinery. In parallel, we have developed a rather sensitive assay to determine the lytic activity of HlyA. Interestingly, the toxin seems to have two modes of action. Obviously, HlyA is capable of lysing human and other mamalian cells at a certain concentration. However, at sub-lytic concentration HlyA induces Ca2+ oscillation and the secretion of cytokines and chemokines. To investigate this further, we have started collaboration with Prof. Rüther and Prof. Köhrer to determine those genes that are induced at sub-lytic concentrations of HlyA.
Based on these results, we will try to answer the following questions: which genes are induced at sub-lytic concentrations of HlyA? Which is the molecular mechanism of cell lysis by HlyA? Does it form a pore such as ?-hemolysin from S. aureus or is it interacting simply with the outer leaflet of the target host cell membrane thereby destroying the integrity of the lipid bilayer or does he toxin bind to a receptor? Only by answering these questions, we will obtain a molecular understanding.
Publications
Jumpertz T, Holland IB, Schmitt L The importance of ATP in membrane transport: from structures and mechanisms in ABC transporters in microorganisms (ed A. Ponte-Sucre), Horizon Academic Press, in press
Jenewein S., Holland I.B., Schmitt L. Bacterial Type I Secretion" in Bacerial protein secretion (ed K. Woolridge), Horizon Publishers (in press).
Oswald C, Jenewein S, Smits S.H., Holland I.B., Schmitt L. Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex, J. Struct. Biol. 2008;162:85- 93.
Jenewein S., Schmitt L. Eine molekulare Analyse der ATP-Hydrolyse, BIOspektrum 2007;1:20-23.
Cooperation within the BMFZ
Prof. U. Rüther and Prof. K. Köhrer, HlyA-induced gene expression in human cells.