Unit: Bacterial Membranes
Director: WANDERSMAN Cécile
Our work concerns membrane transports in Gram negative bacteria with three major research themes: heme acquisition systems requiring an extracellular hemophore, protein secretion by ABC exporters across the two membranes which delimit the Gram negative cells, and a two component system which is involved in iron acquisition. A last research theme on ABC proteins of unknown functions is currently developped by Elie Dassa who joined us in the fall of 2003.
1) Hemophore dependent heme acquisition systems ( Sylvie Létoffé, Laurent Debarbieux, Philippe Delepelaire, Francis Biville, Fréderic Huché, Virginie Roussel)
Iron ions are essential for many metabolic pathways. Yet, iron is not readily available due to its low solubility in the presence of oxygen and to its tight association to iron carrier proteins or to heme in hemoproteins.
Therefore, iron assimilation is an essential function during microbial infection and it represents a potential drug target.
Heme which is a major iron source is uptaken in Gram-negative bacteria by two principal pathways. One involves the direct contact between heme or heme-containing proteins and specific bacterial cell surface receptors. The second requires the secretion of hemophores, a family of proteins discovered in our laboratory in 1994. These proteins present in several Gram negative bacteria such as Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia pestis and Yersinia enterocolitica, capture free heme or extract heme from heme carrier proteins, owing to their higher affinity for heme, and return it to hemophore-specific outer membrane receptors. The S. marcescens hemophore dependent heme acquisition system (Fig. 1) consists of the iron regulated has operon encoding HasR, the hemophore-specific outer membrane receptor, HasA, the hemophore, HasD and HasE, the specific inner membrane hemophore secretion proteins. The last gene of the has operon, hasB encodes a TonB homolog specific for HasR. The activity of HasR is dependent on a protein complex comprising the inner membrane proteins ExbB, ExbD, and TonB or HasB. This is a property shared with several other outer membrane iron receptors whose 3D structures have been elucidated showing an N-terminal domain closing the receptor pore and exposed to the periplasm, where it can make contact with TonB.
The 3D structure of the holo-hemophore and alanine mutagenesis have demonstrated that heme iron atom is ligated by tyrosine 75 and histidine 32 (Fig.2). The 3D structure of apo-hemophore is presently achieved by the NMR laboratory of the Institut Pasteur.
Both heme-free and heme-loaded HasA bind to HasR to the same or overlapping site with the same apparent Kd (5nM). We found that the binding of HasA to HasR is TonB independent and involves two β sheets located on the same side of HasA (stars on Fig. 2) and we propose that this double binding distorts the protein allowing heme transfer to the receptor.
Heme transfer raises several questions. It is not known how heme is transferred from HasA a protein with a very high affinity (Kd : 10-11M) to HasR a protein with a lower affinity ( Kd around 10-6M).
Spectrophotometric analysis of purified HasA, HasR and HasA-HasR complexes indicates that heme transfert from the hemophore to the receptor, occurs "in vitro" and is thus energy independent. We are presently determinating the 3D structure of the heme-loaded complex in collaboration with W. Wellte in Constance, Germany.
Whereas heme is uptaken as a whole through heme receptors, hemophores are not transported and have to be stripped off at the cell surface: only the heme moiety being uptaken, the apo-hemophore remains outside. Since apo and holo-hemophores have the same affinity for the receptor, it is not understood how exchange between the apo- and holo-forms occurs.
We showed that empty-hemophore release from the receptor is energy driven and concomittent with heme uptake. It requires higher TonB-ExbBD complex level than free heme uptake. The step of heme discharge from heme carrier proteins is vital in many cellular functions but poorly understood.
Two regulatory genes hasI and hasS are located upstream to the has operon and encode respectively an extra cytoplasmic function (ECF) sigma factor and its anti-sigma factor. Binding of the heme-loaded hemophore to the outer membrane receptor HasR inactivates the anti-sigma HasS, turning on HasI thereby allowing has operon transcription. Heme alone does not induce. This demonstrates that the inducer and the transported substrate (heme) are different molecules. We are presently trying to define the inducing step at the molecular level using a collection of pentapeptide mutagenized hemophores.
HasI, as the other iron starvation sigma, is iron repressed but not auto regulated. We found an entirely new regulation for the anti-sigma hasS gene, the transcription of which is HasI dependent. This suggests that the has system is both activated and repressed by the availability of external heme. When there is enough heme, the HasS anti-sigma activity is turned off and HasI induces the transcription of HasS. This leads to the storage of inactive HasS molecules which become active when HasR is not occupied by holo-hemophore ligand molecules: as soon as there is a heme shortage transcription is turned off.
2) Protein secretion by Gram negative ABC transporters (Guillaume Sapriel, Sandra Cescau, Laurent Debarbieux and Philippe Delepelaire)
Four secretion pathways have been actually described in Gram-negative bacteria. Type II pathway requires the universal N-terminal signal peptide and the general export pathway to the cross of the inner membrane. Type I and III pathways are independent of the signal peptide and sec system.
The Type I secretion pathway also called the ABC secretion pathway consists of three proteins located in the cell envelope: the ABC protein belonging to the ATP Binding Cassette class of proteins; another cytoplasmic membrane protein and an outer membrane component (Fig.1) .
ATP Binding Cassette (ABC) transporters are implicated in the vectorial movement of solutes across biological membranes. They constitute one of the most abundant family of proteins in the living organisms where they play essential functions as evidenced by the growing number of human health disorders linked to an ABC transporter defect. ABC proteins are also involved in drug efflux in eukaryotes and prokaryotes.
Our work has largely contributed to show that bacterial ABC protein exporters are widespread in Gram-negative organisms. We have shown that in several cases (such as the E. coli hemolysin transporter) the outer membrane component belongs to the TolC family and is a multifunctional protein involved in drug efflux and colicin import. All the proteins following this pathway, including the hemophore, have a C-terminal secretion signal which remains accessible owing to cytoplasmic chaperones which are required for secretion. The signal interacts with the ABC protein, modulates its ATPase activity and induces the formation of a secretion multiprotein complex. The ABC protein has several interaction sites with its substrate that we are currently trying to characterize. Determination of these domains could help to design new drugs able to inhibit ABC proteins involved in multidrug resistance. We are presently purifying the substrate bound ABC transporter multiprotein complex to study it by crystallography and cryomicroscopy This is done in collaboration with the laboratory of Electronic Microscopy of the Institut Pasteur and abroad with Glaeser, Berkeley (USA) and T. Rappoport, Harvard (USA).
3) The two component system YgiX/YgiY (Francis Biville)
This system is present in many Gram negative bacteria. YgiY shares homologies with two component sensor kinases and YgiX with two components transcriptional activators. It appears to be involved in regulation of several E. coli functions such as motility, biofilm formation and iron acquisition. We have shown that inactivation of YgiY allows iron bound to pyrophosphate utilization even in the absence of enterochelin suggesting that YgiY/ YgiX regulates a yet not identified iron transport system. YgiY has a EXXE motif present on several iron binding proteins. We are presently searching whether YgiY binds radioactive iron 55 and trying to chemically characterize the potential iron chelator produced by YgiY mutant.
4) ABC proteins of unknown functions (Elie Dassa, Dorothée Murat and Laurent Goncalves)
In eukaryotes, some ABC proteins lacking transmembrane domains are involved in gene expression regulation. We have shown that a large number of bacterial ORFs of unknown function are homologous to these proteins. We characterized 4 such ORFS in E. coli: uup, ybiT, yjjK and yheS and showed that 3 are involved in the competitiveness of bacteria and in the dynamics of their mobile elements. Our ongoing work aims to:
- analyze the role and the molecular function of these proteins;
- study the evolution of ABC systems and the mechanisms underlying their functional diversity.
Figure 1. : S. marcescens hemophore dependent heme acquisition system
Figure 2 : S.marcescens hemophore 3 D structure
legend : Stars indicate the residues involved in HasA/hasR interactions
Keywords: Membrane transport, iron acquisition, hemophore, ABC protein