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I - Hemophore dependent haem acquisition system
(Sylvie Létoffé, Philippe Delepelaire, Francis Biville, Annick Paquelin, Frédéric Huché et Hélène Cwerman)

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 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. In Gram negative bacteria transport of iron or heme sources through the outer membrane is dependent of the proton motive force and a complex of three proteins TonB, ExbB and ExbD. The periplasmic domain of TonB interacts with conserved sequence close to the N-terminus of these receptors which have a weak primary sequence similarity. In contrast, the X-Ray structure of some of these receptors revealed a common spatial organisation: the C-terminus is forming a tunnel made of 22 anti-parallel b sheets inside of which the N-terminus part is partially located on the periplasmic side.

Hemophores, also called HasA are present in several Gram negative bacteria such as Serratia marcescens, Erwinia carotovora, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia pestis and Yersinia enterocolitica. Their biological role is to capture free heme or extract heme from heme carrier proteins, and return it to hemophore-specific receptors, HasR. While the receptor is absolutely required to heme acquisition, the hemophore is not, but it optimises it by decreasing srequired substrate concentration and making wider the potential substrate spectrum.

We have studied biochemical properties of the HasA hemophore of S. marcescens. It is monomeric protein that bind heme with a very high affinity (10-11M). The 3D structure of the holo-hemophore and alanine mutagenesis have demonstrated that heme iron atom is ligated by tyrosine 75 and histidine 32. This work is done in collaboration with the NMR unit of the Institut Pasteur. We have also shown that none of the residu involved in heme binding is involved in the interaction with the receptor.

Structure cristalline de HasA

HasA-3D-b.jpg

Interactions between hemophore et its receptor do not required heme, and we determined that apo-HasA and holo-HasA have a similar affinity for the receptor (5nM). We also demonstrated that there is most likely a unique site on the receptor for the hemophore (or two overlapping sites). We found that the binding of HasA to HasR is TonB independent and involves two b sheets located on the same side of HasA (in yellow in the figure) 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 indicate 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. Once heme transferred to HasR, HasA remains on the cell surface. 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. Nevertheless, we showed recently that empty-hemophore release from the receptor is energy driven and concomittent with heme uptake. It requires higher TonB-ExbB-ExbD complex level than free heme uptake. The step of heme discharge from heme carrier proteins is vital in many cellular functions in procaryotes and eucaryotes. Heme transfert mecanisms are poorly understood, and we hope that HasA might be used as model for these studies.

While interaction between HasA and HasR is independant of TonB, the heme transport is TonB dependant. S. marcescens has a gene, called hasB, homolog to tonB and localised in the has operon. hasB and tonB genes are only redundant for the heme acquisition via HasR. Only TonB is required for others iron acquisition pathways. TonB homologs more or less specific were also described in Vibrio and P. aeruginosa.

The S. marcescens has operon (hasRADEB) codes for the receptor, the hemophore, two proteins involved in the secretion of the hemophore, and HasB, a TonB homolog. Expression of the operon is repressed by Fur in presence of iron. Just upstream the operon two genes, clustered in an iron regulated transcription unit, hasI and hasS, encode respectively a sigma factor (of the ECF-extra cytoplasmic factor-family) and an 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, on the contrary of 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 as anti-sigma when HasR is not occupied by holo-hemophore ligand molecules: as soon as there is a heme shortage hasS and hasRADEB transcriptions are reduced.

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Working model of the Has system

Le systËme Has


blanc

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