Heme uptake and metabolism

Most organisms have a complete heme biosynthetic pathway. Nevertheless, exogenous heme is taken up by bacteria as an iron source.

Over the last decade, the team has characterized the mechanisms of heme acquisition in gram negative bacteria: secretion of small proteins (hemophores) which bind heme with an extreme affinity and return it to outer membrane heme receptors; mecanism of heme transfer from hemophores to heme receptors. The structure of the whole heme-hemophore-receptor complex has recently been solved. The team has started the study of surface exposed proteins of the porphyrin requiring bacterium Hemophilus influenzae that are involved in heme acquisition from hemopexin. Heme delivery from hemopexin to a surface receptor is achieved by a new mechanism. The team has also studied the mechanism of heme transport through the inner membrane which can be achieve in Escherichia coli by either a heme specific permease or by a heme and dipeptide permease. Recently, we have identified two Escherichia coli paralogs, YfeX and EfeB, involved in a new enzymatic reaction of deferrochelation that allows extraction of iron from heme without breaking the tetrapyrrol ring. EfeB is encoded by an iron regulated operon whose products EfeUOB allow heme iron extraction in the periplasm. This is a first example of an extracytoplasmic heme iron extraction. The EfeUOB proteins have well conserved orthologues in other bacterial species including the Gram-positive Staphylococcus aureus FepABC proteins. We are actually studying the role of the FepABC system in heme-iron extraction in S. aureus, and evaluating contributions of this system to virulence in this important human pathogen.

Protein secretion

Many proteins including hemophores are secreted by the T1SS that consist of three proteins: an inner membrane ABC (ATP Binding Cassette) protein, a periplasmic adaptor and an outer membrane channel of the TolC family. Tripartite assembly of T1SS is not permanent, but is induced upon binding of the substrate to the ABC protein. Proteins secreted by this pathway have a C-terminal sequence required for protein secretion. We have shown that, in addition to the C-terminus signal, the Serratia marcescens hemophore have other regions involved in interaction with the ABC protein. They are distributed throughout HasA, and most likely linear. Together with the C-terminal signal, these elements maximize the secretion of HasA. The C-terminal signal of HasA triggers HasD- driven ATP hydrolysis that precludes disassembly of the complex. Our model for type 1 secretion involves a multistep interaction between the substrate and the ABC protein to maintain the assembly of the export system until the C-terminus is presented.

ABC proteins lacking transmembrane domain

Our team is studying ABC proteins lacking transmembrane domains of unknown function such as E. coliUup, S. aureusVga(A) and streptococcus pneumoniae MsrD. Uup is a DNA binding protein, most likely associated with the DNA replication complex. MsrD and VgaA are involved in macrolide resistance. MsrD is active in E. coliand was recently shown to interact by pull-down experiments with Mef, an integral membrane protein belonging to the MF superfamily involved in multidrug efflux. Moreover an ATPase mutant of MsrD exhibited a trans-dominant negative effect over Mef in terms of resistance to macrolide antibiotics. The BACterial Two-Hybrid technology is currently performed to circumscribe the region(s) of interaction. Topology of Mef with or without MsrD will be next defined using fusions with LacZ and PhoA.

Keywords: Membrane transport, heme, iron acquisition, hemophore, ABC protein, drug efflux, nosocomial infections, meningitis

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