Mechanism of pilus biogenesis by bacterial conjugative transfer systems
Lead Research Organisation:
Birkbeck, University of London
Department Name: Biological Sciences
Abstract
The rise of antibiotic resistance is one of the major threats to public health. Antibiotics have been the standard of care for bacterial infections for decades. Since their discovery, hundreds of millions of humans have successfully been treated by these "wonder" drugs. Unfortunately, the rapid spread of antibiotic resistance genes among bacterial pathogens threatens to undo the remarkable progress made over the years to combat bacterial infections.
Conjugation (or conjugative transfer) is a widespread biological process whereby genes are exchanged horizontally and unidirectionally from a bacterial donor cell to a bacterial recipient cell. Because conjugation is the process via which antibiotic resistance genes propagate among bacterial pathogens, it is in large part responsible for the antibiotic resistance crisis that humanity is presently experiencing. Crucial to conjugative transfer is the prior formation of a pilus, a long hollow filament produced by the donor cell. The pilus is made of 1000s of subunits of a protein called VirB2. The pilus serves not only to make contact with the recipient cell but also as a conduit for the transfer of DNAs from donor to recipient cells. A pilus needs to be generated before DNA transfer can begin.
Pilus biogenesis and DNA transfer are mediated by a very large molecular machine called "conjugative transfer system" which is embedded in the membranes of the donor cell. Although 12 proteins, named VirB1-11 and VirD4, are necessary to carry out pilus biogenesis and DNA transfer, only the VirB2-11 proteins are required for pilus biogenesis. Recently, we have determined the cryo-EM atomic resolution structure of a large complex containing all the 10 proteins involved in pilus biogenesis and, as a result, we were able to propose a plausible mechanism for pilus biogenesis. Here we propose to build on this recent work and elucidate the details of the mechanism of pilus biogenesis by these complex systems. Mechanistic and structural details will lead to a better understanding of how antibiotics resistance genes spread among bacterial populations and therefore will provide the means to block it.
Conjugation (or conjugative transfer) is a widespread biological process whereby genes are exchanged horizontally and unidirectionally from a bacterial donor cell to a bacterial recipient cell. Because conjugation is the process via which antibiotic resistance genes propagate among bacterial pathogens, it is in large part responsible for the antibiotic resistance crisis that humanity is presently experiencing. Crucial to conjugative transfer is the prior formation of a pilus, a long hollow filament produced by the donor cell. The pilus is made of 1000s of subunits of a protein called VirB2. The pilus serves not only to make contact with the recipient cell but also as a conduit for the transfer of DNAs from donor to recipient cells. A pilus needs to be generated before DNA transfer can begin.
Pilus biogenesis and DNA transfer are mediated by a very large molecular machine called "conjugative transfer system" which is embedded in the membranes of the donor cell. Although 12 proteins, named VirB1-11 and VirD4, are necessary to carry out pilus biogenesis and DNA transfer, only the VirB2-11 proteins are required for pilus biogenesis. Recently, we have determined the cryo-EM atomic resolution structure of a large complex containing all the 10 proteins involved in pilus biogenesis and, as a result, we were able to propose a plausible mechanism for pilus biogenesis. Here we propose to build on this recent work and elucidate the details of the mechanism of pilus biogenesis by these complex systems. Mechanistic and structural details will lead to a better understanding of how antibiotics resistance genes spread among bacterial populations and therefore will provide the means to block it.
Technical Summary
Conjugative transfer systems are nano-machines involved in conjugation, the process by which DNAs are transferred from a bacterial donor cell to a bacterial recipient cell. These systems are responsible for the spread of antibiotic resistance genes among bacterial populations. Prior to being able to transfer DNAs, they must elaborate a pilus, a filament produced by the donor cell and made of 1000s of subunits of a membrane-embedded protein called VirB2. The pilus serves not only to mediate contact with the recipient cell but also as a conduit for the transfer of DNAs from donor to recipient cells.
Conjugative transfer systems are made 12 proteins termed VirB1-11 and VirD4. However, for pilus biogenesis, 10 of them are used, VirB2-B11. Among those, two are AAA+ ATPases, VirB4 and VirB11. Our recent cryo-EM structure of a VirB3-10 complex (Nature 2022) revealed several sub-complexes, notably two of novel functional significance: i- "the Stalk", a central structure which contains a recruitment site for pilus subunits, and an assembly site where the recruited VirB2 subunits are translocated to and where the pilus assembles; ii- "the ATPase complex" made of VirB4, which may act as an ATP-driven motor for pilus subunit translocation to the pilus assembly site.
In this proposal, we first propose to study the VirB4/VirB11 interaction, either in the isolated VirB4/VirB11 binary complex or in the context of the entire VirB2-11 machinery. We also propose to study the impact that ATP-binding and hydrolysis has on the whole machine by using various ATP analogues and ATPase mutants. In addition, we propose to probe the pilus subunit-incorporation cycle using amber-suppression technologies and p-benzoyl-L-phenylalanine (Bpa) incorporation followed by crosslinking to trap intermediate states of VirB2 pilus subunit recruitment and assembly. Finally, we propose to initiate experiments aiming to identify the translocation motor and its mechanism of action.
Conjugative transfer systems are made 12 proteins termed VirB1-11 and VirD4. However, for pilus biogenesis, 10 of them are used, VirB2-B11. Among those, two are AAA+ ATPases, VirB4 and VirB11. Our recent cryo-EM structure of a VirB3-10 complex (Nature 2022) revealed several sub-complexes, notably two of novel functional significance: i- "the Stalk", a central structure which contains a recruitment site for pilus subunits, and an assembly site where the recruited VirB2 subunits are translocated to and where the pilus assembles; ii- "the ATPase complex" made of VirB4, which may act as an ATP-driven motor for pilus subunit translocation to the pilus assembly site.
In this proposal, we first propose to study the VirB4/VirB11 interaction, either in the isolated VirB4/VirB11 binary complex or in the context of the entire VirB2-11 machinery. We also propose to study the impact that ATP-binding and hydrolysis has on the whole machine by using various ATP analogues and ATPase mutants. In addition, we propose to probe the pilus subunit-incorporation cycle using amber-suppression technologies and p-benzoyl-L-phenylalanine (Bpa) incorporation followed by crosslinking to trap intermediate states of VirB2 pilus subunit recruitment and assembly. Finally, we propose to initiate experiments aiming to identify the translocation motor and its mechanism of action.
