T4SS_clsums.png

Outer Membrane Vesicle Secretion

Many bacteria secrete outer membrane vesicles (OMVs) from their cell surface into the extracellular milieu. The vesicles act as delivery vehicles to mediate inter-cellular crosstalk between other bacteria, as pathogenic virulence factors against host targets, as inducers of biofilm formation, and as bacterial defence agents against toxicity factors such as lytic phage. Membrane vesicles are often highly immunogenic and represent viable vaccine strategies as demonstrated for reducing meningococcal carriage.

 

Dynamin and OMV secretion in an enterotoxigenic E.coli (ETEC)

LeoA.png

Maximal release of the cholera-like LT toxin in ETEC strain H10407 has been shown to be dependent upon the protein LeoA, whose gene forms part of a mobile chromosomal pathogenicity island thought to encode machinery that aids in the release of outer membrane vesicles (OMVs) from the cell surface. Previously, we observed that LeoA had hallmarks of a DLP by sequence alignment. Now, through purification of LeoA, crystallization and structure solution, we show that LeoA is indeed a true dynamin family member. This finding is exciting as it potentially couples dynamin with vesiculation in bacteria, and provides one of the first tentative hints as to the function of a bacterial dynamin.


The Type IV secretion (T4S) system

The T4S system forms a large macromolecular nanomachine that spans the cell envelope of Gram-negative bacteria. It is critical for initial and sustained infection in human pathogens such as Helicobacter pylori (gastric ulcers/cancer) and Bordetella pertussis (whooping cough), and in the exchange of antibiotic resistance genes between bacteria during conjugation. The canonical T4S system, as found in the plant pathogen Agrobacterium tumefaciens, comprises 12 proteins, VirB1-11 and VirD4, and some systems extend a substantial tubular pilus beyond the cell boundary.

How the T4S system translocates substrate across the cell envelope and delivers it into recipient cells is unknown, and unravelling this complex question has been hindered by the paucity of structural data available for the whole machine.

Work recently completed involved the purification of eight of the eleven VirB proteins from E.coli R388 conjugative plasmid as a ~3 MegaDalton single particle complex. Negative stain electron microscopy was then used to visualise and reconstruct the complex yielding a map of this remarkable molecular machine at around 20 Å. Our work showed that the T4S system has a radically different modular architecture in comparison to other known secretion systems such as the Type III needle injectisome, and provides an unprecedented platform for dissecting its modus operandi in the future.