Neisserial outer membrane vesicles (OMVs) are potent carriers of virulence factors. PorB is a channel-forming protein found in large quantities in the outer membrane of Neisseria species that plays a role in pathogenesis. This study investigated whether OMVs are a major secretion system for PorB and the effects of PorB and OMVs on host cells. The results showed that PorB is highly abundant in and on OMVs isolated from Neisseria gonorrhoeae. Additionally, OMVs were found to have toxic effects on host cells and PorB carried by OMVs was found to co-localize with the mitochondrial outer membrane.

1. Neisserial outer membrane vesicles (OMVs) are potent virulence factor carriers.
1Pankaj Deo, 2Jhih-Hang Jiang, 2Eva Heinz, 1Kip Gabriel & 1Thomas Naderer
1Department of Biochemistry and Molecular Biology, Monash University, Australia.
2Department of Microbiology, Monash University, Australia.
Introduction
1. Neisseria species and Outer membrane vesicles (OMVs).
Pathogenicity of Neisseria spp. is attributed to many virulence
factors. These can be present on the surface membrane or secreted
enzymes & toxins. On the bacterial surface the capsule, lipo-oligosachharides
(LOS), pili, adhesins (Opc, Opa), porins (PorA &
PorB) and Outer membrane vesicles (OMVs) all play major roles.
Gram-negative bacteria characteristically release outer membrane
fragments called outer membrane vesicles or "blebs", during growth.
These range in size from approximately 20-200 nm in diameter.
OMVs are composed of outer membrane and periplasmic
constituents.
OMVs are able to transport a bacterial toxin arsenal to host-cells.
They are also able to transfer proteins and/or genetic material to host
cells or other bacteria.
2. The Porins – PorB.
PorB is a channel forming protein and is found in large quantities in the
outer membrane of Neisseria species. It usually facilitates the uptake of
small nutrient molecules; however it is also associated with pathogenesis.
Structurally, PorB can be characterized as a 16 stranded b-barrel protein
that can form trimers (2). It shares several features with the mitochondrial
voltage-dependent anion channel (VDAC), which is suggested to play a role
in host cell apoptosis (1). An interaction between the two proteins has also
been suggested to occur during Neisserial pathogenesis.
3. Impact of PorB on host cells.
Tanabe et al, 2010
PorB
Other Mechanism
Thin section TEM Image of Neisseia Spp Blebbing Neisseria Spp
Aims
Aims of this project are to investigate whether OMVs are the major secretion system for the pathogenic form of PorB and also to investigate the
effects of Neisserial Porin (PorB) and OMVs on host cells.
Conclusions and Future directions
Pro-apoptotic
Conclusions
We have been able to characterize purified OMVs on protein level and
could show that PorB is highly abundant on/in secreted vesicles.
Additionally we have also been able to show that the protein profile of
OMVs is different to the outer membrane of Neisseria. This suggests
that OMVs are secreted via a specific mechanism and are not “randomly
secreted” segments of outer membrane.
Future directions
Investigate whether OMVs are a major secretion system for a
pathogenic form of PorB.
Study the role of PorB on apoptotic pathways.
Results
Purified OMVs are of uniform morphology PorB is present on OMVs
PorB is targeted to mitochondria
References: 1. Kozjak-Pavlovic et al, 2009, 2. Tanabe et al, 2010, 3. Hannah S.W. Lee, 2007.
Kozjak-Pavlovic et al, 2009,
Anne Müller et al, 2000
Anti-apoptotic
Massari et al., 2010
OMVs are specifically loaded
Comparative immunoblot analysis of
purified OMVs: Purified OMVs
contain outer membrane proteins and
little inner membrane/cytoplasmic
components.
Transmission electron micrograph images of
purified OMVs (N. gonorrhoeae FA1090 strain):
OMVs obtained from optiprep gradient
ultracentrifugation purification technique. Scale bar
C.200nm, D.100nm.
Comparative analysis of protein
content of purified OMVs with
bacterial inner and outer membrane:
Certain outer membrane proteins are
enriched on OMVs, while others are
excluded.
OMVs are toxic to host-cells
Immunofluorescence analysis: PorB,
carried by OMV co-localizes with the
mitochondrial outer-membrane. Green-
PorB, Red-Tom20, Blue-Dapi
MTT analysis: There is loss of cellular
metabolic activity of host cells, when
treated with purified OMVs.
PorB is highly enriched on OMVs
Mass Spectrometric analysis : Porins
are the major components of OMVs.