468695 Pathways Used By Bacteria in Outer Membrane Vesicle Toxicity: A Target for Disease Treatment
Pathways Used by Bacteria in Outer Membrane Vesicle Toxicity: A Target for Disease Treatment
Justin Nice and Angela C. Brown
Department of Chemical and Biomolecular Engineering
Lehigh University, Bethlehem PA 18015
In recent years bacterial resistance to antibiotics has increased; leading to a higher demand for antibiotic alternatives. Our lab is exploring alternative treatment methods which target the bacterial virulence mechanism. One mechanism of toxin delivery to host cells is outer membrane vesicles (OMVs). They are created by gram-negative bacteria as a stress response by blebbing off their outer membrane to form vesicles with a diameter of 10-300nm. OMVs are used by bacteria in delivery and communication. We have investigated the mechanism of delivery for OMVs to their host cells.
Aggregatibacter actinomycetemcomitans (A.a.), a gram-negative bacteria which causes periodontitis and endocarditis in humans, produces outer membrane vesicles. LtxA is a toxin know to be released by A.a. both by secretion and by association with OMVs. Since it is known that free LtxA relies on the LFA-1 receptor and cholesterol for host toxicity we investigated the importance of these factors in OMV associated LtxA.
OMVs were purified as described previously . Purification of OMVs was verified by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Total protein was quantified on a Nanodrop 2000. Toxicity of the OMVs to target cells (THP-1) was measured using the MTT assay.
Purified OMVs had a radius of 83.8nm with a polydispersity index (PDI) of 0.1455 when measured by dynamic light scattering DLS. The size of the vesicles imaged by scanning electron microscopy (SEM) (Figure 1) was consistent with DLS measurements. We showed that LtxA is located on the outside of the vesicle, suggesting it could play a role in OMV targeting and toxicity. Our lab has developed a cholesterol-binding peptide, based on a cholesterol recognition amino acid consensus (CRAC) sequence, which blocks LtxA from recognizing cholesterol and therefore inhibits the toxins activity . Here, we pretreated target cells with this peptide and found that the vesicles were less toxic, indicating that the delivery of A.a. OMVs requires cholesterol (Figure 2).
Figure 1: SEM picture showing several OMVs on surface
Experiments with a scrambled version of the CRAC peptide showed no effect on toxicity (data not shown). The LFA-1 antibody has two subunits, CD11a and CD18; we investigated the effect of blocking each subunit on OMV toxicity. Blocking CD18 decreased OMV toxicity to the cells (Figure 3), but blocking CD11a had no effect on cell viability (Figure 4), demonstrating that LFA-1 plays some role in A.a. OMV delivery.
Figure 2: OMV toxicity on THP-1 cells treated with CRAC peptide (n=3, * p=0.003 compared with no treatment)
Figure 3: OMV toxicity on THP-1 cells pre-treated with anti-CD18 antibody. (n=9, * p=0.00002 for CD18 compared with no antibody)
Figure 4: OMV toxicity on THP-1 cells pre-treated with anti-CD11a antibody. (n=9, no significance seen when compared with no antibody)
Conclusion and Future Directions:
A.a. OMV toxicity, like LtxA toxicity depends on several factors. So far we have shown that cholesterol and the CD18 subunit of LFA-1 are important to OMV toxicity. Our lab will continue to identify mechanisms of A.a. OMV toxicity to host cells with the goal of specifically blocking these mechanisms to treat bacterial infections.
 Masanori Saito, O. T., and Kazuko Takada "Anticytotoxic Effect of Green Tea Catechin on Aggregatibacter actinomycetemcomitans Vesicles." Int J Oral-Med Sci 11(2): 101-105.
 Brown, A. C., et al. (2016). "Inhibition of LtxA toxicity by blocking cholesterol binding with peptides." Mol Oral Microbiol 31(1): 94-105.