(722i) 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

Introduction:

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.

Methods:

OMVs were purified as described previously [1].
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.

Results:

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 toxin’s activity   [2].  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.

References:

[1] 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.

[2] Brown, A. C., et al. (2016).
"Inhibition of LtxA toxicity by blocking
cholesterol binding with peptides." Mol
Oral Microbiol 31(1): 94-105.