Assessment of the Role of Brucella abortus Type-V Auto-secreted Proteins in the Pathogenesis and Host Immunity of Brucellosis

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-SECRETED

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Alyssa Hornay

with Dr. Gerard Andrews Department of Microbiology

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 Brucellosis

 Genitourinary infection  Abortion

 Sterility

 Decreased milk production

 Spread by direct contact with infected tissues and

ingestion of contaminated food

 Infection can become chronic  Zoonotic

BRUCELLA

VACCINES

 First vaccine developed and used  Smooth strain – expresses O side

chain

 False-positives

 Cattle must be slaughtered in order to determine if actually infected

 Not recommended for use in wildlife

 Strain RB51 (RB51)

 Attenuated live vaccine

 Rough strain – no expression of O side chain

 Effectiveness diminishes after three years  Does not induce protection in wildlife

from Sherris, 4th_{ed.. The Gram NEG}

IN-VIVO

INDUCED

ANTIGEN

TECHNOLOGY

(IVIAT)

from Rollins, et al. 2005. Cell Microbiology. 7:1

 IVIAT used to examine in-vivo expressed Yersinia pestis genes  Several clones identified as Clusters of Orthologous Groups (COGs)

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 Searched for orthologs in Brucella abortus based

on loci identified in plague screens

 Twenty Type-V

genes identified

 Type-V secretion

“passenger” domain

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DNA GEL AND

PCR PURIFICATION

AIDA LIGATION

ligated into pETBlue AccepTor Vector

 Then, transformed into

NovaBlue Singles competent cells  Plated on LB media containing carbenicillin tetracycline, X-gal and IPTG

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 Plasmid prep using QIA Spin Miniprep Kit

 Transformed into Tuner(DE3)pLacI competent Escherichia coli cells

 Plated on LB medium

containing chloramphenicol, carbenicillin, X-gal and

IPTG

 Blue/white screening

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 Culture grown-up overnight at 37ᵒC in LB

medium supplemented with carbenicillin and chloramphenicol

 1:10 subculture grown for one hour then IPTG

added

 Samples taken from culture at time 0, 2, 4, 6, and

8 hours Hour OD₆₀₀ 0 0.5 2 1.69 4 3.06 6 3.25 8 3.11

SDS-PAGE A

 A 22kDa protein should be auto-transported out

of the E. coli cell and the amount of protein should increase with incubation time.

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 Successfully induce AidA auto-transportation out

of competent Escherichia coli cells

 Western Blot analysis to determine

immunogenicity of AidA with cattle serum

 Immunize mice with purified recombinant AidA

and challenge with S19 and observe the rate of clearance

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Brucella abortus Strain RB51 Vaccine Licensed for Use in Cattle. APHIS.

http://www.aphis.usda.gov/animal_health/animal_dis_spec/cattle/downloa ds/rb51_vaccine.pdf

Andrews, G.P., Vernati, G., Ulrich, R., Rocke, T.E., Edwards, W.H., and J.J. Adamovicz. 2010. Identification of in vivo-induced conserved sequences (IVICS) from Yersinia pestis during experimental plague infection in the rabbit. Vector-Borne and Zoonotic Diseases. Published on-line, ahead of print, Jan., 2010

Hanfield, M., Brady, L.J., Progulske-Fox, A., and J.D. Hillman. 2000. IVIAT: a novel method to indentify microbial genes expressed specifically during human infections. Trends Microbiology. 8:336-339

Henderson, I.R., Navarro-Garcia, F., and J.P. Nataro. 1988. The great escape: structure and function of the auto transporter proteins. Trends Microbiology. 6:370-378

Urbigkit , C. 2008. Producers say Strain 19 vaccine needs reconsidered. Wyoming Livestock Roundup.

http://www.wylr.net/index.php?option=com_content&view=article&id=202

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 National Science Foundation for contributing to

the EPSCoR Fellowship by providing funding for my research.

 Dr. Gerard Andrews for guiding me in my

research.