Virginia Research Day 2021

Kellie A. King 1 , Angela H. Benton 1 , Kevin K. Lahmers 1 , Lin Kang, Pawel Michalak 2 and Clayton C. Caswell 1 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, 24060, USA. 2 Edward Via College of Osteopathic Medicine, Blacksburg, VA, 24060, USA. A New Regulatory Small RNA, VcrS, is Crucial for Virulence of Brucella abortus

Introduction

Results VcrS: Virulence and Cell wall Regulating sRNA

A

B

178 nt

832 nt

Endonuclease V

GGDEF

hyp

tRNA

vcrS

**

*

*

***

∆ bsr18

(Moreno, Front Microbiol. , 2014)

∆ bsr18-RC

2308

Brucella abortus • Replicates in

*

**

macrophages and dendritic cells • Creates a specialized niche inside the macrophage • It maintains metabolic processes to replicate and promote chronic illness

Bsr18 VcrS

rRNA

5S rRNA

Conclusions and Future Directions A ) Macrophages were harvested from mice and seeded into 96-well plates in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. The following day, macrophages were infected with opsonized brucellae at a multiplicity of infection of 100:1. After 2 h, the infected macrophages were treated with gentamicin (50 μ g · ml − 1 ) for 1 h. The macrophages were then lysed with 0.1% deoxycholate in PBS, and serial dilutions were plated on Schaedler blood agar (SBA). For the 24- and 48-h time points, macrophages were washed with PBS following gentamicin treatment, and fresh cell culture medium containing gentamicin (20 μ g · ml − 1 ) was added to the monolayer. At the indicated time points, macrophages were lysed, and serial dilutions were plated on SBA in triplicates.. B ) There male and three female BALB/c mice (6 total per Brucella strain per experiment) were infected intraperitoneally with ∼ 10 5 CFU of each Brucella strain in sterile PBS. The mice were sacrificed at 4 weeks postinfection, and serial dilutions of spleen homogenates were plated on SBA to determine the CFU per spleen of each strain.

Northern blot analysis

(Celli, Journal of Experimental Medicine , 2003)

Identifying Genes Regulated by VcrS

Small RNAs

Quantitative Proteomics using iTRAQ Analysis

Transmission Electron Microscopy

• 50-500 nucleotides long • Single stranded RNA • Secondary Structures

BAB1_1454 Protein Levels

• Complementary base-pairing with messenger RNA (mRNA) targets

40

Wildtype

30

Gene

Gene

sRNA

20

10

Log Fold Change

• Investigate peptidoglycan composition and architecture in wildtype and ΔvcrS • Confirm proteomic finding • Chromosomal FLAG-tag MurF • Compare protein production in wildtype and ΔvcrS • Artificially overexpress MurF • Compare phenotypic findings

0

Wildtype

ΔvcrS

BAB1_1454: MurF • ATP-dependent amino acid ligase • Involved in cytoplasmic step of peptidoglycan synthesis • Final step in the formation of the pentapeptide precursor • Essential protein

ΔvcrS

Acknowledgments

Funding for this project was provided by the One Health Seed grant provided by VCOM and VMCVM, and NIH AI149124.

(Waters and Storz, Cell , 2009)

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