Virginia Research Day 2021

Removal of a Small, Hypothetical Protein Attenuates B. abortus Survival in Mice and Macrophages

Angela H. Benton , Kellie A. King, and Clayton C. Caswell Department of Biomedical Sciences and Pathobiology Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, 24060, USA.

Results

Introduction

Conclusions

B

A

• BAB1_0245 is a small hypothetical protein that appears to be differentially regulated by FucR. • Removal of bab1_0245 causes a growth defect in GMM +/- 24mM fucose. • Virulence defects were shown in macrophages and mice infected with Δ bab1_0245. • Fucose uptake from GMM + 24mM media was significant in a strain over expressing bab1_0245 (OE bab1_0245 ). Future Directions • Determine the authenticity of BAB1_0245 • Location, condition and stage of growth for optimal expression • Determine if fucose is used for signaling or metabolism • B. abortus do NOT grow well in higher concentrations of fucose

Expression of B. abortus Genes

Brucella abortus • Replicates in macrophages and dendritic cells • Creates a specialized niche inside the macrophage • Preferred carbon (erythritol) is not present in human cells • Utilizes glucose in the macrophage

IclR family transcriptional regulator ( fucR )

Sugar ABC transporter permease

Sugar ABC transporter substrate binding protein ( fucP )

Wildtype in GMM ∆ fucR in GMM

25

BAB1_0239

~155 bp promoter, putative

BAB1_0237

BAB1_0238

20

Maltose ABC transporter permease

Sugar ABC transporter ATPase

L-Rhamnose mutarotase

Mandelate racemase

15

10

BAB1_0243

BAB1_0240

BAB1_0241

BAB1_0242

5

2-Hydroxyhepta- 2,4-diene-1,7-diota isomerase Fuconate deyhdratase

0

Oxidoreductase

Oxidoreductase

Fold Change in Gene Expression

BAB1_0248

BAB1_0247

BAB1_0244

BAB1_0246

bab1_0248

bab1_0244

bab1_0246

bab1_0242

bab1_0245

bab1_0238

bab1_0240

bab1_0243

bab1_0237

bab1_0239

bab1_0241

B. abortus gene

BAB1_0245

Figure B: qPCR data of wildtype B. abortus and the regulator mutant ΔfucR ( Δbab1_0237 ) . Both strains were grown in Gerhardt’s minimal media ( GMM ) for approximately 20 minutes. RNA was extracted, converted to cDNA, and qPCR was done to determine the fold change in gene expression of the genes in the putative fucose operon.

Figure A: Organization of the putative fucose operon in wildtype Brucella abortus strain 2308 .

Growth in GMM Supplemented with 24mM Fucose

Uptake Assay from GMM + 24mM Fucose

D

C

Wildtype Δ bab1_0237 OE bab1_0245 Δ bab1_0245

Wildtype Δ bab1_0245

6

*

(Moreno, Front Microbiol. , 2014)

20 40 60 80 100 Percent of Remaining Fucose in Media

L-fucose

• 6-carbon sugar found in digestive tract of mammals • Also found in the lysosome of macrophages • Leads to increased expression of genes bab1_0237 → bab1_0248

4

Log 10 brucellae/ mL

B. abortus Growth in GMM + 100mM of a Supplemental C Source

Wildtype Wildtype + 100mM glucose Wildtype + 100mM fucose

Figure D: Indicated strains were grown in GMM containing 24mM fucose. At 24 and 48 hours, amount of fucose was remaining in the media was measured relevant to uninoculated GMM + 24mM fucose. 24 48 0

2

6

0

24

48

72

96

120

Time Elapsed (hours)

4

Figure C: Growth of wildtype B. abortus (2308) and Δ bab1_0245 in GMM supplemented with 24mM fucose. CFUs were determined by serial dilutions and plating at indicated timepoints.

2 Log 10 brucellae/ mL

Clearance of B. abortus in C57/BL6 Mice

E

F

Bone-Marrow Derived Macrophages Infected with B. abortus

0

0

24

48

72

96

120

Wildtype Δ bab1_0245 RC Δ bab1_0245

Time Elapsed (hours)

Wildtype Δ bab1_0245

8

8

*

• Purify rBAB1_0245 to determine if this protein can breakdown fucose • Uptake assay data indicates that BAB1_0245 may be involved in uptake or breakdown of fucose.

*

6

6

• Wildtype B. abortus grows significantly better in minimal media supplemented with L-fucose

4

4

B. abortus Growth in GMM +/- Supplemental Carbon

* **

2 Log 10 brucellae/well

2 Log 10 brucellae/spleen

6

Wildtype Wildtype + 24mM glucose Wildtype + 24mM fucose

Acknowledgements

0

2

24

48

0

• Dr. Clay Caswell • Kellie King • Tristan Stoyanof • TRACSS Staff • NIH AI125958

4

8

4

Time Elapsed (hours)

Weeks Post-Infection

Log 10 brucellae/ mL

Figure F: Bacterial recovery from spleens of C57 mice infected with wildtype B. abortus (2308) or Δ bab1_0245. Spleens from infected mice were harvested at the indicated time and homogenized. Homogenates were serially diluted and plated on blood agar plates to determine CFUs.

Figure E: Survival of wildtype B. abortus (2308), Δ bab1_0245, and the reconstructed mutant (RC Δ bab1_0245) in bone-marrow derived macrophages from C57 mice. Macrophages are infected at an MOI of 100. Intracellular bacteria are determined via gentamicin protection, lysis of macrophages at indicated times, and serial dilutions on blood agar plates.

2

0

24

48

72

96

120

Time Elapsed (hours)

89

2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y