Gen Selden Sigma Xi 2015

IDENTIFICATION OF GENETIC REGIONS IN THE YUK OPERON OF BACILLUS SUBTILIS THAT ARE DIFFERENTIALLY REQUIRED FOR SECRETION OF YUKE, A HOMOLOG TO THE VIRULENCE FACTOR, ESXA, IN MYCOBACTERIUM TUBERCULOSIS

Gen Selden Pine Crest School Harvard University

Infectious Diseases

Tuberculosis Second greatest

killer worldwide due to a single infectious agent (WHO, 2014)

2012 – 8.6 million people infected, 1.3 million died

Bacillus subtilis

Model organism for pathogenic bacteria Conserved ESX secretion system

ESX secretion system in M. tuberculosis

YukE Secretion System

YukE – the conserved ESX system in Bacillus subtilis Encoded by the yuk operon

The function of the secretion machinery and the secreted proteins in both the ESX and YukE systems is not well understood

yuk operon with ESX homology

Bacillus subtilis

3610 – “wild-type” YukE secretion

independent of the secretion machinery

PY79 – “domesticated” YukE secretion

dependent on the secretion machinery

168 and 3610 cured – intermediate Have not yet been

analyzed for differences in secretion

3610

168

PY79

3610 cured

Plasmid removal

Genetic alteration

Genetic alteration

Purpose

“Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world.” (Forrellad et al.)

To analyze differences in YukE secretion for variations in molecular signatures in each of the four B. subtilis backgrounds

Methods

Secretion assay Cultures grown in LB media at 37oC Cells were collected and normalized based on cell density measured at OD600nm Cell pellet and supernatant were separated

Protein precipitation Proteins in the supernatant were precipitated using trichloroacetic acid Centrifugation at 4oC at 16,000 rpm separated the proteins and the remaining

liquid Cell lysates

Frozen cell pellets were lysed with lysis buffer and heated at 80oC to release the proteins within the cell

Semi-dry method of western blotting Secretion was observed by blotting the proteins in the cell pellet and the

secreted proteins and probing for YukE Probing for the cytosolic protein, SigA, served as a lysis and loading control to

ensure that the detection of secreted YukE was not due to cell lysis Blots were exposed to chemiluminescence to view the protein bands

Bacillus subtilis

Wild type and deletion strains were compared for YukE secretion

3610 wt

3610 ΔyukE

3610 ΔyukD

3610 ΔyukC

3610 ΔyukBA

3610 ΔyukEDCBAyueB

3610 amyE::yukE

3610 ΔyukEDCBAyueB; amyE::yukE

3610 cured wt

3610 cured ΔyukE

3610 cured ΔyukD

3610 cured ΔyukC

3610 cured ΔyukBA

3610 cured ΔyukEDCBAyueB

3610 cured yhDGH::yukE

3610 cured ΔyukEDCBAyueB; yhDGH::yukE

168 wt

168 ΔyukE

168 ΔyukD

168 ΔyukC

168 ΔyukBA

168 ΔyukEDCBAyueB

168 yhDGH::yukE

168 ΔyukEDCBAyueB; yhDGH::yukE

PY79 wt

PY79 ΔyukE

PY79 ΔyukD

PY79 ΔyukC

PY79 ΔyukBA

PY79 ΔyukEDCBAyueB

PY79 amyE::yukE

PY79 ΔyukEDCBAyueB; amyE::yukE

*

* *

* *

yuk

amyE

yukE

yhDGH

yukE

Bacillus subtilis genome

*

*

α-YueB

Confirmation that the operon was successfully deleted

3610/PY79 Secretion 3610

168

PY79

3610 cured

Plasmid removalGenetic alteration

Genetic alteration

3610 cured/168 Secretion3610

168

PY79

3610 cured

Plasmid removalGenetic alteration

Genetic alteration

ΔyukBA 3610

168

PY79

3610 cured

Plasmid removalGenetic alteration

Genetic alteration

3610/PY79 ΔyukEDCBAyueB

yuk

amyEyukE

3610 cured/168 ΔyukEDCBAyueB

yuk

yhDGH

yukE

Spβ Phage

1 – 168 WT 2-4 – 168, no phage 5 – PY79 WT 6 – PY79 with the phage

Lysis problem in lane 6 inconclusive results High levels of secretion

seen in PY79 could be due to YukE escaping from inside the cell

Doesn’t explain high levels of secretion seen in 168 without the phage

1 2 3 4 5 6

Discussion

3610 secretes YukE independently of the operon

PY79 exhibits strong dependence on the presence of the operon for YukE secretion

3610

168

PY79

3610 cured

3610 cured secretes YukE independently of the operon The plasmid in 3610 is not responsible for YukE

secretion 168 secretes YukE similarly to Py79

Plasmid removalGenetic

alteration

Genetic alteration

Future research

168 and PY79 should be analyzed for genetic differences in the future

Determination of genetic differences in B. subtilis may be able to help us target these areas in pathogenic bacteria and possibly inhibit or reduce secretion of the virulent proteins

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Acknowledgements

Dr. Briana Burton, Associate Professor of Molecular and Cellular Biology, Harvard University

Bram Sterling, Graduate Student, Harvard University

The Burton Lab Jennifer Gordinier, Pine Crest School