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

Infectious diseases are caused by pathogenic organisms such as bacteria, viruses, parasites, and fungi.

They can be spread through human interaction, through contact with animals or insects, or through contaminated food and water.

Infectious diseases kill more people worldwide than any other single cause.

Tuberculosis

Tuberculosis is a major infectious disease that affects people world wide.

According to the World Health Organization, it is the second greatest killer worldwide due to a single infectious agent (WHO, 2014). In 2012 8.6 million people were infected

with tuberculosis, and 1.3 million of those people died from the infection.

Tuberculosis mortality

This figure depicts the estimated tuberculosis mortality rates ar0und the world in 2013

Bacillus subtilis

Nonpathogenic model organism for pathogenic bacteria

Conserved ESX secretion system The ESX system was first discovered in M. tuberculosis Secretes two proteins, EsxA and EsxB that appear to be

required for the virulence of tuberculosis

ESX secretion system in M. tuberculosis

YukE Secretion System

The yuk operon is the conserved ESX system in Bacillus subtilis and secretes the protein YukE Encodes the YukE secretion machinery as well

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

4 different genetic backgrounds of B. subtilis:

3610 – “wild-type” Previous research showed that

YukE secretion was independent of the secretion machinery

PY79 – “domesticated” Previous research showed that

YukE secretion was 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

Genetic alterations

3610 is considered “wild type” because it is genetically closest to Bacillus subtilis that would be found in the soil

3610 cured was created by removing a plasmid from the genome of 3610 because it was suspected that this plasmid encoded an alternate secretion system that could secrete YukE

Small deletions and insertions occurred in 3610 and resulted in the formation of 168

Two large insertions and four large deletions in the 168 genome resulted in the formation of PY79

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.)

The purpose of my research was 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, single deletion strains, strains with the operon removed, strains with YukE inserted at an endogenous location, and strains that were a combination of the two 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

*

*

This figure represents the B. subtilis genome and the endogenous locations at which yukE was inserted into the genome

Wild type with YukE insertion

amyE

yukE

yhDGH

yukE

*

*

Operon deletion with YukE insertion

α-YueB Confirmation that the operon was successfully deleted in

the desired strains, ensuring that the only secreted YukE is coming from the yukE inserted at an endogenous location

Additional Information

SigA is a cytosolic protein and was used as a loading and lysis control to ensure secretion results are not due to cell lysis.

The pellet samples were used to observe and confirm production of YukE within the cells

However, the focus of the results is on the secreted YukE, since YukE is homologous to the secreted M. tuberculosis virulence factor, ESXA.

3610/PY79 Secretion

As previous studies indicated, 3610 deletion strains secreted YukE even with single components of the YukE secretion machinery not present, confirming its independence of the operon for YukE secretion.

However, PY79 exhibited low levels of secretion when components of the secretion machinery were deleted, suggesting a strong dependence on the presence of the operon for secretion.

After the first secretion assay The next step was to analyze the intermediate

strains, 3610 cured and 168, for YukE secretion in order to determine which two of the four B. subtilis backgrounds have the most similar secretion patterns.

In the future, the wild type genomes of the two similar B. subtilis backgrounds identified in this study can be analyzed for specific genetic differences that may be responsible for the observed differences in secretion.

Eventually, due to the homology between B. subtilis and M. tuberculosis, the goal would be to apply this knowledge of genetic differences to M. tuberculosis in order to further research on new drugs to fight the tuberculosis disease.

3610 cured/168 Secretion

I found that 3610 cured showed similar YukE secretion patterns to those of 3610, suggesting only a slight dependence on the presence of the secretion machinery for YukE secretion.

In addition, 168, which is genetically more similar to PY79 than either 3610 or 3610 cured, exhibited similar YukE secretion patterns to those of PY79, suggesting a similar, strong dependence on the secretion machinery for YukE secretion.

ΔyukBA

Analyzing the final single deletion strains confirmed my previous findings:

3610 and 3610 cured have similar secretion patterns – they secrete YukE regardless of the single deletion

168 and PY79 have similar secretion patterns – they secrete much less YukE even when a single component of the secretion machinery is not present

After analyzing single deletions The next step was to analyze YukE secretion

in 3 different strains: deletion of the entire yuk operon, insertion of yukE at an endogenous location, and the combination of these two strains – deletion of the yuk operon complemented with yukE insertion.

These results better represent the dependence of 3610, 3610 cured, 168, and PY79 on the yuk secretion machinery for secretion of YukE

3610/PY79 ΔyukEDCBAyueB

amyEyukE

3610 secreted YukE at similar levels when yukE was inserted into both the wild type strain and and into the operon deletion strain, suggesting that 3610 is completely independent of the operon for YukE secretion

PY79 secreted much less YukE when the secretion machinery was not present, suggesting again a strong dependence on the operon for secretion

yuk

3610 cured/168 ΔyukEDCBAyueByuk

yhDGH

yukE

3610 cured secreted YukE similar to 3610, further suggesting it is not dependent on the operon (secretion machinery) for YukE secretion

Again, 168 secreted YukE similar to PY79 – low levels of YukE in the absence of secretion machinery, suggesting a strong dependence on the presence of the operon (secretion machinery) for YukE secretion

Spβ Phage

After analyzing the results and observing the similarities between YukE secretion in 168 and YukE secretion in PY79, I decided to analyze the effect of the Spβ phage on secretion

The Spβ phage is present in 3610, 3610 cured, and 168, but is absent in PY79

It was speculated that the phage might be creating holes in the cell wall, allowing YukE to leak out of the cells in the absence of the yuk secretion machinery, which could explain some of the observed secretion patterns

Spβ Phage

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

Lysis problem in lane 6 led to inconclusive results High levels of secretion seen in

PY79 (6) could be due to YukE escaping from inside the cell as a result of lysis

However, the lysis problem does not explain the unexpected high levels of secretion seen in 168 without the phage (2-4)

1 2 3 4 5 6

Secreted

Pellet

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

The wild type genomes of 168 and PY79 should be analyzed for genetic differences in the future

In addition, more research should be done on possible alternate routes for YukE secretion, such as the SP phage

Future Research

Determination of the aforementioned genetic differences in 168 and PY79 may be able to help researchers target these areas in pathogenic bacteria such as M. tuberculosis in order to inhibit or reduce secretion of the virulent proteins

Specific inhibition of or reduction in virulence could contribute to the creation of new drugs to fight the disease

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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