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

1. IDENTIFICATIONOF GENETIC
REGIONS INTHE YUK OPERON OF
BACILLUS SUBTILISTHATARE
DIFFERENTIALLY REQUIRED FOR
SECRETION OFYUKE, A HOMOLOG
TOTHEVIRULENCE FACTOR, ESXA,
IN MYCOBACTERIUMTUBERCULOSIS
Gen Selden Pine Crest School Harvard University

2. 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.

3. Tuberculosis
 Tuberculosis is a major infectious disease that
affects people world wide.
 According to theWorld 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.

4. Tuberculosis mortality
 This figure depicts the estimated tuberculosis mortality rates
ar0und the world in 2013

5. 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

6. YukE Secretion System
 The yuk operon is the conserved ESX system in
Bacillus subtilis and secretes the proteinYukE
 Encodes theYukE secretion machinery as well
 The function of the secretion machinery and the
secreted proteins in both the ESX andYukE
systems is not well understood
yuk operon with ESX homology

7. 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

8. 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 secreteYukE
 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

9. 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 inYukE secretion for variations in
molecular signatures in each of the four B. subtilis
backgrounds

10. 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 forYukE
 Probing for the cytosolic protein, SigA, served as a lysis and loading control to ensure
that the detection of secretedYukE was not due to cell lysis
 Blots were exposed to chemiluminescence to view the protein bands

11. Bacillus subtilis
 Wild type, single deletion strains, strains with the operon removed,
strains withYukE inserted at an endogenous location, and strains that
were a combination of the two were compared forYukE 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
*
* *
* *

12. 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 withYukE insertion
amyE
yukE
yhDGH
yukE
*
*
Operon deletion withYukE insertion

13. α-YueB
 Confirmation that the operon was successfully deleted in the desired
strains, ensuring that the only secretedYukE is coming from the yukE
inserted at an endogenous location

14. 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 ofYukE within the cells
 However, the focus of the results is on the
secretedYukE, sinceYukE is homologous to the
secreted M. tuberculosis virulence factor, ESXA.

15. 3610/PY79 Secretion
 As previous studies indicated,
3610 deletion strains secreted
YukE even with single
components of theYukE
secretion machinery not
present, confirming its
independence of the operon
forYukE 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.

16. After the first secretion assay
 The next step was to analyze the intermediate strains,
3610 cured and 168, forYukE 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.

17. 3610 cured/168 Secretion
 I found that 3610 cured
showed similarYukE 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 similarYukE
secretion patterns to those of
PY79, suggesting a similar,
strong dependence on the
secretion machinery forYukE
secretion.

18. ΔyukBA
 Analyzing the final single
deletion strains confirmed my
previous findings:
 3610 and 3610 cured have
similar secretion patterns –
they secreteYukE regardless
of the single deletion
 168 and PY79 have similar
secretion patterns – they
secrete much lessYukE even
when a single component of
the secretion machinery is not
present

19. After analyzing single deletions
 The next step was to analyzeYukE 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 ofYukE

20. 3610/PY79 ΔyukEDCBAyueB
amyE
yukE
 3610 secretedYukE 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 lessYukE
when the secretion machinery
was not present, suggesting
again a strong dependence on
the operon for secretion
yuk

21. 3610 cured/168 ΔyukEDCBAyueB
yuk
yhDGH
yukE
 3610 cured secretedYukE similar
to 3610, further suggesting it is
not dependent on the operon
(secretion machinery) forYukE
secretion
 Again, 168 secretedYukE similar
to PY79 – low levels ofYukE in the
absence of secretion machinery,
suggesting a strong dependence
on the presence of the operon
(secretion machinery) forYukE
secretion

22. Spβ Phage
 After analyzing the results and observing the
similarities betweenYukE 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, allowingYukE to
leak out of the cells in the absence of the yuk
secretion machinery, which could explain some of
the observed secretion patterns

23. Spβ Phage
 1 – 168WT
 2-4 – 168, no phage
 5 – PY79WT
 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 toYukE 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

24. Discussion
 3610 secretesYukE
independently of the operon
 PY79 exhibits strong
dependence on the presence of
the operon forYukE secretion
3610
168
PY79
3610
cured
 3610 cured secretesYukE independently of the operon
 The plasmid in 3610 is not responsible forYukE secretion
 168 secretesYukE similarly to Py79
Plasmid
removalGenetic alteration
Genetic alteration

25. 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 forYukE secretion, such
as the SP phage

26. 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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28. Acknowledgements
 Dr. Briana Burton, Associate Professor of
Molecular and Cellular Biology, Harvard University
 Bram Sterling, Graduate Student, Harvard
University
 The Burton Lab
 JenniferGordinier, Pine Crest School