Selected to be presented at the 2014 CURO Symposium

1. A targeted and an unbiased screen for genetic suppressors of the Legionella pneumophila
effector protein LegC7
Chetan N. Hebbale*, Kevin M. O’Brien*, and Vincent J. Starai*†
Departments of *Microbiology and †Infectious Diseases, University of Georgia, Athens, GA 30602.
Background
This work is made possible by the National Science Foundation grant number
1062589, the Starai Lab grant from National Institutes of Health-Allergy and Infectious
Disease (R01-Al 100913), and the University of Georgia Microbiology and Infectious
Diseases Departments. Personal thanks to Vinny Starai and Kevin O’Brien.
Acknowledgments
University of Georgia
Athens, GA
Microbiology
Department
Conclusions
pYES2/NTC
Method – Unbiased Screen
Abstract
Legionella pneumophila is a Gram-negative bacterium that causes a severe
form of pneumonia known as Legionnaires’ disease. During infection, L.
pneumophila secretes nearly 300 effector proteins into host cells in order to
evade lysosomal degradation by modulating vesicle trafficking pathways. One
of these effector proteins, LegC7, has been shown to be toxic upon
expression in the budding yeast, Saccharomyces cerevisiae. Upon LegC7
expression, S. cerevisiae accumulates membranous structures reminiscent of
so called “class E” compartments, which result from defects in multivesicular
body function. The proteins which comprise the Class E VPS family are
members of the endosomal sorting complex required for transport proteins
(ESCRT) which are responsible for recognizing, sequestering and packaging
membrane proteins into vesicles for vacuolar degradation. Because the Class
E phenotype was produced in yeast during LegC7 expression, we hypothesize
that LegC7 interacts with one or more of the yeast Class E gene products. We
therefore continued a targeted screen of the yeast Class E genes by
transforming a plasmid encoding LEGC7 into yeast strains with deletions
of vps23, vps28, snf7, or bro1 and found that deletion of these genes did not
suppress LegC7 toxicity. We then performed an unbiased screen in an attempt
to find genetic suppressors of LegC7 toxicity using ethyl methanesulfonate
(EMS) mutagenesis to isolate a strain that exhibits a toxicity reversal due to a
genomic mutation. We will sequence the genome from this strain to identify
the gene products that LegC7 might require for toxicity.
Future Directions
Class E Vacuolar Protein Sorting
Multivesicular Body: receives and sorts ubiquitinated membrane
proteins from Golgi or plasma membrane to the vacuole
ESCRT (endosomal sorting complex required for transport): proteins
found on MVB that identify membrane proteins bound for destruction
and establish intraluminal vesicles containing these proteins
Class E Mutants Screened:
VPS23, VPS28, SNF7, BRO1
Western Blot:
LegC7
• LegC7 is highly toxic when
expressed in Saccharomyces
cerevisiae
• Causes an accumulation of
vesicular material and aberrantly
secretes CPY Invertase – resembles
Class E VPS Mutants
• pYES2/NTC (pYES) plasmid allows
for selection and expression
• pGAL1 : Promoter that induces
LegC7 expression if galactose is
present
• URA3: Involved in the
biosynthesis of uracil. BY4742
yeast will not grow in CSM-uracil
media without pYES being
present – ensures the plasmid
enters yeast
Results – Targeted Screen
Current Class E VPS Mutants:
SEY6210
vps23∆ + LEGC7 +
vps28∆
vps28∆ + LEGC7 +
snf7∆
snf7∆ + LEGC7 +
bro1∆
bro1∆ + LEGC7 +
SEY6210 +LEGC7+
vps23∆
Targeted screen yielded no new genetic
suppressors of LegC7
BY4742
BY4742 + LEGC7+
vps27∆
vps27∆ + LEGC7 +
Previous Class E VPS Mutants:
2% Glucose 2% Galactose
Results – Unbiased Screen
Legionella pneumophila
• Causative agent of Legionnaire's disease
• Obligate intracellular pathogen, utilizes a type IV
secretion system to inject effector proteins that
manipulate host physiology
Deletion of vps27 resulted in a partial
toxicity reversal of LegC7
EMS Mutant Strains
BY4742
β - EMS
BY4742 β–EMS plasmid
LEGC7+
α - EMS
2% Galactose
BY4742 α–EMS plasmid
BY4742
ε – EMS plasmid
BY4742 +LEGC7+
γ – EMS plasmid
Δ – EMS plasmid
2% Galactose
• Wild type yeast (BY4742) are treated with ethyl
methanesulfonate (EMS) - inducing random point mutations
throughout the genome
• LEGC7 plasmid is transformed into mutated yeast cells and
plated onto selective media containing galactose, any
colonies that grow are selected for further screening
• Growth could be due to a plasmid mutation or a genomic
mutation
EMS
Mutagenesis
• LEGC7 plasmid is extracted from mutated yeast cells
Yeast Plasmid
Prep
• Extracted LEGC7 plasmid is transformed into E.coli to
amplify copy number
• LEGC7 plasmid is then purified from the E.coli
Bacterial
Transformation
and Mini-Prep
• LEGC7 plasmid is transformed into BY4742 and spot
plated to see if the LEGC7 plasmid is still inhibitory
Yeast
Transformation
and Spot Plate
• EMS Mutants are grown up in media containing
galactose
• Proteins are separated using SDS PAGE and
transferred to nitrocellulose membrane
• Blot is probed with α-LegC7 serum
• Demonstrates if LegC7 is being expressed by
plasmids that are no longer inhibitory
Known
LegC7
ε
EMS
γ
EMS
Δ
EMS
• Because there is no expression of LegC7 from non-
inhibitory plasmids possible genetic mutations are:
1) Mutation in the GAL1 promoter region
2) Mutation resulting in a stop codon in LEGC7 ORF
θ
EMS
α LegC7
deFelipe 2009
https://science.nichd.nih.gov/conf
luence/display/machner/Home
GFP-Leg C7 Localization
GFP- LegC7 FM4-64
Deletion of Class E genes does not affect
LegC7 localization
Hansen, Annu. Rev. Cell Dev. Biol. 2012.
vacuole
Class E
compartments
vps4∆ + LEGC7+
did4∆ + LEGC7+
ESCRT – 0
Vps27,Hse1
ESCRT – I
Vps23, Vps28,Srn2,
Mvb12
ESCRT – II
Vps36, Snf8,
Vps25
ESCRT – III
Vps20, Snf7, Vps24,
Did4, Bro1
Class E VPS Mutants
1) Deletion of vps23, vps28, snf7 and bro1 do not result in a
toxicity reversal of LegC7
2) Deletion of vps27 is unique among the ESCRT pathway as
it is the only one that shows a reversal phenotype
3) Deletion of representative class E genes do not affect
LegC7 localization at the vacuole
EMS Mutagenesis
1) EMS strains α and β exhibited a toxicity reversal of LegC7
likely due to a genomic mutation
2) EMS strains γ, Δ, ε and θ exhibited a toxicity reversal of
LegC7 due to a plasmid mutation
Genomic Mutation Sequence:
• EMS strains α and β will have the whole genome
sequenced and screened for specific genetic mutations
• After mutations are identified they will be reconstructed in a
clean background and have LegC7 transformed to see if
there is a toxicity reversal
Over-Expression Screen:
• Perform an overexpression screen to locate any genes that
when overexpressed result in a decrease of LegC7 toxicity
in order to find a biochemical interacting partner with LegC7
2% Galactose