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An overexpression screen to determine how Legionella pneumophila
effector protein LegC7 disrupts Class C tethering Complex Formation
Chetan N. Hebbale*, Kevin O’Brien*, 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 Vincent Starai and Kevin O’Brien.
Acknowledgments
University of Georgia
Athens, GA
Microbiology
Department
Conclusions
pYES2/NTC
Yeast Homologous Recombination
Abstract
Future Directions
CORVET/HOPS Complex
• Essential for early-to-late endosome transition and endolysosomal
trafficking pathways
• Works to tether membranes, activate and proof-read SNARE
assembly and interact with Rab GTPases
• Set of universally conserved proteins from yeast to humans
Class C Core Vacuole/Endosome Tethering (CORVET):
• Binds to vps21 and promotes early endosomal fusion reactions
• vps21 is a Rab GTPase that recruits tethering complexes
• Tethering complex brings membranes into close proximity to
promote SNARE mediated membrane fusion
Homotypic Fusion and Vacuole Protein Sorting (HOPS):
• Binds to ypt7 and promotes late endosome/vacuole fusion
reactions
• ypt7 is also a Rab GTPase that recruits tethering complexes
pYES2NTC Restriction Digest
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
BY4742
BY4742 + LEGC7+
BY4742 + LEGC7N242I
N242I Point Mutation
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
Mutation of the 242nd amino acid resulted in
a toxicity reversal of LegC7deFelipe 2009
https://science.nichd.nih.gov/conf
luence/display/machner/Home
GFP – N242I Localization
• Deletions of vps41 and vps21 in BY4742 with the
N242I mutation resulted in a different phenotypic
expression than deletions of those genes exposed to
LegC7 - thus toxicity reversal is not due to
mislocalization but could indicate functional
interaction of LegC7 with one of these genes
Yeast Homologous Recombination
• Extremely toxic to develop constructs for these
genes, but it appears that we have proper gene
insertion for vps16 and vps21
• Isolate plasmid containing vps8 and vps41 genes that are
property inserted into pYES2NTC
• Transform LEGC7 into BY4742 wild type yeast containing
pYES2NTC overexpressing CORVET/HOPS gene of
interest and determine what effect it has on LegC7 toxicity
• Determine what binding interactions LegC7 may have
within the yeast endocytic pathway
2% Galactose
• Mutation of the 242nd amino acid from asparagine to isoleucine
• Does not induce trafficking defects – thus identifying a critical
domain of Leg C7 functionality
2% Glucose
• Forward and reverse primers are designed to amplify genes
of interest
• PCR product is transformed into BY4742 wild type yeast
with a cut pYES2NTC vector
• If there are more colonies with the PCR + cut vector versus
the cut vector alone, insertion was likely successful
PCR
Amplification
and Insertion
Screen
• pYES2NTC + gene insert is extracted from yeast cells
Yeast Plasmid
Prep
• Extracted plasmid is transformed into E.coli to amplify
copy number
• pYES2NTC plasmid is then purified from the E.coli
• Restriction digest is performed to confirm gene is
properly inserted
Bacterial
Transformation
and Mini-Prep
• pYES2NTC plasmid is transformed into BY4742 with
LegC7 and spot plated to see if the LEGC7 is still toxic
Yeast
Transformation
and Spot Plate
Kuwamayama, Hidekazu, "Cell Interaction“, 2012
• Method to over-express CORVET/HOPS genes
• Gene of interest is inserted into a digested plasmid – the gene
product contains 40 base pairs of homology to the plasmid
• Yeast cell machinery will recombine the gene into the sites of
homology on the plasmid
• Preferable method to standard cloning techniques because yeast
machinery is more efficient than gel extraction and ligation
CORVET HOPS
vps16 vps21
(5576 kb/5886 kb)
(2852 kb)
(778 kb)
• vps16 cut with EcoRV and PmeI
• vps21 cut with KpnI and PmeI
• vps16 and vps21 are likely to be correctly
inserted into pYES2NTC given the size of
fragments
GFP – N242I Localization
vps41∆
GFP + N242I DIC
vps8∆
vps21∆
GFP + LegC7
GFP +
LEGC7
GFP +
N242I
Yeast Endocytic Pathway
• LegC7 expression disrupts the localization of model cargo that
transits through the endosomal system
• LegC7 doesn’t disrupt alternate vacuolar targeting pathways
• It is hypothesized that one or more endosomal genes is required for
LegC7 toxicity O’Brien, Kevin. “The Legionella pneumophila effector protein,
LegC7, disrupts Class C Tethering Complex Formation”, 2014.
O’Brien, Kevin. “The Legionella pneumophila effector protein,
LegC7, disrupts Class C Tethering Complex Formation”, 2014.
Legionella pneumophila is a Gram-negative bacterial species that causes a
severe pneumonia known as Legionnaires’ disease. During infection, L.
pneumophila secretes nearly 300 effector proteins into host cells in order to
evade lysosomal degradation by remodeling the host’s membrane trafficking
pathways. One of these effector proteins, LegC7, has been shown to be lethal
upon expression in the budding yeast, Saccharomyces cerevisiae, by
disrupting normal vesicle trafficking and vacuole morphology. It is
hypothesized that LegC7’s toxicity is dependent on an interaction with one or
more endosomal genes due to its disruption of cargo localization that transits
through endosomes to the vacuole. I examine the relationship between LegC7
and the Class C Tethering Complexes – CORVET (class C Core
vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole
protein sorting). The CORVET and HOPS complexes have been identified as a
set of universally conserved proteins which are essential for early-to-late
endosome transition and endo-lysosomal trafficking pathways by tethering
membranes, activating and proof-reading SNARE assembly to drive
membrane fusion and interact with Rab GTPases. Using homologous
recombination, 4 different genes will be overexpressed in yeast: vps8, vps16,
vps21 and vps41. By assaying whether overexpression of CORVET/HOPS
subunits can suppress LegC7 toxicity we can determine which specific
subunit(s) LegC7 may have binding interactions with.

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An overexpression screen to determine how Legionella pneumophila effector protein LegC7 disrupts Class C tethering Complex Formation

  • 1. An overexpression screen to determine how Legionella pneumophila effector protein LegC7 disrupts Class C tethering Complex Formation Chetan N. Hebbale*, Kevin O’Brien*, 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 Vincent Starai and Kevin O’Brien. Acknowledgments University of Georgia Athens, GA Microbiology Department Conclusions pYES2/NTC Yeast Homologous Recombination Abstract Future Directions CORVET/HOPS Complex • Essential for early-to-late endosome transition and endolysosomal trafficking pathways • Works to tether membranes, activate and proof-read SNARE assembly and interact with Rab GTPases • Set of universally conserved proteins from yeast to humans Class C Core Vacuole/Endosome Tethering (CORVET): • Binds to vps21 and promotes early endosomal fusion reactions • vps21 is a Rab GTPase that recruits tethering complexes • Tethering complex brings membranes into close proximity to promote SNARE mediated membrane fusion Homotypic Fusion and Vacuole Protein Sorting (HOPS): • Binds to ypt7 and promotes late endosome/vacuole fusion reactions • ypt7 is also a Rab GTPase that recruits tethering complexes pYES2NTC Restriction Digest 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 BY4742 BY4742 + LEGC7+ BY4742 + LEGC7N242I N242I Point Mutation 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 Mutation of the 242nd amino acid resulted in a toxicity reversal of LegC7deFelipe 2009 https://science.nichd.nih.gov/conf luence/display/machner/Home GFP – N242I Localization • Deletions of vps41 and vps21 in BY4742 with the N242I mutation resulted in a different phenotypic expression than deletions of those genes exposed to LegC7 - thus toxicity reversal is not due to mislocalization but could indicate functional interaction of LegC7 with one of these genes Yeast Homologous Recombination • Extremely toxic to develop constructs for these genes, but it appears that we have proper gene insertion for vps16 and vps21 • Isolate plasmid containing vps8 and vps41 genes that are property inserted into pYES2NTC • Transform LEGC7 into BY4742 wild type yeast containing pYES2NTC overexpressing CORVET/HOPS gene of interest and determine what effect it has on LegC7 toxicity • Determine what binding interactions LegC7 may have within the yeast endocytic pathway 2% Galactose • Mutation of the 242nd amino acid from asparagine to isoleucine • Does not induce trafficking defects – thus identifying a critical domain of Leg C7 functionality 2% Glucose • Forward and reverse primers are designed to amplify genes of interest • PCR product is transformed into BY4742 wild type yeast with a cut pYES2NTC vector • If there are more colonies with the PCR + cut vector versus the cut vector alone, insertion was likely successful PCR Amplification and Insertion Screen • pYES2NTC + gene insert is extracted from yeast cells Yeast Plasmid Prep • Extracted plasmid is transformed into E.coli to amplify copy number • pYES2NTC plasmid is then purified from the E.coli • Restriction digest is performed to confirm gene is properly inserted Bacterial Transformation and Mini-Prep • pYES2NTC plasmid is transformed into BY4742 with LegC7 and spot plated to see if the LEGC7 is still toxic Yeast Transformation and Spot Plate Kuwamayama, Hidekazu, "Cell Interaction“, 2012 • Method to over-express CORVET/HOPS genes • Gene of interest is inserted into a digested plasmid – the gene product contains 40 base pairs of homology to the plasmid • Yeast cell machinery will recombine the gene into the sites of homology on the plasmid • Preferable method to standard cloning techniques because yeast machinery is more efficient than gel extraction and ligation CORVET HOPS vps16 vps21 (5576 kb/5886 kb) (2852 kb) (778 kb) • vps16 cut with EcoRV and PmeI • vps21 cut with KpnI and PmeI • vps16 and vps21 are likely to be correctly inserted into pYES2NTC given the size of fragments GFP – N242I Localization vps41∆ GFP + N242I DIC vps8∆ vps21∆ GFP + LegC7 GFP + LEGC7 GFP + N242I Yeast Endocytic Pathway • LegC7 expression disrupts the localization of model cargo that transits through the endosomal system • LegC7 doesn’t disrupt alternate vacuolar targeting pathways • It is hypothesized that one or more endosomal genes is required for LegC7 toxicity O’Brien, Kevin. “The Legionella pneumophila effector protein, LegC7, disrupts Class C Tethering Complex Formation”, 2014. O’Brien, Kevin. “The Legionella pneumophila effector protein, LegC7, disrupts Class C Tethering Complex Formation”, 2014. Legionella pneumophila is a Gram-negative bacterial species that causes a severe pneumonia known as Legionnaires’ disease. During infection, L. pneumophila secretes nearly 300 effector proteins into host cells in order to evade lysosomal degradation by remodeling the host’s membrane trafficking pathways. One of these effector proteins, LegC7, has been shown to be lethal upon expression in the budding yeast, Saccharomyces cerevisiae, by disrupting normal vesicle trafficking and vacuole morphology. It is hypothesized that LegC7’s toxicity is dependent on an interaction with one or more endosomal genes due to its disruption of cargo localization that transits through endosomes to the vacuole. I examine the relationship between LegC7 and the Class C Tethering Complexes – CORVET (class C Core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting). The CORVET and HOPS complexes have been identified as a set of universally conserved proteins which are essential for early-to-late endosome transition and endo-lysosomal trafficking pathways by tethering membranes, activating and proof-reading SNARE assembly to drive membrane fusion and interact with Rab GTPases. Using homologous recombination, 4 different genes will be overexpressed in yeast: vps8, vps16, vps21 and vps41. By assaying whether overexpression of CORVET/HOPS subunits can suppress LegC7 toxicity we can determine which specific subunit(s) LegC7 may have binding interactions with.