This part of my PhD work was to figure out what a particular bacterial gene does. We knew that it was important for E. coli to cause disease, but we didn't understand how it did this. This presentation outlines how we figured out the role of this gene in E. coli.
Given to the Bacterial Ultra Group (BUG) of Singapore on 11 May 2017.
Gallbladder Double-Diverticular: A Case Report المرارة مزدوجة التج: تقرير حالة
The genotoxic potential of the RqlH helicase is curbed by RqlI activity in extraintestinal pathogenic Escherichia coli
1.
2. Escherich T. The intestinal bacteria of the
neonate and breast-fed infant. 1884. Rev
Infect Dis. 1988;10(6):1220-5. PubMed
PMID: 3060950.
Bacterium coli
commune
3.
4. GI tract
• non-pathogenic
• 70% of individuals carry B2 E. coli
• Fecal-oral route of transmission
Extraintestinal Pathogenic Escherichia coli
(ExPEC) inhabit various niches
Urinary Tract Infections (UTIs)
• majority caused by ExPEC
• ½ of all women during lifetime
• ~8 million bladder infections/year
• 250,000 pyelonephritis cases/year
Blood and brain infections
• 20-40% of cases of neonatal meningitis
• Primary cause of bloodstream infections
5. Intrahost transit of ExPEC is
from the gut to other sites
1) Gut colonization
2)Extraintestinal infection
Same pattern applies to other
extraintestinal niches, such as
the bloodstream
The gut is the primary
ExPEC niche
6. RqlI (RecQ-like helicase interactor) is required
for colonization of several host niches
• ΔrqlI exhibits defect in:
• Zebrafish pericardial cavity
• Zebrafish bloodstream
• Mouse urinary tract
• Mouse bloodstream
Wiles TJ, Norton JP, Russell CW, Dalley BK, Fischer KF, Mulvey MA. Combining quantitative
genetic footprinting and trait enrichment analysis to identify fitness determinants of a bacterial
pathogen. PLoS Genet. 2013;9(8):e1003716.
• Expressed in human UTIs
Hagan EC, Lloyd AL, Rasko DA, Faerber GJ, Mobley HL. Escherichia coli global gene
expression in urine from women with urinary tract infection. PLoS Pathog.
2010;6(11):e1001187.
7. The ability of the ΔrqlI mutant to colonize the
mouse gut is reduced in several ExPEC isolates
Russell CW, Mulvey MA. The Extraintestinal
Pathogenic Escherichia coli Factor RqlI
Constrains the Genotoxic Effects of the RecQ-
Like Helicase RqlH. PLoS Pathog.
2015;11(12):e1005317.
Gut Colonization
8. The ΔrqlI mutant exhibits a growth defect that is
accentuated by low oxygen conditions
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
RqlI
homology to DNA
protection protein
(DprA)
homology to molybde-
num cofactor carrier
protein
Helix turn helix
domain of the CRP
superfamily
9. The ΔrqlI mutant has an increased cell length,
suggesting induction of the SOS response
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
10. The ΔrqlI mutant has an increased cell length,
suggesting induction of the SOS response
DNA Damage SOS Response
• DNA Repair
• Error-Prone Replication
• Inhibition of Cell Division via SulA
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
11. The SOS response is induced in the ΔrqlI mutant
indicating that it experiences genotoxic stress
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
12. A genetic screen uncovers RqlH as the source of
genotoxicity in the ΔrqlI mutant
High LacZ activity = pink to white colony
Low LacZ activity = red colony
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
14. RqlH is a helicase with a PRTase domain,
and is encoded in an operon with RqlI
• RqlH is a RecQ homolog
• M. smegmatis RqlH unwinds DNA 3’ to 5’
• The phosphribosyltransferase (PRTase) domain is not required for unwinding
DNA
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
Ordonez H, Unciuleac M, Shuman S. Mycobacterium smegmatis RqlH defines a novel clade of bacterial
RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.
Nucleic Acids Res. 2012;40(10):4604-14.
ATPase
Helicase
Zn PRTase
15. Phosphoribosyltransferases add phosphoribose
groups to nucleobases and nucleotides
For example, Apt (adenine phosphoribosyltransferase):
Phosphoribosyltransferases are involved in:
• Nucleotide salvage pathways
• NAD biosynthesis
• Histidine biosynthesis
16. The ΔrqlHI DKO exhibits WT levels of SulA
expression and microaerobic growth
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
17. The ΔrqlHI DKO exhibits a WT phenotype
in vivo
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
ΔrqlHI DKO
ΔrqlI KO
Gut Colonization
18. The ΔrqlHI DKO exhibits a WT phenotype
in vivo
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
UTI
Sepsis
19. The PRTase domain of RqlH is the source of
toxicity in the absence of RqlI
1. Overexpress various RqlH proteins in the absence of RqlI
2. Measure growth (CFU/mL), cell lengths, and SOS induction (sulA-GFP)
3. Normalize all measurements to the empty vector control
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
ATPase
Helicase
Zn PRTase
DIVD663EVME
D663A
(empty vector control)
CFU/mL
1.00
0.01
1.00
0.98
0.46
0.16
Cell Lengths
WT
Long
WT
WT
WT
WT
sulA-GFP
No
Yes
No
No
No
No
RqlH
1-509
509-698
DIVD663EVME
D663A
None
Protein
20. The N-terminus of RqlI is required for
microaerobic growth while the HTH
domain can have inhibitory effects
Growth (norm to WT)
0.16
0.61
0.50
0.92
0.08
0.10
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
rqlI mutant carrying various plasmids during microaerobic growth
21. RqlH and RqlI physically interact in a manner
dependent on the PRTase domain of RqlH
75
50 IB: HA (RqlH)
IB: FLAG (RqlI)
IB: LacI
37
37
5%
input
IP: FLAG
(R
qlI)
IP: H
A
(R
qlH
)
5%
input
IP: FLAG
(R
qlI)
IP: H
A
(R
qlH
)
RqlH RqlH 1-509
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
22. Transcription of rqlHI is not affected by
oxygen levels
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
23. Gram-negatives tend to carry both rqlH and rqlI,
while Gram-positives tend to have only rqlH
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
Total=1062
Gram-negative
26
103
933
Total=1083
1079
4
Gram-positive
Both RqlH & RqlI
Only RqlH
Only RqlI
24. Model: RqlH and RqlI work together to modify
DNA, perhaps as part of an unknown repair
process
A
RqlI
RqlH
Hel
PRTase
B
resolution
RqlI
RqlH
Hel
PRTase
C
RqlH
Hel
PRTase
toxicity
SOS response
cell elongation
inhibition of growth
sensitivity to DNA damage
mutations
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
25. Model: RqlH and RqlI work together to modify
DNA, perhaps as part of an unknown repair
process
RqlI
Hel
RTase
RqlI
Hel
RTase
C
RqlH
Hel
PRTase
toxicity
SOS response
cell elongation
inhibition of growth
sensitivity to DNA damage
mutations
Remaining Questions
• Does the PRTase domain of RqlH modify DNA as hypothesized?
• What is the function of the RqlHI system?
• Does it do something similar to RecQ (e.g. block illegitimate
recombination, clear blocked replication forks, induce SOS)?
• Would a Gram-positive RqlH be toxic to a Gram-negative organism?
Russell CW, Mulvey MA. PLoS Pathog.
2015;11(12):e1005317
26. Acknowledgements
Mulvey Lab
Matt Mulvey
Adam Lewis
Alan Stenquist
Alex Chang
Alex Tran
Amanda Richards
Amelia Barber
Betsy Ott
Bijaya Dhakal
June Round
Kael Fischer
Adam Barker
Joel Griffitts
Mary Bronner
Tim Mosbruger
Others
Brittany Fleming
Grant Donovan
Irina Debnath
Kevin Nelson
Liz Enrico
Matt Blango
Paul Norton
Phung Chinh
Travis Wiles
Editor's Notes
Genetically diverse group
Effect nearly 9 million/year in US alone.
70-95% of UTIs, 50% of nosocomial UTIs,
~8 million bladder infections, 250,000 pyelonephritis cases
20-40% of neonatal meningitis cases,
19-40% of sepsis cases (2nd after Group B Strep in neonates and Stap aureus in adults) causing 40,000 deaths/year (127,000 severe sepsis cases in 2001)
#1 cause of bloodstream infections (35/100,000 population)
Antibiotic resistance increasing rapidly.
How is it that ExPEC colonize so many diverse niches? One way we are approaching this question is by using zfish.
536: pyelonephritis
24 h growth, shaking +/- O2, then titer
24 h growth, shaking
Aerobic growth
Microaerobic, 24 h growth
Remind them of the rqlI SKO phenotype
Remind them of the rqlI SKO phenotype
Sepsis: IP injection, 12 h time point, 10^7
UTI: 3 d time point, 10^8
MG1655 attTn7::PsulA-GFP grown 6 h in M9 with arabinose (pBAD33 based vectors)
F11 rqlI mutant grown 24 h microaerobically with different rqlI expression vectors (pRR48-based)
MG1655 carrying pRR48 and pBAD33 plasmids. Grown 4 h, then induced, then 1 h more before lysis and CoIP