Download Tn917 insertion site in the 2C4 mutant

Fast Killing of C. elegans by Enterococcus
faecium is mediated by Hydrogen Peroxide
Terence Moy
Lab meeting #2
February 25, 2003
What are Enterococci?
-Gram-positive enteric bacteria
-they are considered a “hardy” bacteria
-2nd or 3rd leading source of nosocomial infections
-causes endocarditis, bacteremia, urinary-tract infections,
surgical wound infections
-are intrinsically resistant to many antibiotics & they
are capable of acquiring antibiotic resistance
-increasingly, enterococci are becoming vancomycin-resistant
- 80% of infections are caused by E. faecalis and
15-20% are caused by E. faecium
- VREF (Vancomycin Resistant E. faecium): a larger % of
clinical isolates are VRE compared to faecalis
E. faecium that is grown anaerobically
quickly kills C. elegans
Killing Assay Protocol:
1. Grow E. faecium in liquid BHI media at 37° (5h-O/N)
2. Spread culture onto BHI plates
]
ANAEROBIC
Conditions
3. Incubate plates at 37° O/N in a GasPack
4. Remove plates from GasPack, cool to RT
5. Add C. elegans. Watch’em die.
Atmospheric
Air
]
Atmospheric
Air
grown anaerobically
Killing of C. elegans by E. faecium strain E7
100
90
80
Percent Killing
70
60
E7-1
50
E7-2
OG1RF-1
40
30
20
10
0
0
100
200
300
400
Time (minutes)
Mobility
decreased
500
600
Characteristics of E. faecium Fast Killing
 Growth under Anaerobic conditions leads to toxicity
 Killing efficiency depends on bacterial cell density,
the bacterial strain, & the age of the nematode
 Toxicity can be inactivated by autoclaving the plate
 Toxicity does not required direct bacteria to worm contact;
The toxin is diffusible.
The toxin decreases worm mobility. Most worms die within
24 hours upon incubation with the toxin.
Goal of my project: Identify the mechanism of fast killing.
Approach: Transposon mutagenize E. faecium. Identify
and characterize mutants produce more toxin or less toxin.
Transposon Mutagenesis of E. faecium SE34
• plasmid pTV1OK was transformed into SE34
• pTV1OK contains Tn917 marked w/ Ermr,
kanr on vector backbone, and a
temperature sensitive origin of replication
• SE34 w/ pTV1OK is maintained at 28° on
BHI-kan 2 mg/ml, erm 50 µg/ml
• Transposition is selected at 46-47° on BHI-erm 2 µg/ml
• Transposition frequency is 4  10-4
• Qbot transferred approx. 13,000 colonies to 35- 382 well plates
containing BHI-erm50+15% glycerol, cultures were grown
O/N at 45°, and then stored at -80°
Screening Method
Bacteria:
1. liquid cultures were inculated by frogging frozen stocks and
grown at 37° O/N under semi-aerobic conditions
2. saturated cultures were either frogged to 96 well plates or 20 µl
of culture was spotted onto 24 well plates c BHI-erm50-agar,
3. Each strain was spotted onto two plates, one was incubated
aerobically and the other anaerobically (in a gaspak)
Worms: approx 500,000 L2 worms and 100,000 L4/young adults
were used to screen a 382 well plate. Worms were resuspended in
M9 buffer and pipetted onto each well.
After ~ 2 hours, I examined the Anaerobically grown plates and
I identified the bacteria that did not impair worm mobility.
After ~ 5 hours, I examined the aerobically grown plates and identified
the bacteria that impaired worm movement
Results of the Screen
3,120 E. faecium clones were screened
3 strains were less toxic to C. elegans
2F22
3C23
11M12
1 strain was more toxic to C. elegans
2C4
Mutants are altered in C. elegans killing
Strain
Percent of Worms Alive after 24 hrs
(number of worms)
Bacterial Growth Condition
Anaerobic
Aerobic
SE34
0% (n=102)
100% (92)
2C4
1% (102)
35%* (95)
2F22
31%* (99)
100% (95)
E. faecalis
VS583
100% (109)
99% (98)
Tn917 insertion site in the 2C4 mutant
Tn917
p1955 is an amino acid aminotransferase
p1954 NADH peroxidase (npr), a 1379 bp gene
Tn917 is inserted 16 bp upstream of the npr start codon
p1953 is similar to the transposase of IS1077E, ISSmu1, & IS861
p1952 is a conserved hypothetical ORF of unknown function
p1947 thru p1951 are ORFs encoded in the opposite direction of p1954
NADH peroxidase reduces
hydrogen peroxide to water
NADH+H+ + H2O2
Npr
NAD+ + 2H2O
Npr is a FAD containing reductase with a
sulfonic acid derivative of cysteine at its active site
In contrast, heme containing Catalase converts 2H2O2
to 2H2O and O2 (gas). E. faecalis possess a catalase
while E. faecium does Not
In E. faecalis, Npr activity is induced by aerobic growth
and a putative OxyR binding site upstream of npr may
regulate its expression
Tn917 insertion site in the 2F22 mutant = 3C23
Tn917
p172 Fur family (Ferric Uptake Regulator)
similar to B. subtilis PerR (peroxide stress response regulator)
p173 NADH oxidase (nox)
nox is a 1305 bp gene, Tn917 is inserted at position +925 bp in 2F22
p174 is a 300 bp hypothetical ORF with no homology to known genes
p175 is a 567 bp ORF w/ homology to MarR & NapB
p176 & p177 are putative tetracycline efflux pumps
NADH Oxidase reduces oxygen to form
water or hydrogen peroxide
2NADH+H+ + O2
NADH+H+ + O2
Nox-2
Nox-1
2NAD+ + 2H2O
NAD+ + H2O2
Nox contains a cysteine-sulfenic acid redox center & a FAD
A S. pneumoniae nox-2 mutant grows poorly in aerobic conditions
unless glucose is present. This mutant & a S. mutans mutant are
attenuated in mouse infection models.
E. faecalis V583 has 3 nox genes.
E. faecium DO has 7 nox-like genes
The metabolic role of the water forming
NADH oxidase
Nox-2
The aerobic growth of 2C4 & 2F22 is impaired
Aerobic
Anaerobic
SE34
2C4
2F22
Growth of the mutants under semi-aerobic conditions
1.00E+10
1.00E+09
CFU/ml
1.00E+08
1.00E+07
SE34
2C4
1.00E+06
2F22
1.00E+05
0
10
20
30
40
Time (hours)
50
60
70
80
Hydrogen Peroxide levels in culture supernatants
Strain
Culture conditions- (culture density in CFU/ml)
Anaerobic to
semi-aerobic aerobic
Anaerobic aerobic (2hrs)
SE34
BDL<30µM
(1.1E9)
0.62 mM
(8.8E8)
BDL
0.35 mM
(1.1E9)
2C4
0.22 mM
(9.4E8)
1.71 mM
(3.8E8)
BDL
0.59 mM
(1.5E9)
2F22
BDL
0.04 mM
(2.2E8)
BDL
0.12 mM
(1.4E9)
E. fs VS583
BDL
BDL
(3.8E9)
BDL
BDL
(2.8E9)
Killing of C. elegans by Hydrogen Peroxide
killing curve from Jansen et al. (2002) Infect Immun 70:5202.
Sources of Hydrogen Peroxide
1. NADH oxidase-1
2. Dismutation of superoxide
3. oxidoreductases of glucose, alcohols, malate, choline, amino acids,
sugars, nucleosides, lactate, etc
4. -glycerophosphate oxidase (GlpO)
Glycerol
metabolism in
E. faecalis
M. Huycke
Physiology of
Enterococci
Addition of Glucose to E. faecium cultures
lowers Hydrogen peroxide concentrations
Strain
Liquid Culture conditions
aerobic
Anaerobic (o/n) to aerobic (2h)
no glu +0.5% glu
no glu
+0.2% glu
SE34
0.63 mM
BDL (<50µM)
2C4 (npr)
1.8 mM
BDL
2F22 (nox)
BDL
BDL
1.17 mM
BDL (<0.3mM)
Effect of Glycerol and Glucose of the toxicity
of E. faecium towards C. elegans
Glucose decreases the toxicity of E. faecium
0.5-1% glucose prevents the toxicity of E7 and 2C4 grown
anaerobically & delays 2C4’s effect when grown aerobically
Glycerol increases the toxicity of E. faecium (mobility assay):
E7 and 2F22 become toxic when grown aerobically in the
presence of 0.2% glycerol
The onset of the toxicity from 2C4 grown aerobically occurs
earlier in the presence of 0.02% glycerol
Sucrose (1.7%) prevents toxicity of E7, delays 2C4’s effect
In contrast, addition of sorbitol, DTT, DMSO, succinate,
formate, or NaCl does not effect toxicity
Catalase rescues E. faecium Toxicity
*Culture supernatants from SE34 is toxic to C. elegans when the
bacteria are grown aerobically but not when grown semi-aerobically
Addition of 150 U/ml catalase rescues effect of faecium toxicity
**qualification: fresh, untreated BHI liquid can kill C. elegans
*** Fast killing assay on plates ± 1,000 U/ml catalase
strain
SE34
2C4
2F22
Percent of live worms after 26.5 hrs (number of worms)
reg AN plates
+ catalase
62% (21)
100% (43)
14% (22)
100% (24)
94% (31)
100% (39)
The 11M12 mutant is less toxic to C. elegans
In mutant screen- mobility assay:
> 75% of L2 worms retain normal mobility in presence of 11M12
> 50% of adult worms retain normal mobility “
“
Versus WT toxin producing strain which causes
complete immobility in >99% L2 worms and ~70% of adult worms,
the adult worms continue to move do so at a much slower pace
• Sequencing the Tn917 insertion site in the 11M12 mutant
was problematic.
Tn917 insertion site in 11M12 is near a copA-like gene
11M12
copA
tblastx using NCBI produced one hit:
S. epidermidis copA score = 126 bits
expect = 10-27
The translated 11M12 ORF aligns to the N terminus
of a E. faecalis putative cation transporter
11M12
EF0758
CopA is a Copper influx pump
Copper can generate hydroxyl radicals
from hydrogen peroxide
O2- + Cu2+
H2O2 + Cu+
O2 + Cu+
•OH + OH- + Cu2+
Hydrogen peroxide production is decreased in
the 11M12 mutant
strain
SE34
11M12
SE34
11M12
concentration of H2O2
17 hrs aerobic culture
1.5-1.6 mM
BDL ( < 0.25 mM)
15 hr Anaerobic culture
followed by 2 hrs of aeration
0.8 mM
0.5-0.6 mM
Summary of the Evidence that E. faecium kills
Worms with Hydrogen Peroxide
1. The E. faecium toxin is diffusible and does not require direct
contact with the bacteria.
2. The NADH peroxidase mutant produces more H2O2 and
has enhanced C. elegans killing activity
3. The NADH oxidase and the potential Copper transport mutants
produce less H2O2 and have decreased C. elegans killing activity
4. The concentration of H2O2 produced by WT faecium is sufficient
to kill C. elegans. The timing of the toxicity and effect on worm
mobility by the faecium toxin can be duplicated with purified H2O2.
5. Glycerol stimulataes faecium killing while
Glucose inhibits faecium killing.
6. Changes in Oxygen levels leads to faecium toxin production
7. Addition of Catalase completely rescues faecium toxicity
Possible Reasons for Hydrogen Peroxide Production
1. Byproduct of oxidase reactions
2. Deleterious consequence of aerobiosis
3. Inhibition the growth of competing bacteria
4. Promotes pathogenicity: host tissue damage, induce
an inflammatory response or interfere with an immune response
5. The H2O2 forming NADH oxidase (nox-1) may protect against
reactive oxygen species. Alternatively, its function may be to
actively produce hydrogen peroxide.
6. Acts as a signaling molecule?
Young Larval C. elegans feeding off of
Enterococci appear to be Starving
Adult C. elegans feeding off of E. faecalis or E. faecium does not
give rise to viable progeny. Progeny does not develop past the
L2-L3 stage.
I plated E. faecium strains SE34, 2C4, & 2F22
E. faecalis strain VS583
B. subtilis (didn’t grow well because of antibiotics)
I added either eggs, synchronized L1, L2, or young adults
Development of the worms
Eggs grown on E. coli OP50 or B. subtilis at 25 degress
after 24 hrs, reach L2-L3 stage
after 48 hrs, become young adults laying eggs
and L1 progeny are present
after 72 hrs, they are old adults, progeny are L1-L3 stage
Eggs grown on Enterococcus resemble eggs grown on
bacteria free media
after 1 day, reach L1
after 3 days, mostly L2
stay L2-L3 for up to 20 days
There is some killing of young larval worms on E. faecalis
but the number of worms alive on E faecium is similar to the
number on bare media
Future Plans
1. Repeat killing assays w/ catalase, the 11M12 mutant, other mut.
2. Rescue the 2C4 and 2F22 mutant phenotypes by expressing
plasmid encoded npr and nox genes.
3. Clone the SE34 copA locus.
4. Testing the 2C4 and 2F22 mutants in a mouse sepsis model in
collaboration with Joahnnes Huebner
5? Produce recombinant NADH oxidase. Determine if it produces
water or hydrogen peroxide?
6? Determine the effect of changing oxygen levels on the
expression of npr & nox?
Acknowledgments
Fred Ausubel
Lefterios Mylonakis
Danielle Garsin
Joahnnes Huebner
Markus
Stephanie
Ausubelites