1. Role of a Moraxella
catarrhalis Two Component
System in Polymyxin B
Resistance
Laura M. Jenkins
Dr. Randall Harris, Thesis Advisor
School of Natural Sciences and Mathematics
Department of Biology
Claflin University
Orangeburg, South Carolina
5. Moraxella catarrhalis
• Colonizes nasopharynx
soon after birth
– 75% of children are
colonized by age 2
• Colonization decreases
with age
Source: Unknown
6. Upper Respiratory Tract Infections
(URTIs)
• Respiratory viruses
– release inflammatory mediators
– increase bacterial adherence to
epithelial cells
– decrease immune system efficiency
– prevent ET from equalizing middle
ear pressure with environmental
pressure
– destroy ET epithelium
7.
8. Otitis Media
• Most commonly diagnosed
pediatric disease
– ¾ of children under the age of 3
– 20 million doctor visits annually in
the US
10. Zasloff, J. Antimicrobial peptides of multicellular
organisms. Nature. 415 (24): 389-395
(Mammalian cell)
w/ negatively charged
lipopolysaccharides
11. Bacterial Response to
CAMP Attacks
• Developed mechanisms that
sense and respond to protein
misfolding caused by
peptide-induced cell wall
stress
– Periplasmic Stress Sensor DegS
12. Escherichia coli Periplasmic
Stress Sensor DegS
• Encodes a serine protease that
senses periplasmic stress in
E. coli
• Transmits signals across the
cytoplasmic membrane
• Controls expression of genes for
maintaining the integrity of
periplasm
13. CAMP of Choice: Polymyxin
B (PxB)
• Antibiotic produced by Bacillus
polymyxa
• Contains a cyclic amphipathic
heptapeptide modified by an
isooctanoyl group
• Net charge of +5
14. Polymyxin B sensitive M. catarrhalis mutant
-polymyxin B +polymyxin B
degS+
degS- degS-degS+
Source: Dr. Randall Harris, 2012
One polymyxin B sensitive strain had a mutation in a gene
whose product was 58% homologous to Escherichia coli DegS.
15. Summary of degS Results
• Deletion of the degS gene in MCAT
RH4 or 035E (using the RH4 ΔdegS
fragment) did not cause increased
sensitivity to PxB.
• The 035E degS transposon mutant,
from previous data, showed small but
reproducible sensitivity to PxB than
035E after retesting it.
16. Previous Laboratory Data
• M. catarrhalis was exposed to sub lethal PxB
concentration.
• 211 M. catarrhalis genes were identified by
microarray analysis whose gene expression
was altered by at least 1.5 fold after peptide
exposure.
• One gene MCR_180 showed 1.70 fold increase
in expression after PxB exposure. It is
predicted to encode a response regulator in
the OmpR subfamily that forms the second
component of two component systems.
17. Two Component System (TCS)
ATP
ADP
P
Gram Negative Bacterial
Cell Interior
1. Autophosphorylation: Upon receiving the proper signal, the C
terminal domain of the HPK transfers the γ–phosphoryl group to the
side chain of a conserved histidine within the C-terminal domain.
18. Two Component System (TCS)
P
Gram Negative Bacterial
Cell Interior
2. Phosphotransfer: RR catalyzes the transfer of this phosphoryl group
from the phospho-His residue in the HPK to the side chain of a
conserved aspartate residue within the receiver domain.
19. Two Component System (TCS)
Gram Negative Bacterial
Cell Interior
P
RR phosphorylation enhances the binding of RR to its specific recognition site
within the promoters of regulated genes and ultimately leads to a particular
response that in pathogenic bacteria often contributes to virulence.
20. Hypothesis
It is hypothesized that 1) deletion of
the RR and/or HPK genes will result in
increased sensitivity to PxB and 2) a
set of M. catarrhalis genes is
controlled by the two component
system in response to PxB exposure.
M. catarrhalis TCS
21. Experimental Goal #1
Determine the in vitro
susceptibility of defined
M. catarrhalis RH4 TCS
mutants to PxB
23. Experiment 1. TCS Recombinant Gene Construction
A = Purified M. catarrhalis gDNA
B = Ladder
C = kanR gene w/ primers 1 & 2
D = hpk upstream flanking
DNA w/ primers A & B
E = hpk downstream flanking
DNA w/ primers C & D
A
B
C
D
kanR1
kanR2
Upstream flanking
DNA of M. catarrhalis
RH4 hpk gene
Downstream flanking
DNA of M. catarrhalis
RH4 hpk gene
kanR selectable marker
PCR 1
Fig. 1. Verification of
fragment creation by
PCR & AGE.
400bp
500bp
A B C D E
kb
10
1.5
1
0.7
0.5
1000bp
~50Kbp
0.3
24. Experiment 1. TCS Recombinant Gene Construction
Overlap PCR: Mix three complementary fragments (no primers)
Purification PCR: Add A and D primers
A
Source: http://openwetware.org/wiki/PCR_Overlap_Extension
B
C
D
kanR1
kanR2
Upstream flanking
DNA of M. catarrhalis
RH4 hpk gene
Downstream flanking
DNA of M. catarrhalis
RH4 hpk gene
kanR selectable marker
PCR 1
PCR 2A
PCR 2B
A
D
26. M. catarrhalis Natural Transformation
5 ml of the overlap constructs were mixed with a colony of M. catarrhalis RH4 on BHI agar
making a slurry. The plates were incubated overnight 37oC with 5% CO2. Following the growth
of the bacteria, a wire loop was used to streak for isolation onto BHI agar with 20 mg/ml of
kanamycin. Plates were incubated overnight at 37oC with 5% CO2. Genomic DNA was isolated
from M. catarrhalis RH4.
27. Allelic Exchange Confirmation
1900bp (hpk-)
2500bp (hpk)
1900bp (hpk/rsr-)
3000bp (hpk/rsr)
Figure 2. Agarose gel electrophoresis of PCR confirming hpk and hpk/rsr mutant
construction. Lanes 1 and 6, DNA Ladder. Lane 2, hpk gene. Lane 3, hpk::KnR. Lane 4,
hpk/rsr genes. Lane 5, D (hpk/rsr)::KnR.
28. Figure 3. Organization of the Two Component System MCR_0179 (histidine protein kinase,
hpk) and MCR_0180 (response regulator, rsr) in wild type, and rsr, hpk and hpk/rsr deletion
mutants. The DNA flanking the 5’ end or 3’ end of each gene was amplified with A and B or C
and D primer pairs respectively. The Tn903 kanamycin resistance (KnR) gene in pUC4K was PCR
amplified using primers kan1 and kan2. A second PCR reaction was set up containing the DNA
fragments flanking the RR or HPK, the KnR cassette, and primers A and D.
30. PxB Sensitivity: Disk Diffusion Assay
Figure 4. Zones of inhibition for in vitro sensitivity of M. catarrhalis RH4 strains to 25 mg/ml
PxB. Strains were suspended in BHI broth and plated onto BHI agar. Filter disks saturated with
15 uL of 25 mg/ml PxB were placed in the center quadrant of each plate. Plates were
incubated overnight at 37oC in 5% CO2. Diameters for the zones of inhibition were measured
for each strain in millimeters. Experiment conducted at least three times.
31. 27.8 27.5
29.2
21.4
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
hpk- rsr- (hpk/rsr)- RH4
Zone of Inhibition (mm)
Strain
p ≤ 0.05
p ≤ 0.05
p ≤ 0.05
Figure 5. Disk diffusion assays for in vitro sensitivity of M. catarrhalis RH4 strains to 25 mg/ml
PxB. Strains were suspended in BHI broth and plated onto BHI agar. Filter disks saturated with
15 uL of 25 mg/ml PxB were placed in the center quadrant of each plate. Plates were
incubated overnight at 37oC in 5% CO2. Diameters for the zones of inhibition were measured
for each strain in millimeters. Differences in zones of inhibition with a P value of ≤ 0.05 by
Tukey’s HSD analysis were considered significant. Experiment conducted three times.
32. PxB Sensitivity: MIC & MBC
Table 1. Minimum PxB Inhibitory & Bactericidal
Concentrations for M. catarrhalis rsr, hpk and
hpk/rsr Deletion Mutants
M. catarrhalis
Mutant
Minimum Inhibitory
Concentration (MIC) (mg/ml)
Minimum Bactericidal
Concentration (MBC)
(mg/ml)
HPK 0.5-1.0 0.5-1.0
RR 0.5-1.0 0.5-1.0
TCS
(HPK + RR)
0.25-1.0 0.25-1.0
RH4 2.0 2.0-4.0
33. Experimental Goal #2
Determine the global gene
expression in response to
PxB exposure using
microarrays
35. 1.4
1.2
1
0.8
0.6
0.4
0.2
0
RH4
rsr-hpk-
(hpk/rsr)-
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
OD 600 nm
Time (hr)
Figure 6. Growth curve of M. catarrhalis wild type, rsr, hpk and hpk/rsr deletion mutants. M.
catarrhalis RH4 and rsr, hpk and hpk/rsr deletion mutants were resuspended in BHI broth to
an O.D. 600 nm = 1. The bacteria were diluted 1:10 in BHI broth and grown in the shaking
incubator at 37oC and 220 rpm. The absorbance of each strain was measured at regular
intervals. Experiment conducted at least three times.
36. 1.4
1.2
1
0.8
0.6
0.4
0.2
0
RH4
rsr- SUP
(hpk/rsr)- SUP
hpk- SUP
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
OD 600 nm
Time (hr)
Figure 7. Suppression of growth defect in M. catarrhalis mutants. At the end of the initial
growth curve, mutants were plated and individual colonies selected for an additional growth
curve. Colonies were resuspended in BHI broth to an O.D. 600 nm = 1. The bacteria were diluted
1:10 in BHI broth and grown in the shaking incubator at 37oC and 220 rpm. The absorbance of
each strain was measured at regular intervals. Experiment conducted at least three times.
37. 24.1
25.8
27.7
21.4
30.0
25.0
20.0
15.0
10.0
5.0
0.0
p ≤ 0.05
p ≤ 0.05
p ≤ 0.05
hpk- SUP rsr- SUP (hpk/rsr)- SUP RH4
Zone of Inhibition (mm)
Sample
Figure 8. Effect of growth defect suppression on in vitro sensitivity of M. catarrhalis wild type, rsr, hpk and
hpk/rsr deletion mutants to PxB. Strains were suspended in BHI broth and plated onto BHI agar. Filter
disks saturated with 15 mL of 25 mg/ml PxB were placed in the center quadrant of each plate. Plates were
incubated overnight at 37oC in 5% CO2. Diameters for the zones of inhibition were measured for each
strain in millimeters. Differences in zones of inhibition with a P value of ≤ 0.05 by Tukey’s HSD analysis
were considered significant. Experiment conducted at least three times.
39. 0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
4ug/ml
2ug/ml
1ug/ml
0.5ug/ml
Control
0 0.5 1 1.5 2 2.5 3 3.5
OD 600 nm
Time (hr)
Figure 9. A time course experiment was conducted to determine the sublethal concentration
of PxB by measuring the absorbance at 600 nm of M. catarrhalis RH4 incubated with
increasing concentrations of peptide. Experiment conducted at least three times.
40. 0.6
0.5
0.4
0.3
0.2
0.1
0
4ug/ml
2ug/ml
1ug/ml
0.5ug/ml
Control
0 0.5 1 1.5 2 2.5 3 3.5
OD 600 nm
Time (hr)
Figure 10. A time course experiment was conducted to determine the sublethal concentration
of PxB by measuring the absorbance at 600 nm of rsr deletion mutant incubated with
increasing concentrations of peptide. Experiment conducted at least three times.
41. Conclusion
• Deletion of rsr, hpk, or both genes
resulted in increased sensitivity of the
resulting M. catarrhalis mutants to
PxB.
• The mutants grew poorly in broth
culture but suppression of the growth
defect occurred without affecting
susceptibility to PxB.
• The sublethal concentration of PxB was
determined to be 0.5 mg/ml for the
microarray.
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45. GOD
Family & Friends
Committee Members: Dr. Leslie Wooten, Dr. Leonard
Pressley and Dr. Charlie Holman
Claflin University Department of Biology
Claflin University School of Natural Sciences and
Mathematics
South Carolina IDeA Networks of Biomedical
Research Excellence (INBRE):
National Center for Research Resources
(5 P20 RR016461) & NIH National Institute of General
Medical Sciences (8 P20 GM103499)
The middle ear takes sound waves > vibrations > inner ear. Ossicles: malleus (mal-e-us), stapes (rhymes with tapes), incus: three most delicate bones in body; transport vibrations to wave pool/hairs/nerve-endings in cochlea (cock-lea). In order for this process to work, your ET must equalize the pressure between the external environment and middle ear
If something were to compromise the system, it would be more successful in infants than in adults since the tube is shorter and less angled.
Mcat generally likes to stay in the nasopharynx, but when it does migrate it is more often than not coupled with an upper respiratory virus from an URTI.
Mcat counts for about 1/3 of infections.
ET epithelial cells trigger inflammation which signals & recruits neutrophils from bloodstream to clear infection
Major class of effectors that aid neutrophils are neutrophil-derived cationic antimicrobial peptides (CAMPs), Disrupt bacterial outer membrane and permeabilize the membrane to other peptide molecules in a process known as self-promoted uptake (interfere with the electron transport chain, target cytoplasmic components)
degS+ = Parent strain, degS- = transposon mutants; transposon interrupted a gene that was 58% homologous to E. coli degS genes which made it sensitive to polmyxin b, as a result, this is how we found DegS in M.cat.
Kan marker used to 1) ensure the bacteria had successful transformation, 2) serve as a functioning placeholder for the gene sequence removed when the bacteria is exposed to Polymyxin B to confirm whether or not the gene sequence that was removed is involved with Polymyxin B resistance.
A bacterial transformation was then performed using 5 ml of the overlap construct mixed with a colony of M. catarrhalis RH4 on BHI agar. Plates were incubated overnight and streaked for isolation. Exp done at a minimum of three times.
The desired MIC difference is at least 2 ug/ml for bacterial strains.
