4thfinalthesisdefense_powerpoint_laurajenkins

Role of a Moraxella catarrhalis Two Component System in Polymyxin B

Resistance

Laura M. JenkinsDr. Randall Harris, Thesis Advisor

School of Natural Sciences and Mathematics Department of Biology

Claflin University Orangeburg, South Carolina

Source: http://www.umm.edu/imagepages/19596.htm

Moraxella catarrhalis

• Gram-negative diplococcus

• Nonhemolytic, round opaque colonies

• Capnophile Source:http://www2.truman.edu/~jherrera/microbiology05/mcatarrhalis.htm

Moraxella catarrhalis• Colonizes nasopharynx

soon after birth– 75% of children are

colonized by age 2• Colonization decreases

with age

Source: Unknown

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

POLYMICROBIAL OTITIS MEDIA

INFECTION

Otitis Media

• Most commonly diagnosed pediatric disease – ¾ of children under the age of 3 – 20 million doctor visits annually in

the US

Source: healthandphysicaleducationteacher.com

Zasloff, J. Antimicrobial peptides of multicellular organisms. Nature. 415 (24): 389-395

(Mammalian cell)

w/ negatively charged lipopolysaccharides

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

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

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

Polymyxin B sensitive M. catarrhalis mutant

One polymyxin B sensitive strain had a mutation in a gene whose product was 58% homologous to Escherichia coli DegS.

+polymyxin B-polymyxin B

degS- degS-

degS+ degS+

Source: Dr. Randall Harris, 2012

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.

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.

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.

ATP

ADP

P

Gram Negative Bacterial Cell Interior

Two Component System (TCS)

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.

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.

P



HypothesisIt 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

Experimental Goal #1

Determine the in vitro susceptibility of defined

M. catarrhalis RH4 TCS mutants to PxB

Experiment 1 Flow Diagram

PxB Sensitivity Analysis

Natural Transformation & Allelic Exchange Confirmation

TCS Recombinant Gene Construction

Experiment 1. TCS Recombinant Gene Construction

A = Purified M. catarrhalis gDNAB = LadderC = kanR gene w/ primers 1 & 2 D = hpk upstream flanking DNA w/ primers A & BE = 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 genekanR selectable markerPCR 1

Fig. 1. Verification of fragment creation by

PCR & AGE.

400b

p

500b

p

A B C D E

1

kb

1.5

10

0.5

1000

bp

~50K

bp

0.3

0.7

Source: http://openwetware.org/wiki/PCR_Overlap_Extension

Overlap PCR: Mix three complementary fragments (no primers)

Purification PCR: Add A and D primers

A

B

C

D

kanR1

kanR2

Upstream flanking DNA of M. catarrhalis

RH4 hpk gene

Downstream flanking DNA of M. catarrhalis

RH4 hpk genekanR selectable markerPCR 1

PCR 2A

PCR 2B

A

D

Experiment 1. TCS Recombinant Gene Construction

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.

Allelic Exchange Confirmation

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.

1900

bp (hpk-)

2500

bp (hpk

)

1900

bp (hpk

/rsr-

)

3000

bp (hpk/rsr)

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.

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.

hpk- rsr- (hpk/rsr)- RH40.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

27.8 27.529.2

21.4

Strain

Zone

of I

nhib

ition

(mm

)

p ≤ 0.05

p ≤ 0.05p ≤ 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.

PxB Sensitivity: MIC & MBC

M. catarrhalisMutant

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 0.5-1.0TCS

(HPK + RR) 0.25-1.0 0.25-1.0

RH4 2.0 2.0-4.0

Table 1. Minimum PxB Inhibitory & Bactericidal Concentrations for M. catarrhalis rsr, hpk and

hpk/rsr Deletion Mutants

Experimental Goal #2

Determine the global gene expression in response to

PxB exposure using microarrays


Growth Curve

Microarray & qPCR

Sub-lethal PxB Time Course Analysis

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.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

OD

600

nm

Time (hr)

RH4

rsr-

hpk-

(hpk/rsr)-

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.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

OD

600

nm

Time (hr)

RH4

rsr- SUP

(hpk/rsr)- SUP

hpk- SUP

hpk- SUP rsr- SUP (hpk/rsr)- SUP RH40.0

5.0

10.0

15.0

20.0

25.0

30.0

24.125.8

27.7

21.4

Sample

Zone

of I

nhib

ition

(mm

)

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.

p ≤ 0.05p ≤ 0.05p ≤ 0.05


Growth Curve

Microarray & qPCR


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.

0 0.5 1 1.5 2 2.5 3 3.50

0.1

0.2

0.3

0.4

0.5

0.6

0.7

4ug/ml2ug/ml1ug/ml0.5ug/mlControl

Time (hr)

OD

600

nm

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.

0 0.5 1 1.5 2 2.5 3 3.50

0.1

0.2

0.3

0.4

0.5

0.6

4ug/ml2ug/ml1ug/ml0.5ug/mlControl

Time (hr)

OD

600

nm

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.


Growth Curve

Microarray & qPCR


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Thank You Dr. Randall Harris!

Acknowledgements

GODFamily & Friends

Committee Members: Dr. Leslie Wooten, Dr. Leonard Pressley and Dr. Charlie Holman

Claflin University Department of BiologyClaflin 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)

Thank You!Questions?

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