Mul3-drug

Transporter
Khadijah Suhaimi

Peterhouse

 Overall the aim was to explore the mechanisms of mul.drug transporter lmrP and to see if it t proposed model of transport* (gure 6) LmrP is a plasma encoded H+ an3port transport protein in Lactococcus Lac.s. We inves3gated: 1. If Calcein, Calcein.AM or EtBr are substrates for export and if there is substan3al eux 2. The requirement of metabolic energy for transport through produc3on of pmf (p) 3. The importance of Asp-235 and Glu-327 acidic residues in facilita3ng transport 4. If Calcein is ac3vely taken up by LmrP

CLEAVAGE

Types of Cells: Hydrophilic + Non Polar (Acetomethoxy deriva3ve) i. WT LmrP ii. DE mutant LmrP - muta3on in Asp-235 (D) and Glu-327 (E) iii. 8048 (Control no LmrP) Figure 1 1. Eux of Calcein.AM and Calcein Fluorometer was used to measure uorescence of calcein (non-membrane permeable) (gure 1) in cells and supernatant. Centrifuge to separate. Readings were taken at 0, 10 and 20 mins a^er Calcein.AM or buer (control) added. Amount of calcein recorded over 3me. 2. Eux of EtBr and Growth Rate of Lactococcus Lac.s Spectrometer was used to measure op3cal density of the dierent cells. All cells made up to 0.1 absorbance. Measurements taken every hour to observe growth rate. 3. EtBr Eux Assay of Ini.ally De-energised Cells EtBr intercalates in DNA and uoresces. Fluorometer used to measure amount of EtBr in cell. Glucose added a^er set 3me for EtBr satura3on (vary for each cell type).

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Is Calcein.AM a substrate for export by LmrP? DE m utant Calcein.AM Figure 2 shows WT and DE Mutant Calcein-AM 15 LmrP have the same uorescence level DE mutant but less than control indica3ng some DMSO 10 8048 Calcein- eux. Absence of LmrP results in AM Calcein accumula3on. Calcein.AM 5 8048 DMSO eux further supported in Figure 3 - DMSO solvent as nega.ve control. 0 uorescence was no3ceably larger in non-expressing LmrP cells sugges3ng Time (mins) eux of Calcein.AM out of cell by Figure 3: Time Dependence of control LmrP before it has 3me to be cleaved 400 Calcein Eux from Cells (8048) cells (Ac.on shown in gure 1). 350 Is there a substan.al eux of Calcein control 300 (8048) by LmrP? supernatant 250 We found there was no signicant LmrP cells dierence of Calcein (error bars) in 200 Cal-AM cells compared to supernatant. 150 Fluorescence levels (Calcein) remained LmrP 100 supernatant steady in LmrP expressing cells. Cal-AM 50 Calcein levels decreased in control cells which may be due to endogenous 0 0 min 10 min 20 min Calcein transporter in L. Lac3s. Time (min)
20

Figure 2: Calcein.AM Eux Assay

wtLmrP Calcein-AM wtLmrP DMSO

Fluorescence (A.U)

Fluorescence (a.u)

1 13 25 37 49 61 73 85 97 109 121 133 145 157 169 181 193 205 217

EtBr which is a intercala3ng mutagen inhibits growth of L.Lac3s through inducing muta3ons. If cells are able to extrude EtBr this will increase growth rate. High op.cal density = greater number of cells = Higher growth rate. Therefore WT LmrP must be ac3vely extruding EtBr. 4: Lactococcus Lac.s Growth Figure D.E Mutant
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time (h) 0

Does LmrP extrude EtBr to allow growth of L.Lac.s?

In Presence of EtBr
Control Wt LmrP DE Mutant LmrP

shows lihle dierence to control (no LmrP). This indicates that Asp-235 and Glu-327 might be necessary for EtBr eux.

Op.cal Density at 600nm

We now know from gure 4 that EtBr is substrate of LmrP. Is metabolic energy required for transport? EtBr enters cells through facilitated diusion, intercalates into DNA and uoresces. Upon glucose addi3on in WT LmrP shows immediate eux highligh3ng need for ATP for produc3on of pmf (p). DE Mutant shows lower rate of eux. This indicates DE residues are sucient for eux but not essen3al. Control lacks EtBr Glucose Figure 5:EtBr Eux Assay with 250 LmrP but ATP LmrP powers H+ Glucose 200 Glucose ATPase which pumps H+ out 150 crea3ng nega.ve 100 wtLmrP poten.al which Demutant LmrP could ahracts 50 8048 EtBr in cell 0 leading to an increase of EtBr SATURATION entry. Time (s)
Fluorescence (A.U) 1 87 173 259 345 431 517 603 689 775 861 947 1033 1119 1205 1291 1377 1463 1549 1635

Our data (gure 4+5) suggests that LmrP is able to extrude EtBr (+vely charged). This concept ts Figure 6 whereby low anity, nega3vely charged, hydrophobic binding pocket of LmrP is facing inwards. Opposite charges allows interac3on and subsequent binding. Figure 5 also suggests metabolic energy is required in the form of pmf as well as a H+ gradient. Proposed model suggests high anity outward facing side necessita3ng the need for pmf (H+ an3port) to power substrate release. H+ associa3on could indicate pH dependent transport as it will inuence protonated state of residues and thus binding poten3al to substrate. Further experiments would need to be conducted to be certain. However mutant in gure 5 s3ll shows eux. DE residues are not necessary for transport. This is further reinforced in gure 2 with Calcein.AM eux in DE mutant LmrP being similar to WT LmrP. However as EtBr was not extruded to the same extent it could be said that EtBr is more dependent on DE residues. Calcein.AM and EtBr are clearly a substrates for eux but Calcein was not shown to be. The ques3on remains if LmrP is capable of substrate Figure 6: Proposed mechanism inux. Interes3ngly EtBr satura3on is fastest in WTLmrP. (gure 5) for transport by LmrP.