Fig 1.
E. cloacae displays inoculum-dependent dynamic survival in the presence of cecB.
Overnight cultures were diluted (A) 10-fold or (B) 100-fold into fresh LB containing 20 μg/mL cecB, and cells were grown at 37°C for 24 hours. OD600 measurements were taken every 5 minutes. Error bars represent standard deviation (n = 3). (C) Cultures were prepared as in (B), but samples were taken at indicated timepoints to quantify colony forming units (CFU) per mL. Error bars represent standard deviation (n = 3). (D) Inoculum effect in different growth media. MIC assays were conducted at the indicated dilution of an overnight culture of E. cloacae.
Fig 2.
E. cloacae exhibits population heterogeneity in its response to cecropin B.
(A) Cells were grown to mid-exponential phase in liquid medium and then placed on 0.8% agarose pads containing the indicated amounts of cecropin and incubated at 37°C. Representative, cropped frames from a time series are shown. In the 20 μg/mL condition, red arrows indicate examples of lysing cells. (B) Percentage of cells that divided during the time lapse, grouped by drug concentration (0, 10, 20, 200 μg/mL). Cells are binned by birth frame, i.e., the frame of the time lapse during which the cell was first identified by SuperSegger [85]. Blue represents cells that underwent a successful division event throughout the course of the timelapse, red indicates cells which did not divide. Missing bars indicate that the initial population of cells was unable to divide.
Fig 3.
PhoPQ partially contributes to cecB resistance.
Overnight cultures were diluted (A) 10-fold or (B) 100-fold into fresh LB containing 20 μg/mL cecB, and cells were grown at 37°C for 24 hours. For the complement strain, expression of phoPQ from pMMB was induced with 200 μM IPTG. OD600 measurements were taken every 5 minutes. Error bars represent standard deviation (n = 3). (C) Mid-exponential phase cells were placed on 0.8% agarose pads containing the indicated amounts of cecropin and incubated at 37°C. Representative, cropped time frames are shown. Red arrow indicates phase-light cell, white arrow indicates a cell that stays intact.
Fig 4.
The Rcs phosphorelay partially contributes to resistance.
Overnight cultures were diluted (A) 10-fold or (B) 100-fold into fresh LB containing 20 μg/mL cecB, and cells were grown at 37°C for 24 hours. OD600 measurements were taken every 5 minutes. Bars at each point represent standard deviation (n = 3). (C) Mid-log cells were placed on 0.8% agarose pads containing indicated amounts of cecropin and incubated at 37°C. Representative, cropped frames from a timelapse movie are shown.
Fig 5.
OmpT contributes to cecB resistance via proteolytic cleavage of cecB.
Overnight cultures were diluted (A) 10-fold or (B) 100-fold into fresh LB containing 20 μg/mL cecB, and cells were grown at 37°C for 24 hours. Where applicable, expression of ompT from pBAD was induced with 0.1% arabinose. OD600 measurements were taken every 5 minutes. Error bars represent standard deviation (n = 3). (C) Mid-exponential cells were placed on 0.8% agarose pads containing the indicated amounts of cecropin and incubated at 37°C. Representative, cropped frames from a timelapse movie are shown. Red arrow indicates a cell which initially divided, but later succumbed to cecropin and lysed. (D) Effects of OmpT active site mutations were tested using conditions in (A).
Fig 6.
PhoPQ, Rcs, and OmpT are collectively required for cecB resistance.
Overnight cultures were diluted (A) 10-fold into fresh LB containing 20 μg/mL cecB, and cells were grown at 37°C for 24 hours. OD600 measurements were taken every 5 minutes. Bars at each point represent standard deviation (n = 3). (B) Cultures were diluted 100fold into fresh medium with or without cecropin; samples were taken at indicated timepoints to quantify colony forming units (CFU) per mL. Error bars represent standard deviation (n = 3). (C) Mid-exponential phase cells were placed on 0.8% agarose pads containing indicated amounts of cecropin and incubated at 37°C. Representative, cropped time frames are shown. (D) Percentage of cells that divided during the time lapse, grouped by drug concentration (0, 10, 20, 200 μg/mL). Cells are binned by birth frame, i.e., the frame of the time lapse during which the cell was first identified by SuperSegger [85]. Blue represents cells that underwent a successful division event throughout the course of the timelapse, red indicates cells which did not divide. Missing bars indicate that the initial population of cells was unable to divide.
Fig 7.
Model of Enterobacter cloacae cecropin B dynamic survival.
(A) Mechanisms of AMP resistance discussed in this study. 1) OM stress induces the Rcs phosphorelay, which phosphorylates the response regulator RcsB. Among other systems, RcsB upregulates capsule production. 2) AMPs and envelope stress induces the PhoPQ signaling system, which upregulates transcription of enzymes which modify the outer membrane. 3) OmpT, bound in the outer membrane, may be able to cleave AMPs before they act on the cell surface. (B) Model of E. cloacae growth vs concentration of cecB over time. Black line represents hypothetical population density (indicated by OD600), solid red line represents the hypothetical concentration of cecropin in the sample, and the dotted red line represents the hypothetical concentration of cecropin necessary for lysis. In this model, the population must bring the amount of cecropin below the effective killing concentration before all cells in the population are lysed. Factors like initial starting concentration or protease production may modify the rate at which bacteria can reduce cecropin concentration, while other factors like PhoPQ and RcsB may raise the concentration of cecropin necessary for lysis.