Letters pubs.acs.org/acschemicalbiology
Small Molecule Downregulation of PmrAB Reverses Lipid A Modification and Breaks Colistin Resistance Tyler L. Harris,† Roberta J. Worthington,† Lauren E. Hittle,‡ Daniel V. Zurawski,§ Robert K. Ernst,‡ and Christian Melander*,† †
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States Department of Microbial Pathogenesis, University of Maryland, Baltimore, Baltimore, Maryland 21201, United States § Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, United States ‡
S Supporting Information *
ABSTRACT: Infections caused by multi-drug resistant bacteria, particularly Gram-negative bacteria, are an ever-increasing problem. While the development of new antibiotics remains one option in the fight against bacteria that have become resistant to currently available antibiotics, an attractive alternative is the development of adjuvant therapeutics that restore the efficacy of existing antibiotics. We report a small molecule adjuvant that suppresses colistin resistance in multidrug resistant Acinetobacter baumannii and Klebsiella pneumoniae by interfering with the expression of a two-component system. The compound downregulates the pmrCAB operon and reverses phosphoethanolamine modification of lipid A responsible for colistin resistance. Furthermore, colistin-susceptible and colistin-resistant bacteria do not evolve resistance to combination treatment. This represents the first definitive example of a compound that breaks antibiotic resistance by directly modulating two-component system activity.
A
problem, and the ever increasing isolation of carbapenem resistant strains means that colistin, a polymyxin antibiotic that disrupts bacterial membrane integrity, is now essentially the last line of defense against MDR Gram-negative infections, especially in wounded soldiers returning from combat operations in Iraq/Afghanistan.4,5 With the advent of colistin therapy, however, colistin-resistant mutants of A. baumannii and K. pneumoniae are also now being isolated.6,7 To address the rising tide of MDR infections, we have been pursuing an approach based upon the development of small molecule adjuvants that do not target essential bacterial processes but suppress the ability of MDR bacteria to respond to antibiotics via their two-component signaling systems (TCS).8,9 Several TCSs are known to play a role in antibiotic
mong the greatest current global health problems are bacterial infections that stem from multidrug resistant (MDR) organisms.1 Infections that arise from the so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are considered the greatest threat to human health.2 Along with a slowing in the development of antibiotics over the past several decades, this has created a “perfect storm” that is threatening the vast strides in human medicine that have been enabled due to the advent of antibiotics, such as surgery, premature infant care, cancer chemotherapy, and organ transplantations. The only new classes of antibiotics to be introduced to the clinic in the last two decades, daptomycin and linezolid, are active solely against Gram-positive bacteria, while the development of therapeutics to treat Gram-negative bacterial infections has met with much less success.3 The dearth of new antibiotics to treat infections caused by MDR Gram-negative bacteria is a considerable © 2013 American Chemical Society
Received: July 3, 2013 Accepted: October 16, 2013 Published: October 16, 2013 122
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Figure 1. Structure and activity of compound 1. (A) Structure of active compound 1 and inactive compound 2. (B) Effect of compound 1 on colistin MIC. (Values reported in μg/mL). (C) Colistin MIC90 values for 29 A. baumannii isolates and 14 K. pneumoniae isolates when dosed with varying concentrations of 1. (Values reported in μg/mL.)
colistin resistance as ≥8 μg/mL. We then determined the colistin MIC of each strain in the presence of 10, 20, and 30 μM compound 1 (3, 6, and 9 μg/mL respectively, Figure 1B). We observed a dose responsive effect, with colistin MICs reduced to 0.5 − 4 μg/mL at 30 μM compound 1. To quantify this effect, time kill curves were constructed for one representative isolate (4106) (Supporting Information) for colistin alone, compound 1 alone, and the combination of colistin and compound 1. Compound 1 alone had a bacteriostatic effect on early bacterial growth at 30 μM; however, bacterial growth was identical to untreated bacteria by 8 h (1024 μg/mL, while repetition of the experiment in the presence of 10 μM compound 1 completely inhibited resistance evolution with the MIC remaining at 1 μg/mL. Serially passaging the bacteria in the presence of compound 1 at 5 μM (1.5 μg/mL) slowed the evolution such that after seven days the colistin MIC had only risen to 16 μg/mL. The strain evolved against colistin only was also susceptible to the action of compound 1, as we observed suppression of colistin MICs to 128 μg/mL and 16 μg/mL in the presence of compound 1 at concentrations of 2.5 μM (0.75 μg/mL) and 5 μM, respectively. After establishing that compound 1 reduced colistin MICs 128−1024-fold against the clinical isolates obtained from WRAIR, we sought to further establish the activity of compound 1 by screening against 24 additional A. baumannii isolates with varying colistin MICs (Supporting Information). We determined the MIC of colistin against each strain in the presence of 10, 20, or 30 μM compound 1. MIC90 values for the collection (including the five original WRAIR strains) are summarized in Figure 1C. Compound 1 substantially reduced the colistin MIC against each strain. It has also been reported that colistin resistance in K. pneumoniae is driven by TCS signaling.20 To further establish the effect of compound 1 upon colistin resistance, we studied the ability of compound 1 to lower colistin MICs against 14 K. pneumoniae clinical isolates with varying degrees of colistin resistance (Supporting Information). MIC90 values for the collection are summarized in Figure 1C, and compound 1 significantly suppressed the colistin MIC against each strain. In conclusion, there is no doubt that the rise of MDR bacteria is placing a large burden on our healthcare system and infections caused by these bacteria lead to significant patient morbidity and mortality. Novel approaches to treat these infectious are sorely needed, regardless of whether they come from the development of novel antibiotics or antibiotic adjuvants. In this vein, we have demonstrated that a 2aminoimidazole adjuvant (compound 1) has the ability to downregulate pmrCAB expression, suppress lipid A modification, and in the process restore colistin sensitivity to colistin resistant A. baumannii and K. pneumoniae. To the best of our knowledge, this is the first example of a small molecule that directly modulates TCS signaling, reverses an antibioticresistance phenotype, and in the process breaks antibiotic resistance in Gram-negative bacteria. Serially passaging of either colistin-susceptible or colistin-resistant bacteria in the presence of compound 1 and colistin failed to deliver a resistant bacterial population, indicating that selection pressure to evolve resistance to this combination is severely reduced in comparison to conventional antibiotics. Finally, compound 1 establishes that small molecule suppression of TCS activity is a viable approach to reversing antibiotic resistance and scaffolds based upon compound 1 or other small molecules that suppress TCS function can potentially be employed as adjuvants to restore antibiotic activity.
Letters
METHODS
Bacterial Strains, Media, and Antibiotics. A. baumannii strains were obtained from WRAIR (3941, 3942, 4106, 4112, 4119) or The University of Maryland, Baltimore (UMB) (A2, A3, A6, A7, B2, B5, D4, D5, D9, E1, E3, E4, E5, E6, E8, E9, F2, F3, F8, F9, G2, G3, G9, H1) or ATCC (19606), K. pneumoniae strains were obtained from UMB (A2, A5, B2, B3, B5, B6, B8, B9, C2, C3, C4, C5, D4, D7) and grown on nutrient agar. Mueller-Hinton II broth cation adjusted (CAMHB) (cat. no. 212322) and nutrient agar (cat. no. 213000) were purchased from BD Diagnostics. Colistin sulfate salt (cat. no. C4461) was purchased from Sigma-Aldrich. All assays were run in duplicate and repeated at least two separate times. All compounds were synthesized as previously reported18 and dissolved as their HCl salts in molecular biology grade DMSO as 100 mM or 10 mM stock solutions. Broth Microdilution Method for Determination of Minimum Inhibitory Concentrations. Bacteria were grown in CAMHB for 6− 8 h then subcultured (5.0 × 105 CFU/mL), aliquoted into culture tubes and compound added. Inoculated media not treated with compound served as the control. From each sample, 1 mL was transferred to a new culture tube and antibiotic added. Rows 2−12 of a 96-well microtiter plate were filled (100 μL/well) from the remaining inoculated media, allowing the concentration of compound to be kept uniform throughout the antibiotic dilution procedure. The samples containing antibiotic were then aliquoted (200 μL) into the corresponding first row wells of the microtiter plate. Row 1 wells were mixed 6 to 8 times then 100 μL was transferred to row 2. This procedure was repeated to serially dilute the rest of the rows of the microtiter plate, with the exception of the final row (to check for growth of bacteria in the presence of compound alone). The plate was then covered and incubated under stationary conditions at 37 °C. After 16 h, MIC values were recorded as the lowest concentration of antibiotic at which no visible growth of bacteria occurred. Evolution of Colistin Resistance in A. baumannii. A. baumannii was grown in CAMHB overnight then subcultured (5 × 105 CFU/ mL), aliquoted into culture tubes and compound and colistin at 0.5×, 1.0×, and 2.0× MIC were added. Growth at the highest colistin concentration was carried forward and MICs determined every onetwo days. This was repeated for a period of seven days. Time Kill Curves. A. baumannii was grown in CAMHB overnight then subcultured (5 × 105 CFU/mL), aliquoted into culture tubes and compound and/or colistin were added, untreated inoculated media served as the control. Tubes were incubated at 37 °C with shaking, samples taken at 2, 4, 8, and 16 h, serially diluted and plated on nutrient agar. Plates were incubated at 37 °C overnight and colonies enumerated. The experiment was repeated four times for each data point. Bacterial Membrane Permeabilization Assay. A. baumannii was grown overnight in CAMHB then subcultured 1:40 and grown to an OD600 of ∼1.0. The cultures were centrifuged (10 000g, 15 min), and the cell pellet washed with sterile water, resuspended to 1/10 of the original volume and diluted 1:20 into either water or water containing test compounds. Suspensions were incubated at 37 °C with shaking for 1 h then centrifuged (10 000g,10 min) washed and resuspended in water. A 1:1 mixture of SYTO-9 and propidium iodide were added to the suspension (3 μL/mL). 100 μL of the suspension was added to each well of a 96-well plate and incubated in the dark for 15 min at RT. Green fluorescence (SYTO-9) was read at 530 nm, and red fluorescence (propidium iodide) was read at 645 nm (excitation wavelength 485 nm). The ratio of green to red fluorescence was compared to the control. RNA Extraction Protocol and rtPCR. A. baumannii was grown overnight in CAMHB then subcultured 1:100, grown to an OD600 of 0.6, then treated with compound for 1 h. The cultures were mixed with bacterial RNA protect solution (Qiagen, cat. no. 76506). Samples were centrifuged (3000g, 20 min), cell pellets collected and suspended in 1 mL TRIzol reagent (Invitrogen, cat. no. 15596-026). Cell lysis was accomplished using a Branson sonifier 450. RNA was extracted and purified using a RNeasy mini kit, following the manufacturer’s instructions (Qiagen, cat. # 74104). RNA was quantified using a 125
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NanoDrop ND-1000 spectrophotometer. Reverse transcriptase polymerase chain reaction (rtPCR) was accomplished using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen, cat. # 11736−059) according to manufacturer’s instructions. 16S rRNA gene was used as a reference gene in all experiments and relative levels of gene expression were calculated using Bio-Rad CFX Manager Software. All reactions were performed in triplicate using a C1000 Touch Thermal Cycler, CFX96 Real-Time System (Bio-Rad) on three independent samples. Forward and reverse primers for pmrA, pmrB, pmrC, and 16S rRNA are listed in the Supporting Information.10 MALDI-ToF Mass-Spectrometry Analysis of Lipid A (A. baumannii 4106). A. baumannii was grown to 1.0 × 107 CFU then either left untreated, or treated with compound. After 8 h cells were pelleted and Lipid A was extracted by the method previously described by Caroff21 and subjected to negative-ion matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis. Cell pellets were resuspended in isobutyric acid (250 μL) and 1 M ammonium hydroxide (150 μL) and incubated at 100 °C for 1 h, with occasional vortexing. Samples were cooled on ice and centrifuged (2000g, 15 min). Supernatants were transferred to new tubes, diluted with equal volumes of water, and lyophilized. Dried samples were washed with methanol and centrifuged (2000g, 15 min). Lipid A, was solubilized in 120 μL chloroform, 60 μL methanol, and 10 μL water. Norharmane (1 μL) followed by 1 μL of sample were spotted on a MALDI plate. Analyses were performed on a Bruker UltrafleXtreme MALDI-TOF mass spectrometer in negative mode. Each spectrum was an average of 300 shots. ESI peptide calibration standard (Bruker Daltonics) was used for calibration
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Supporting information and methods, including RT-PCR primers, MIC tables, and time kill curves. This material is available free of charge via the Internet at http://pubs.acs.org.
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Letters
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): C. Melander is cofounder, chief research officer, and board member of Agile Sciences, a commercial entity that is commercializing the potential of 2-aminoimidazole derivatives as antibiotic adjuvants.
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ACKNOWLEDGMENTS We thank the DOD DMRDP program (W81XWH-11-2-0115 to CM), the Army Research Laboratory and the U.S. Army Research Office under Contract/Grant No. W911NF-11-10274 (RKE) and multiple grants from the Military Infectious Diseases Research Program (MIDRP) and the Defense Medical Research and Development Program (DMRDP) (DVZ) for their support. The DMRDP program is administered by the Department of Army; The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702-5014 is the awarding and administering office. The content of this manuscript does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. Furthermore, the findings and opinions expressed by Dr. Zurawski belong to him and do not necessarily reflect the official views of the WRAIR, the U.S. Army, or the Department of Defense. 126
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(18) Worthington, R. J., Bunders, C. A., Reed, C. S., and Melander, C. (2012) Small molecule suppression of carbapenem resistance in NDM-1 producing Klebsiella pneumoniae. ACS Med. Chem. Lett. 3, 357−361. (19) Hilliard, J. J., Goldschmidt, R. M., Licata, L., Baum, E. Z., and Bush, K. (1999) Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems. Antimicrob. Agents Chemother. 43, 1693−1699. (20) Cheng, H. Y., Chen, Y. F., and Peng, H. L. (2010) Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J. Biomed. Sci. 17, 60. (21) El Hamidi, A., Tirsoaga, A., Novikov, A., Hussein, A., and Caroff, M. (2005) Microextraction of bacterial lipid A: Easy and rapid method for mass spectrometric characterization. J. Lipid Res. 46, 1773−1778.
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