Intracellular activity of a membrane-active ...

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JGAR-216; No. of Pages 4 Journal of Global Antimicrobial Resistance xxx (2016) xxx–xxx

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Short Communication

Intracellular activity of a membrane-active glycopeptide antibiotic against meticillin-resistant Staphylococcus aureus infection Venkateswarlu Yarlagadda, Sandip Samaddar, Jayanta Haldar * Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India

A R T I C L E I N F O

Article history: Received 6 October 2015 Received in revised form 18 November 2015 Accepted 22 December 2015 Keywords: Glycopeptides Membrane-active antibiotic Intracellular activity Meticillin-resistant Staphylococcus aureus MRSA

A B S T R A C T

Staphylococcus aureus is a facultative intracellular pathogen and there are limited options for the treatment of severe intracellular bacterial infections. The membrane-active glycopeptide antibiotic VanQC8 is a permanent positively charged lipophilic vancomycin analogue that demonstrates high activity against clinically relevant drug-resistant Gram-positive bacteria both in vitro and in vivo. In this study, the intracellular activity of Van-QC8 was evaluated against meticillin-resistant S. aureus (MRSA) infection in RAW macrophages. Furthermore, the mechanism of intracellular uptake of Van-QC8 was investigated. Van-QC8 showed time- and concentration-dependent bactericidal activity against intracellular MRSA. Van-QC8 displayed significantly higher intracellular activity compared with vancomycin and linezolid. Cellular uptake of Van-QC8 was found to be through clathrin-dependent and independent and caveolin-dependent and -independent endocytic pathways. The findings of this study suggest that Van-QC8 could be translated clinically for the treatment of intracellular infections due to MRSA. ß 2016 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.

1. Introduction Meticillin-resistant Staphylococcus aureus (MRSA) remains one of the major causes of healthcare-associated infections, ranging from simple and uncomplicated skin and wound infections to more serious lethal infections such as endocarditis, osteomyelitis and pneumonia [1,2]. In addition to this, MRSA has become a community-associated pathogen in recent years, which is a matter of concern [2]. Often considered an intracellular organism, S. aureus is capable of surviving within phagocytes, which is probably an important factor in the persistent, recurrent and relapsing nature of these infections [3,4]. Antibiotics such as vancomycin, linezolid and daptomycin, which exhibit poor intracellular activity, are commonly recommended for infections caused by resistant strains of S. aureus (i.e. MRSA) [5,6]. Extensive use of these antibiotics for the treatment of MRSA infections has resulted in reduced susceptibility among MRSA [7]. Therefore, new antibacterial agents that remain active against drug-resistant bacteria and that also exhibit bactericidal activity both against extracellular and intracellular bacteria are needed. Lipoglycopeptides such as telavancin, oritavancin and

dalbavancin have been shown to possess significant activity both against extracellular and intracellular S. aureus [8,9]. The membrane-active glycopeptide antibiotic Van-QC8 has been developed recently by us. It is a permanent positively-charged lipophilic vancomycin analogue wherein 3-amino-N-octyl-N,Ndimethylpropan-1-aminium chloride is appended to the carboxyl group of vancomycin (Fig. 1A) [10]. Van-QC8 displays rapid extracellular bactericidal activity compared with vancomycin against MRSA and other Gram-positive organisms and remains active against vancomycin-resistant bacteria [10,11]. Furthermore, Van-QC8 has been shown to be highly effective in mouse models of drug-resistant staphylococcal infections [11]. The potent activity of Van-QC8 has been attributed to its intrinsic bactericidal activity due to the presence of strong membrane disruption properties. In the present study, the intracellular activity of Van-QC8 was determined and was compared with the activities of vancomycin and linezolid against MRSA using mouse macrophages (RAW 264.7). Furthermore, the mechanism of cellular uptake of Van-QC8 was investigated. 2. Materials and methods 2.1. Antimicrobial agents

* Corresponding author. Tel.: +91 8022082565. E-mail address: [email protected] (J. Haldar).

The membrane-active glycopeptide Van-QC8 was synthesised following previously published protocols and was purified to >95%

http://dx.doi.org/10.1016/j.jgar.2015.12.007 2213-7165/ß 2016 Published by Elsevier Ltd on behalf of International Society for Chemotherapy of Infection and Cancer.

Please cite this article in press as: Yarlagadda V, et al. Intracellular activity of a membrane-active glycopeptide antibiotic against meticillin-resistant Staphylococcus aureus infection. J Global Antimicrob Resist (2016), http://dx.doi.org/10.1016/j.jgar.2015.12.007

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Fig. 1. (A) Chemical structures of vancomycin, linezolid and the membrane-active glycopeptide Van-QC8. (B) Intracellular activity of vancomycin, linezolid and Van-QC8 at 100 MIC against MRSA as determined by viable bacterial counts after 6 h of incubation. Data are expressed as the mean standard deviation (error bars). MIC, minimum inhibitory concentration; MRSA, meticillin-resistant Staphylococcus aureus.

purity using high-performance liquid chromatography (HPLC) [10]. Vancomycin and linezolid were obtained from commercial sources. 2.2. Bacterial strains MRSA ATCC 33591 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). 2.3. Cells and cell cultures All experiments were performed with RAW 264.7 macrophages (ATCC1 TIB-71TM). Cells were grown in complete growth medium containing Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% v/v decomplemented fetal bovine serum at 37 8C in humidified air containing 5% CO2. The cells were scraped to avoid cell confluence. 2.4. In vitro susceptibility studies The antibacterial activities of vancomycin, linezolid and VanQC8 were measured by the broth microdilution method, and the minimum inhibitory concentrations (MICs) were calculated according to previously published protocols [10–13]. 2.5. Intracellular antibacterial activity The intracellular activities of vancomycin, linezolid and Van-QC8 were determined according to previously published protocols with slight modifications [9,14,15]. Briefly, ca. 105 RAW 264.7 cells were seeded in 24-well tissue culture plates containing complete growth medium at 37 8C in humidified air containing 5% CO2 for 12 h to ensure cell adherence. Next, MRSA cells (ca. 107 CFU/mL) were suspended in complete growth medium for 1 h for opsonisation. Then, 0.5 mL of opsonised bacterial suspension was added to the 24well tissue culture plates containing RAW 264.7 cells and the plates were incubated further for 1.5 h at 37 8C in humidified air containing 5% CO2 to allow phagocytosis. Following incubation, macrophages were washed twice with phosphate-buffered saline (PBS) (pH 7.2) and were treated with gentamicin (50 mg/mL) for 1 h in complete growth medium to remove any extracellular or non-phagocytosed bacteria. RAW 264.7 macrophages were then washed twice with PBS to remove the gentamicin. Test compounds at 100 MIC (vancomycin at 60 mM, linezolid at 230 mM and Van-QC8 at 30 mM) in

growth medium (0.5 mL) were added and were incubated for 6 h at 37 8C in humidified air containing 5% CO2. The multiplicity of infection (MOI) (number of bacterial cells per infected macrophage) was determined at pre-treatment stage by lysing the cells with 0.5 mL of ice cold water for 1 h and the lysates were then collected and centrifuged. Dilutions of lysates in sterile PBS were plated on nutrient agar plates. The plates were then incubated at 37 8C for 24 h and viable bacterial colonies were counted the next day. After treatment with test compounds, macrophages were washed twice with PBS to eliminate extracellular drug. The cells were then lysed and bacterial counts in the lysates were determined as described above. 2.6. Intracellular bactericidal time–kill kinetics MRSA were subjected to phagocytosis by macrophages as described above. The infected macrophages were then treated with the test compounds at 100 MIC (vancomycin at 60 mM, linezolid at 230 mM and Van-QC8 at 30 mM) and were incubated at 37 8C in humidified air containing 5% CO2. At specified time intervals (0, 1, 3, 6 and 24 h), cells were washed, lysed and analysed for bacterial counts as described above. Another intracellular bactericidal time–kill kinetic study (incubation with compound for different time periods of 0, 1, 3, 6 and 24 h) was performed with Van-QC8 at different concentrations (1, 10, 20 and 100 MIC) as mentioned above. 2.7. Cellular uptake of Van-QC8 in RAW 264.7 macrophages MRSA were subjected to phagocytosis by macrophages as described above. The infected macrophages were then treated with Van-QC8 at 100 MIC with or without pharmacological inhibitors and were incubated at 37 8C for 6 h in humidified air containing 5% CO2. The pharmacological inhibitors chlorpromazine (inhibitor of clathrin-dependent endocytosis at 25 mM), methyl-b-cyclodextrin (MBC) (inhibitor of clathrin-independent endocytosis at 100 mM), genistein (inhibitor of caveolin-dependent endocytosis at 100 mM), filipin (inhibitor of caveolin-independent endocytosis at 10 mM) and ethyl isopropyl amiloride (EIPA) (inhibitor of macropinocytosis at 25 mM) were used to investigate the pathway of cellular uptake of Van-QC8. Initially, the infected macrophages were incubated for 1 h with one of the abovementioned inhibitors followed by Van-QC8 treatment for 6 h. Then the cells were washed, lysed and analysed for bacterial counts as described above.


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3. Results and discussion 3.1. In vitro antibacterial activity The MICs of Van-QC8, vancomycin and linezolid were determined against MRSA ATCC 33591. The activity of Van-QC8 was found to be ca. two-fold higher than that of vancomycin (MICs of 0.3 mM vs. 0.6 mM). On the other hand, linezolid showed good activity against MRSA with an MIC of 0.8 mg/mL (2.3 mM). 3.2. Intracellular antibacterial activity As S. aureus is known to invade and survive in macrophages, MRSA was chosen as a model to study the intracellular bactericidal effect of the membrane-active glycopeptide Van-QC8 in comparison with linezolid and vancomycin at 100 MIC using RAW 264.7 macrophages. Initially, macrophages (ca. 105 macrophages/well) were subjected to phagocytosis with MRSA (ca. 107 CFU/well) for 1.5 h and the infected macrophages were then treated with test compounds. The bacterial count prior to initiation of treatment was found to be 6.5 0.5 log10 CFU, indicating a MOI of ca. 30 bacteria per mammalian cell. After 6 h of treatment with the test compounds, the intracellular antibacterial activity was determined by bacterial counts in infected macrophages. In saline-treated controls (growth control), the bacterial count increased to 10.6 0.6 log10 CFU in 6 h (Fig. 1B). On the other hand, vancomycin and linezolid resulted in bacterial growth of ca. 7.4 log10 CFU and ca. 8.2 log10 CFU, which are, respectively, ca. 3.2 log10 CFU and ca. 2.4 log10 CFU lower than the saline-treated controls. In contrast to vancomycin and linezolid, VanQC8 showed significantly higher activity, producing an ca. 5 log10 CFU reduction in bacterial count compared with the growth control (Fig. 1B). 3.3. Intracellular bactericidal time–kill kinetics Next, the effect of incubation time on drug efficacy was determined against MRSA infection in RAW 264.7 macrophages. Infected macrophages were treated with vancomycin, linezolid and Van-QC8 at 100 MIC for 1, 3, 6 and 24 h. At specified time intervals, macrophages were lysed with cold water treatment and were analysed for bacterial counts. The pre-treatment bacterial count was ca. 6.7 log10 CFU. In growth controls, the bacterial count increased to 12 0.9 log10 CFU within 24 h. Vancomycin and linezolid caused a minimal response wherein they were able to

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reduce the bacterial count by ca. 4 log10 CFU compared with the growth controls (Fig. 2A). In contrast to vancomycin and linezolid, Van-QC8 was able to lower the bacterial burden by ca. 8.5 log10 CFU compared with growth controls and, furthermore, it showed timedependent intracellular bactericidal activity (Fig. 2A). In another study, the effect of dose–response and incubation time on the efficacy of Van-QC8 was studied against intracellular MRSA infection. Infected macrophages were incubated with VanQC8 at different concentrations (1, 10, 20 and 100 MIC) for 1, 3, 6 and 24 h. At specified time intervals, macrophages were lysed with cold water treatment and were analysed for bacterial counts. The bacterial count was found to be 12 0.2 log10 CFU over 24 h incubation in growth controls. Van-QC8 produced comparable doseand time-dependent reductions in the bacterial count at each of four dosing regimens (Fig. 2B). At 1 MIC, Van-QC8 showed a bacteriostatic effect that was similar to the intracellular activity of vancomycin at 100 MIC. These results suggest that Van-QC8 displays rapid bactericidal activity against intraphagocytic forms of MRSA. This observation is in contrast to vancomycin, which displays only a bacteriostatic effect even at 100 MIC against intracellular bacteria. Although Van-QC8 shares the pharmacophore of vancomycin that causes inhibition of cell wall biosynthesis, it also comprises an additional permanent positively charged lipophilic moiety that confers strong membrane destabilisation properties in bacteria. This may explain the rapid bactericidal activity of Van-QC8 compared with vancomycin both against extracellular and intracellular bacteria. 3.4. Cellular uptake of Van-QC8 To study the pathway of internalisation of Van-QC8 by RAW 264.7 macrophages, different inhibitors were used to inhibit various entrance routes for the drug and its intracellular activity was then evaluated. The inhibitors chlorpromazine (clathrin-mediated endocytosis), genistein (caveolin-mediated endocytosis), MBC (clathrinindependent endocytosis), filipin (caveolin-independent endocytosis) and EIPA (macropinocytosis) were used. Infected macrophages were incubated with one of the above inhibitors for 1 h before treatment with Van-QC8. The results demonstrated that the intracellular activity of Van-QC8 was reduced in the presence of endocytosis inhibitors (chlorpromazine, genistein, filipin and MBC), indicating that Van-QC8 enters the mammalian cell through several endocytosis pathways (Fig. 3). Conversely, in presence of the macropinocytosis inhibitor EIPA, the activity of Van-QC8 remained unchanged, indicating that inhibition of the macropinocytosis path

Fig. 2. Intracellular bactericidal time–kill kinetics against MRSA. (A) Time–kill bactericidal properties of vancomycin, linezolid and Van-QC8 at 100 MIC. (B) Dose-dependent time–kill bactericidal properties of Van-QC8. Data are expressed as the mean standard deviation (error bars). MRSA, meticillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration.


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Ethical approval Bacterial experiments were approved by the Institutional Human Bioethics and Biosafety Review Committee of JNCASR [Reg. No. JNC/IBSC/2013/12-1500]. Acknowledgment The authors thank Prof. C.N.R. Rao [Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, India] for his constant support and encouragement. References

Fig. 3. Intracellular activity of Van-QC8 at 100 MIC against MRSA with or without cell entry inhibitors. Data are expressed as the mean standard deviation (error bars). MIC, minimum inhibitory concentration; MRSA, meticillin-resistant Staphylococcus aureus; MBC, methyl-b-cyclodextrin; EIPA, ethyl isopropyl amiloride.

does not affect the activity of Van-QC8. These findings suggest that Van-QC8 enters the cell through clathrin-dependent and -independent and caveolin-dependent and -independent endocytic pathways rather than macropinocytosis. In summary, the present study confirms that the membraneactive glycopeptide antibiotic Van-QC8 has the ability to display potent activity against MRSA in infections where not only the killing of extracellular bacteria but also the eradication of intracellular forms is vital. Achieving both the objectives may reduce persistence and recurrence, two well known features of many staphylococcal infections. Unlike vancomycin, Van-QC8 is a rapidly bactericidal agent against intracellular bacteria. However, further studies using in vivo models are required to confirm the improved intracellular efficacy of Van-QC8. Funding The Department of Science and Technology, Government of India, awarded a Ramanujan Fellowship to JH [SR/S2/RJN-43/ 2009]. The Council of Scientific and Industrial Research (CSIR) awarded a research fellowship to VY. The Sheikh Saqr Laboratory at JNCASR awarded a postdoctoral fellowship to SS. Conflict of interest JNCASR has filed a patent application based on the work described in this manuscript. All of the authors declare no competing interests.

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