Resistant Staphylococcus Aureus Potentiates Killing by Reactive Oxygen Species
Total Page：16
File Type：pdf, Size：1020Kb
FULL PAPER Phototherapy www.advancedscience.com Photolysis of Staphyloxanthin in Methicillin-Resistant Staphylococcus aureus Potentiates Killing by Reactive Oxygen Species Pu-Ting Dong, Haroon Mohammad, Jie Hui, Leon G. Leanse, Junjie Li, Lijia Liang, Tianhong Dai, Mohamed N. Seleem,* and Ji-Xin Cheng* 1. Introduction Confronted with the severe situation that the pace of resistance acquisition is faster than the clinical introduction of new antibiotics, health organiza- Staphylococcus aureus causes a variety tions are calling for effective approaches to combat methicillin-resistant of diseases ranging from skin and soft Staphylococcus aureus (MRSA) infections. Here, an approach to treat tissue infections to life-threatening bacte- MRSA through photolysis of staphyloxanthin, an antioxidant residing in remia.[1] Moreover, S. aureus has acquired the microdomain of S. aureus membrane, is reported. This photochemistry resistance to multiple antibiotic classes that were once effective.[2] A classic process is uncovered through transient absorption imaging and example is the emergence of clinical iso- quantitated by absorption spectroscopy, Raman spectroscopy, and mass lates of methicillin-resistant S. aureus spectrometry. Photolysis of staphyloxanthin transiently elevates the (MRSA) strains in the 1960s that exhib- membrane permeability and renders MRSA highly susceptible to hydrogen ited resistance to β-lactam antibiotics.[3] peroxide attack. Consequently, staphyloxanthin photolysis by low-level More recently, some MRSA strains have 460 nm light eradicates MRSA synergistically with hydrogen peroxide and exhibited reduced susceptibility to newer antibiotics such as daptomycin and to other reactive oxygen species. The effectiveness of this synergistic therapy last-resort antibiotics such as vancomycin is well validated in MRSA planktonic culture, MRSA-infected macrophage and linezolid.[4] Besides the acquired cells, stationary-phase MRSA, persisters, S. aureus biofilms, and two resistance through mutational inactiva- mice wound infection models. Collectively, the work demonstrates that tion, S. aureus develops other strategies to staphyloxanthin photolysis is a new therapeutic platform to treat MRSA undermine the effect of antibiotics, such as residing inside host immune cells,[5] infections. forming biofilms, and becoming dormant P.-T. Dong, Prof. J.-X. Cheng Dr. L. G. Leanse, Prof. T. Dai Department of Chemistry Wellman Center for Photomedicine Boston University Massachusetts General Hospital Boston, MA 02215, USA Harvard Medical School E-mail: [email protected] MA 02114, USA Dr. H. Mohammad, Prof. M. N. Seleem Dr. L. Liang Department of Comparative Pathobiology State Key Laboratory of Supramolecular Structure and Materials College of Veterinary Medicine Institute of Theoretical Chemistry Purdue University Jilin University West Lafayette, IN 47907, USA Changchun 130012, China E-mail: [email protected] Prof. J.-X. Cheng Dr. J. Hui, Dr. J. Li Department of Biomedical Engineering Prof. J.-X. Cheng, Department of Electrical and Computer Engineering Boston University Boston University Boston, MA 02215, USA Boston, MA 02215, USA Prof. J.-X. Cheng The ORCID identification number(s) for the author(s) of this article Photonics Center can be found under https://doi.org/10.1002/advs.201900030. Boston University Boston, MA 02215, USA © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/advs.201900030 Adv. Sci. 2019, 6, 1900030 1900030 (1 of 12) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com persisters.[6] Those situations pose an appalling challenge to microscope, the strong signal measured at zero delay between developing new ways to treat MRSA infections. the 520 nm pump and 780 nm probe pulses quickly attenuated On the verge of post-antibiotic area, researchers are taking over second scale (Figure 1a; Movie S1, Supporting Informa- several approaches to tackle MRSA-caused infections. Repur- tion). We hypothesized that a specific chromophore in MRSA posing existing anticancer, antifungal, and anti-inflammatory is prone to photobleaching under the abovementioned settings. drugs, has been pursued by harnessing their established feasi- To verify this, we fitted the time-course curve with a photo- bility and antibacterial properties.[7] Therapeutic application of bleaching model developed for photosensitizers[17] (Figure 1b, bacteriophages offers another promising alternative to combat see Methods in the Supporting Information ) staphylococcal infections.[8] In addition, novel approaches are developed through targeting MRSA-specific virulence fac- t exp− tors. More than 90% of all S. aureus clinical isolates generate τ 1 [9] yy=+0 A * (1) a golden pigment, staphyloxanthin (STX). STX condenses τ 1 t [10] 1*+−1exp − in the functional membrane microdomain of S. aureus, ττ21 endowing S. aureus with membrane integrity and excellent antioxidant property.[11] Ever since Nizet and co-workers eluci- where t is the duration of light irradiation, y is the signal inten- dated the pivotal role of STX, the virulence factor which pro- sity, y0 and A are constants, τ1 and τ2 are the time constants tects S. aureus from neutrophil-based killing,[12] stripping this for the first- and second-order photobleaching, respectively. The important pigment off MRSA has become a novel therapeutic first-order bleaching occurs at low concentration of chromo- [13] approach. A range of synthetic cholesterol inhibitors have phores (τ2 = ∞). The second-order bleaching takes place when been harnessed to inhibit STX biosynthesis.[12,13b] Chen et al. quenching within high-concentration surrounding chromo- found that naftifine, an FDA-approved antifungal drug, blocked phores dominates (τ1 = ∞, Figure S2, Supporting Information). the biosynthesis of STX through inhibition of diapophytoene Derivation of Equation (1) is detailed in the Supporting Infor- desaturase.[14] Jabra-Rizk et al. demonstrated that sesquiterpene mation. Strikingly, this photobleaching model fitted well with farnesol, a natural plant metabolite, effectively suppressed the the raw time-course curve (τ1 = ∞, τ2 = 0.15 ± 0.02 s). Moreover, production of STX through binding the active domain of the oxygen depletion (Na2S2O4: oxygen scavenger) showed neg- dehydrosqualene synthase, thus compromising the membrane ligible effect on the photobleaching speed (τ2 = 0.14 ± 0.01 s, integrity.[15] However, administration of exogenous agents only Figure S3a, Supporting Information). The same phenomenon achieved limited efficacy possibly due to off-targeting.[16] There- was observed in methicillin-susceptible S. aureus (Figure S3b, fore, drug-free approaches to eradicate STX have been press- Supporting Information). To determine whether oxygen plays ingly desired. an essential role during this photobleaching process, we kept Here, through label-free transient absorption imaging of the extracted chromophore solution bubbling with nitrogen gas nonfluorescent chromophores in S. aureus, we accidentally for 2 h in order to deplete the oxygen.[18] We found that oxygen find that STX is prone to photolysis and this photolysis process depletion did not affect the photobleaching process (Figure strongly depends on the excitation wavelength. By absorption S3c, Supporting Information). Collectively, these data support a spectroscopy, we identify that the optimal wavelength for STX second-order photobleaching process. photolysis is around 460 nm. We also unveil that 460 nm light Next, we aimed to deduce the specific chromophore inside induces STX CC bond breakdown by employing Raman spec- MRSA that accounts for the observed photobleaching phe- troscopy and mass spectrometry. We then find that STX photo- nomenon. It is known that carotenoids are photosensitive lysis transiently elevates the membrane permeability and ren- due to the conjugated CC bonds.[19] Therefore, we hypoth- ders MRSA highly susceptible to reactive oxygen species attack. esized that STX, the major carotenoid pigment residing in Based on these findings, we developed a highly effective syn- the cell membrane of MRSA, underwent photobleaching in ergy between STX photolysis and low-concentration hydrogen our transient absorption study. To test this hypothesis, we peroxide, which is well established in eliminating stationary- treated MRSA with naftifine to block the synthesis of STX.[14] phase MRSA, MRSA persisters, S. aureus biofilms, and MRSA- The treated MRSA exhibited lower signal intensity (Figure 1c) infected mice wound models. We also find that STX photolysis and slower photobleaching speed (Figure 1d). Specifically,τ 2 could assist macrophage cells to eliminate intracellular MRSA, of naftifine-treated MRSA (0.39 ± 0.07 s) is 2.5 times of that whereas high-concentration antibiotic fails. Our findings open of MRSA (0.15 ± 0.02 s), in consistence with second-order new opportunities for treating MRSA infections. photobleaching. Furthermore, no transient absorption signal was observed in S. aureus strain with mutation in dehydros- qualene synthase (CrtM) (Figure 1e) that is responsible for [11] 2. Results and Discussion STX biosynthesis. To avoid the systematic error aroused by single bacterium measurement, we repeated the same anal- 2.1. STX