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Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

ABSTRACT: We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus. Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 μM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.

SUBMITTER: Mussini A

PROVIDER: S-EPMC9026257 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

Mussini Andrea A Uriati Eleonora E Hally Cormac C Nonell Santi S Bianchini Paolo P Diaspro Alberto A Pongolini Stefano S Delcanale Pietro P Abbruzzetti Stefania S Viappiani Cristiano C

Bioconjugate chemistry 20220310 4

We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with <i>Staphylococcus aureus</i> protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation sp ...[more]

PMID: 35266706

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Project description:A promising alternative to antibiotics for treatment of Staphylococcus aureus infections is photodynamic inactivation (PDI), which employs a photosensitizer (PS) that produces cytotoxic reactive oxygen species (ROS) when exposed to molecular oxygen and antimicrobial blue light in the spectrum of 400-470 nm. Although the precise mechanistic basis of PDI has not been defined, the formation of ROS and free radicals that oxidize a number of cellular targets, including membrane lipids, damage to proteins and nucleic acids result in inactivation of essential cellular functions and subsequent cell death. Because PDI is non-selective and affects multiple cellular targets, development of resistance or tolerance to PDI has been considered to be unlikely and attempts to induce S. aureus resistance or tolerance upon repeated sub-lethal doses of PDI have not succeeded. However, multiple aspects of PDI suggest that development of tolerance is highly probable, in particular when PDI is used for treatment of infections where the environment at the infection site prevents penetration of PDI at a level sufficient to cause death of all bacteria and with tolerant phenotypes emerging from the surviving bacteria. In this study, we sought to identify the mechanisms that contribute to PDI tolerance in S. aureus. S. aureus HG003 and the isogenic HG003DmutSL strain with defects in DNA mismatch repair were used to evaluate the response of S. aureus and the roles of DNA mismatch repair and gene regulatory networks to repeated sublethal doses of PDI. Global transcriptome and genome analyses were used in an agnostic approach to identify the underlying transcriptional responses and genetic adaptations that occur as a result of repeated PDI and contribute to PDI tolerance. Our results reveal multiple metabolic, transport and cell wall biogenesis pathways that contribute to PDI tolerance, and a S. aureus regulatory gene likely responsible for the adaptive transcriptional response to PDI.

Project description:The evolving threat of antibiotic resistance development in pathogenic bacteria necessitates the continued cultivation of new technologies and agents to mitigate associated negative health impacts globally. It is no surprise that infection prevention and control are cited by the Centers for Disease Control and Prevention (CDC) as two routes for combating this dangerous trend. One technology that has gained great research interest is antimicrobial photodynamic inactivation of bacteria, or APDI. This technique permits controllable activation of antimicrobial effects by combining specific light excitation with the photodynamic properties of a photosensitizer; when activated, the photosensitizer generates reactive oxygen species (ROS) from molecular oxygen via either a type I (electron transfer) or type II (energy transfer) pathway. These species subsequently inflict oxidative damage on nearby bacteria, resulting in suppressed growth and cell death. To date, small molecule photosensitizers have been developed, yet the scalability of these as widespread sterilization agents is limited due to complex and costly synthetic procedures. Herein we report the use of brominated carbon nanodots (BrCND) as new photosensitizers for APDI. These combustion byproducts are easily and inexpensively collected; incorporation of bromine into the nanodot permits photosensitization effects that are not inherent to the carbon nanodot structure alone-a consequence of triplet character gained by the heavy atom effect. BrCND demonstrate both type I and type II photosensitization under UV-A irradiation, and furthermore are shown to have significant antimicrobial effects against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and Listeria monocytogenes as well. A mechanism of "dark" toxicity is additionally reported; the pH-triggered release of reactive nitrogen species is detected from a carbon nanodot structure for the first time. The results described present the BrCND structure as a competitive new antimicrobial agent for controllable sterilization of bacteria.

Project description:Antimicrobial photodynamic inactivation (aPDI) and antimicrobial blue light (aBL) are considered low-risk treatments for the development of bacterial resistance and/or tolerance due to their multitargeted modes of action. In this study, we assessed the development of Staphylococcus aureus tolerance to these phototreatments. Reference S. aureus USA300 JE2 was subjected to 15 cycles of both sub-lethal aPDI (employing an exogenously administered photosensitizer (PS), i.e., rose Bengal (RB)) and sub-lethal aBL (employing endogenously produced photosensitizing compounds, i.e., porphyrins). We demonstrate substantial aPDI/aBL tolerance development and tolerance stability after 5 cycles of subculturing without aPDI/aBL exposure (the development of aPDI/aBL tolerance was also confirmed with the employment of clinical MRSA and MSSA strain as well as other representatives of Gram-positive microbes, i.e. Enterococcus faecium and Streptococcus agalactiae). In addition, a rifampicin-resistant (RIFR) mutant selection assay showed an increased mutation rate in S. aureus upon sub-lethal phototreatments, indicating that the increased aPDI/aBL tolerance may result from accumulated mutations. Moreover, qRT-PCR analysis following sub-lethal phototreatments demonstrated increased expression of umuC, which encodes stress-responsive error-prone DNA polymerase V, an enzyme that increases the rate of mutation. Employment of recA and umuC transposon S. aureus mutants confirmed SOS-induction dependence of the tolerance development. Interestingly, aPDI/aBL-tolerant S. aureus exhibited increased susceptibility to gentamicin (GEN) and doxycycline (DOX), supporting the hypothesis of genetic alterations induced by sub-lethal phototreatments. The obtained results indicate that S. aureus may develop stable tolerance to studied phototreatments upon sub-lethal aPDI/aBL exposure; thus, the risk of tolerance development should be considered significant when designing aPDI/aBL protocols for infection treatments in vitro and in clinical settings.

Project description:It is known that multiple cationic charges are required to produce broad-spectrum antimicrobial photosensitizers (PS) for photodynamic inactivation (aPDI) or photodynamic therapy of bacteria and fungi. In the present study we describe the synthesis and aPDI testing of a set of derivatives prepared from the parent pheophytin molecule with different numbers of attached side arms (1-3) each consisting of five quaternized cationic groups (pentacationic), producing the corresponding [Zn2+]pheophorbide-a-N(C2N+C1C3)5 (Zn-Phe-N5+, 5 charges), [Zn2+]chlorin e6-[N(C2N+C1C3)5]2 (Zn-Chl-N10+, 10 charges) and [Zn2+]mesochlorin e6-[N(C2N+C1C3)5]3 (Zn-mChl-N15+, 15 charges). Moreover, a conjugate between Zn-Phe-N5+ and the antibiotic vancomycin called Van-[Zn2+]-m-pheophorbide-N(C2N+C1C3)5 (Van-Zn-mPhe-N5+) was also prepared. The aPDI activities of all compounds were based on Type-II photochemistry (1O2 generation). We tested these compounds against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Escherichia coli, and the fungal yeast Candida albicans. All three compounds were highly active against MRSA, giving eradication (≥6 logs of killing) with <1.0 μM and 10 J cm-2 of 415 nm light. The order of activity was Zn-Phe-N5+ > Zn-Chl-N10+ > Zn-mChl-N15+. In the case of E coli the activity was much lower (eradication was only possible with 50 μM Zn-mChl-N15+ and 20 J cm-2). The order of activity was the reverse of that found with MRSA (Zn-mChl-N15+ > Zn-Chl-N10+ > Zn-Phe-N5+). Activity against C. albicans was similar to E. coli with Zn-mChl-N15+ giving eradication. The activity of Van-Zn-mPhe-N5+ was generally lower than that of Zn-Phe-N5+ (except for E. coli). Red (660 nm) light was also effective as might be expected from the absorption spectra. An initial finding that Van-Zn-mPhe-N5+ might have higher activity against vancomycin resistant Enterococcus fecium (VRE) strains (compared to vancomycin sensitive strains) was disproved when it was found that VRE strains were also more sensitive to aPDI with Zn-Phe-N5+. The minimum inhibitory concentrations of Van-Zn-mPhe-N5+ were higher than those of Van alone, showing that the antibiotic properties of the Van moiety were lessened in the conjugate. In conclusion, Zn-Phe-N5+ is a highly active PS against Gram-positive species and deserves further testing. Increasing the number of cationic charges increased aPDI efficacy on C. albicans and Gram-negative E. coli.

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Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

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Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

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