Hava Ladan

The binding and photosensitization effects of tetrabenzoporphyrins and texaphyrin in bacterial cells

A new class of photosensitizers which inactivate efficiently Gram-positive bacterial cells is described. The photosensitizers used in this study were Mg and Zn-tetrabenzoporphyrin (MgTBP and ZnTBP), which possess strong absorption bands at 630 nm and Cd-texaphyrin (CdTX), whose longest absorption band is at 770 nm. These sensitizers thus have optical properties that are superior to those of haematoporphyrin. It is demonstrated that MgTBP and CdTX have similar photosensitizing efficiencies as haematoporphyrin on Gram-positiveStaphylococcus aureus cells but are better on the Gram-negativeEscherichia coli in the presence of polymyxin nonapeptide, a membrane permeabilizing material. It seems, from the elemental content of the photosensitized Gram-positive and Gram-negative bacterial cells, that there is a difference in the killing mechanism of haematoporphyrin as compared to that of MgTBP or CdTX.

The bactericidal activity of a deuteroporphyrin—hemin mixture on gram-positive bacteria. A microbiological and spectroscopic study

The combined antibacterial activity of various porphyrins with hemin on Gram-positive bacteria was studied. Protoporphyrin, hematoporphyrin derivative and deuteroporphyrin show only a marginal inhibitory effect in the dark. However, hemin has a strong cytotoxic effect which is independent of illumination and is equally strong in the dark. The disadvantage of hemin treatment is that it is temporary. In this study, we have demonstrated that a combination of deuteroporphyrin and hemin has a unique cytotoxic activity on Staphylococcus aureus, Streptococcus faecalis and Bacillus cereus. The effect of the combined compound is stronger than that of the separate constituents, and is as strong in the dark as in the light. Only 0.005% of the initial S. aureus population survive after a 2 h treatment. Absorption and fluorescence spectra of hemin-deuteroporphyrin mixtures in water and liposomes suggest the formation of a species with spectroscopic properties which are different from those of the two constituents.

Spectroscopy, photokinetics, and cellular effect of far-red and near-infrared absorbing photosensitizers

Two types of photosensitizers are compared here for their singlet oxygen production and in vivo sensitizing capacity: Mg2+ and Zn2+-tetrabenzoporphyrin, which have a strong Q band at the HeNe laser line; and Cd-texaphyrin, with absorption at 770 nm. The singlet oxygen quantum yields in homogeneous solutions were determined by employing a singlet oxygen-destroyable fluorescent target. Photosensitizers of bacterial and human leukemic cells were carried out with these sensitizers, in comparison with HPD and Photofrin II. The new sensitizers were found to have high quantum yields for production of singlet oxygen in solutions. They also have comparable photosensitizing capacities of bacterial cells.

In vivo effects of porphyrins on bacterial DNA

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.

Mesosomal structures and antimicrobial activity induced by hemin oxidation or porphyrin photodynamic sensitization inStaphylococci

The porphyrin-dependent inactivation ofStaphyloccocus aureus and the induction of mesosomal structures are described. The antimicrobial activity of different photoactivated porphyrins was compared with the dark cytotoxic effect of hemin. Deuteroporphyrin, protoporphyrin, hematoporphyrin, and hematoporphyrin derivative (Hpd) markedly reduced cell growth upon irradiation with light. Photofrin II, the polymerized fraction of Hpd, and other synthetic porphyrins had no effect on staphylococcal growth. Hemin immediately inhibited cell viability in the dark and induced the development of an irregular cell wall, analyzed by scanning electron microscopy (SEM). Inside the cytoplasm a multivesicular mesosome was formed near the septum, as detected by transmission electron microscopy (TEM). The mesosomal volume and its frequency in the cells was increased in a time-dependent manner. The mesosome appearance was not related to fixation by glutaraldehyde or to post-fixation by osmium tetroxide. Glycosyl moieties stained by ruthenium red revealed the formation of membrane-like structures in the mesosome. It is concluded that oxygen-dependent reactions potentiated by porphyrins may induce disturbances in the synthesis of staphylococcal membrane and cell wall, revealed as mesosomes.

Growth-inhibitory Effect of Hemin on Staphylococci

The antibacterial activity of hemin onStaphylococcus aureus is described. Hemin binding to bacteria was a rapid process, and each cell accumulated 5×105 to 1×106 molecules within 5 min. Bacterial growth was stopped completely after 30 min from addition of low concentration of hemin (3–10 μg/ml). Cell viability was reduced by 99.9% in 1 h of exposure, and the effect was consistent at any stage of the growth curve whenever hemin was added. Glucose utilization was arrested immediately after hemin addition, and no CO2 was produced. The survivors of hemin treatment regrow in a time-related kinetics depending on the dose of hemin to which the cells were exposed. The recovered bacteria were again sensitive to hemin, similar to an untreated culture. We suggested that the recovery phenomenon is a result of an “on-off” mechanism regulating sensitivity to hemin, rather than a selection mechanism giving rise to hemin-resistant mutants.

Antimicrobial and antiviral activity of porphyrin photosensitization

In order to photosensitize Gram (-) bacteria such as Pseudomonas aeuruginosa and Escherichia coli, we introduced the small peptide polymyxin-B nona-peptide (PBNP) which stimulated the translocation of porphyrin through the outer membrane of these bacteria and makes PDT possible. Gram negative cell killing by the use of PBNP and DP broadens the antibacterial spectrum of photodynamic inactivation and opens new horizons for this modality as a wide spectrum drug when antibiotic resistance is the main concern. Plasmidial and chromosomal DNA damage in S. aureus and E. coli cells was mediated by DP photosensitization. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected and its degradation products were detected after treatment.