Caracterização química e quantificação das porfirinas produzidas por amostras do complexo Corynebacterium diphtheriae isoladas de casos de difteria e infecções invasivas e sua utilização como fotossensibilizador em terapia de fotoinativação

Abstract

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis are Gram-positive bacteria of the Corynebacterium diphtheriae complex, known to produce diphtheria toxin and other virulence factors that contribute to their pathogenicity in different clinical contexts. In addition to diphtheria, caused by C. diphtheriae and C. ulcerans, systemic infections, usually by non-toxigenic strains of the C. diphtheriae complex, are increasingly observed, including cases with resistance to antibiotic therapy. Elucidating the virulence mechanisms of these pathogens, in addition to diphtheria toxin, is fundamental for the development of new drugs and technologies for prevention and treatment. Porphyrins are macrocyclic molecules involved in the metabolism of the heme group. Eukaryotic and bacterial cells share a common precursor, aminolevulinic acid (ALA), which, when added to the bacterial culture medium, increases heme metabolism, causing the secretion of porphyrins, useful as endogenous photosensitizers. Photodynamic inactivation using endogenous porphyrins has emerged as a promising approach to fighting bacterial infections, since these molecules, when interacting with light, produce reactive oxygen species that induce microbial death. In this study, the chemical characterization and quantification of porphyrins produced by C. diphtheriae, C. ulcerans and C. pseudotuberculosis, isolated from cases of diphtheria and invasive infections, was carried out using emission spectrometry and high-performance liquid chromatography (HPLC) coupled with fluorescence (FL), ultraviolet (UV) and mass spectrometry (MS) detectors. Three porphyrins were identified: uroporphyrin I, protoporphyrin IX and coproporphyrin III, with the latter predominating. Quantification showed variations between the samples. The influence of ALA, iron and hydrogen peroxide on the porphyrin profile in C. diphtheriae strains was also investigated. The addition of ALA (2 mmol/L) increased porphyrin production, while the addition of iron (2 μg/mL) and H₂O₂ at subinhibitory concentrations (subMIC) reduced production. The porphyrin profile was not altered. The study demonstrated the potential of endogenous porphyrins from C. diphtheriae as photosensitizers for bacterial photoinactivation in planktonic and biofilm cultures. The experiments showed a significant reduction in colony-forming units/mL (CFU/mL) in all samples after exposure to LED light (402 nm). The efficiency of photoinactivation correlated directly with the amount of porphyrins produced. ALA supplementation increased photoinactivation efficiency, and planktonic cells were slightly more susceptible to light than biofilm cells. Biofilm formation correlated positively with porphyrin production, suggesting a relationship between heme metabolism and biofilm formation.