Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Antibacterial photosensitization through activation of coproporphyrinogen oxidase.

Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of CgoX induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, CgoX activation reduces bacterial burden in murine models of SSTI. Thus, small-molecule activation of CgoX represents an effective strategy for the development of light-based antimicrobial therapies.

Exogenous coproporphyrin III production by Corynebacterium aurimucosum and Microbacterium oxydans in erythrasma lesions.

Erythrasma is a superficial skin disease caused by Gram-positive Corynebacterium species. Coral-red fluorescence under Wood's light, strongly suggestive of erythrasma, can be attributed to the presence of porphyrins. Fractionated porphyrin analysis in erythrasma lesions is yet to be reported. We attempted to investigate erythrasma lesions by isolating the responsible bacteria and determining their exogenous porphyrin production by HPLC analysis. We observed a 78-year-old woman with erythrasma who had a well-demarcated slightly scaling patch on her left foot, between the fourth and fifth toes. Two kinds of colonies on 5 % sheep blood agar were obtained from this lesion. Analysis of the 16S rRNA sequence revealed the colonies to be Corynebacterium aurimucosum and Microbacterium oxydans. HPLC analysis demonstrated that coproporphyrin III (Copro III) levels were clearly elevated, although the amounts of protoporphyrin were diminished. These results indicate that the fluorescent substance was Copro III. This study supports the view that excess Copro III synthesis by C. aurimucosum and M. oxydans leads to accumulation of porphyrin in cutaneous tissue, which emits a coral-red fluorescence when exposed to Wood's light.

Induction of a chemoattractive proinflammatory cytokine response after stimulation of keratinocytes with Propionibacterium acnes and coproporphyrin III.

BACKGROUND: The inflammation in acne vulgaris is widely thought to be induced by an immunological reaction, but the role of Propionibacterium acnes is unclear. OBJECTIVES: To examine the local host response mechanism of a keratinocyte cell line 3 h and 6 h after stimulation with viable and heat-killed P. acnes. METHODS: The quantitative expression of cytokines was measured at the mRNA level by real-time reverse transcription-polymerase chain reaction. RESULTS: The coincubation of a keratinocyte cell line with viable, but not heat-killed, P. acnes modulated an adequate cytokine response for interleukin (IL)-1beta, granulocyte/macrophage colony-stimulating factor and IL-8. High-performance liquid chromatographic analysis of the in vivo porphyrin pattern secreted by P. acnes revealed a predominance of coproporphyrin III in acne lesions. This same porphyrin fraction also modestly induced IL-8 expression by keratinocytes. CONCLUSIONS: This cytokine pattern may favour a chemotactic response and implicates P. acnes and coproporphyrin III in the recruitment of inflammatory cells to the site of infection and in the development of acne lesions.

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures.

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.

Increased urinary coproporphyrin excretion observed in patients with differently staged Hodgkin's disease treated with chemotherapy.

It has been reported that patients with Hodgkin's disease (HD) show altered porphyrin metabolism, and suggested that the cause is the neoplastic process itself. If this is true, disease progression should be associated with higher levels of porphyrin excretion. The aim of this study was to evaluate urinary coproporphyrin levels in patients with Hodgkin's disease at different stages. As many of the patients received chemotherapy, another aim was to verify experimentally whether chemotherapeutic agents might increase porphyrin levels in rabbits. All of the patients had above-normal urinary coproporphyrin levels. On the other hand, rabbits receiving the porphyrin precursor 5-aminolevulinic acid (5-ALA), and also treated with doxorubicin, showed very high plasma porphyrin levels. The increased levels of urinary coproporphyrins seem to be due to the disease itself, since the patients in stages III and IV had higher excretion values, presumably due to biochemical heme synthesis lesions that lead to the availability of the porphyrin precursor, as well as coproporphyrin accumulation and excretion. The altered porphyrin synthesis may be attributable to the cytotoxic oxygen species generated in the presence of NADH and iron. As the patients also received extensive chemotherapy regimes, the altered porphyrin metabolism may be affected by antineoplastic treatment generating oxygen reactive radicals. The alterations in porphyrin metabolism induced by chemotherapeutic agents appear to be demonstrated in rabbits in which doxorubicin increases porphyrin synthesis after porphyrin precursor treatment.

delta-Aminolaevulinic acid-induced porphyrin synthesis and photodynamic inactivation of Escherichia coli B.

The possibility and conditions for the induction of porphyrin synthesis by exogenous delta-aminolaevulinic acid (ALA) and its applicability for the inactivation of Gram-negative bacteria Escherichia coli B. by photodynamic therapy (PDT) have been studied. The bacteria are supplemented with ALA in the log phase of growth, and are grown in a synthetic medium at 37 degrees C in the dark. The efficiency of porphyrin synthesis is detected by fluorescence spectroscopy performed on the isolated bacterial cells and the medium, respectively, and compared with results of high-performance liquid chromatography (HPLC) analysis. ALA stimulates the synthesis of protoporphyrin in the bacteria by a factor of five to six, and an increased amount of the more hydrophilic derivatives with a significant contribution of mesoporphyrin by a factor of two to three is observed in the culturing medium. The optimal conditions of ALA treatment with respect to PDT are 10-15 min of incubation of a bacterial culture of 2 x 10(7) cells ml-1 with (5-9) x 10(-3) mol l-1 ALA. The ALA-treated cells are irradiated by white light of 80 mW cm-2 under growth conditions and a decrease to 0.6% of the number of colony-forming units (CFUs ml-1) is observed after 90 min of irradiation.

The Alcaligenes eutrophus hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase, is required for heme biosynthesis during anaerobic growth.

The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite. When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis. With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment. Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa. In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified. The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN. HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX. Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A. eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector. Likewise, the A. eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth. Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A. eutrophus is nitrate-independent and repressed by oxygen.

Excretion pattern of faecal coproporphyrin isomers I-IV in human porphyrias.

The relative proportions of the four coproporphyrin isomers I-IV were analysed in faeces of 20 healthy subjects and 60 patients suffering from one of the seven common types of hepatic or erythropoietic hereditary porphyrias. A newly developed, reliable method for sample preparation was applied, using reversed-phase thin layer chromatography for the isolation of naturally occurring coproporphyrin free carboxylic acids. Accurate separation and quantitation of the individual isomers I-IV were achieved with the help of ion-pair high-performance liquid chromatography. The four coproporphyrin isomers I-IV were positively identified by on-line scanning of their fluorescence spectra in the emission and excitation modes. Recovery rates with this new analytical procedure were between 90 and 100%, and coefficients of variation varied between 0.8 and 5.7% (N = 7). Diagnostically important findings were greatly increased proportions of isomer I and decreased proportions of isomers III, II and IV in erythropoietic porphyrias, such as congenital erythropoietic porphyria and protoporphyria. Significantly increased proportions of isomers III, II and IV, on the other hand, were observed in acute hepatic porphyrias, e.g. acute intermittent porphyria and porphobilinogen synthase deficiency porphyria, as compared with porphyria cutanea tarda (p < 0.005 and p < 0.03, respectively). Inversion of the faecal coproporphyrin III to I ratios and markedly elevated percentages of the atypical isomers II and IV were important diagnostic markers for variegate porphyria and hereditary coproporphyria. The highest proportions of isomer III were found in hereditary coproporphyria, where the amount of the isomers II and IV exceeded that of isomer I. Asymptomatic carriers of the relevant gene defect in families with hereditary coproporphyria could be detected by an increased faecal coproporphyrin III to I ratio. Our results clearly demonstrate the potential of faecal coproporphyrin I-IV isomer ratios for the diagnosis and differential diagnosis of hereditary porphyrias.

Endogenous porphyrins in murine skin and transplanted PAM-212 squamous cell carcinoma tissues after injection of delta-aminolevulinic acid.

After intraperitoneal (IP) injection of delta-aminolevulinic acid (ALA), the endogenous porphyrins in murine skin and tumor tissues were determined by a method involving solvent and acid extractions. The results showed that the total amount of porphyrins in the tumor tissues after ALA injection was much higher than that in the skin from the same mice, although the amount of porphyrins in the skin from the ALA-injected mice was higher than that from the saline-injected (control) mice. The porphyrins in the tumor were mostly protoporphyrin and coproporphyrin, with only a small amount of uroporphyrin. The optimum period for porphyrin accumulation in the tumor as well as in the skin was 1 hour after the injection of ALA. As the period was extended to 3 and 6 hours, the amount of porphyrins in these tissues decreased considerably. These findings could be valuable for further application of ALA in the photodynamic therapy of skin cancer.

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis.

Aluminum (0.74 mM) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with delta-aminolevulinic acid (delta-ALA, 1.2 mM) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. delta-ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 +/- 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.

The production of porphyrins from delta-aminolaevulinic acid by Haemophilus parainfluenzae.

Porphyrin production by the haemin-independent Haemophilus parainfluenzae in the diagnostic porphyrin test, which determines the X-factor requirement in Haemophilus spp., was analysed quantitatively by applying modern high-performance liquid chromatographic (HPLC) methods. Ion-pair reversed-phase HPLC enabled the simultaneous separation of all porphyrin intermediates and their isomers of haem biosynthesis produced by the bacteria. The pH-dependence of porphyrin production and the respective composition of the porphyrins within the bacterial cells and in the supernate were investigated. A pH optimum of 6.9-8.0 for the production of porphyrins was found and there were marked differences in the porphyrin profiles at different pH values.

Coproporphyrin isomers II and IV are normal constituents of human urine.

We describe for the first time the detection of small amounts of the atypical coproporphyrin isomers II and IV in urine from healthy subjects. Efficient sample preparation procedures together with a highly selective isocratic ion-pair high-performance liquid chromatographic method enabled the determination of these unexpected compounds. Formation of the atypical coproporphyrin isomers was shown to result mostly from non-enzymatic isomerization at the coproporphyrinogen level within the human body, whereas only traces were formed in vitro during collection and storage of the urine samples under the conditions applied.

Destruction of erythroleukaemic cells by photoactivation of endogenous porphyrins.

Selective destruction of Friend erythroleukaemic cells (FELC) was potentiated by stimulation of endogenous porphyrin synthesis followed by light sensitization. Endogenous porphyrin biosynthesis in FELC was induced by supplementation of 5-amino levulinic acid (5-ALA) at a concentration of 5 X 10(-4) M. The main accumulated product, after 4 days culture, was uroporphyrin, while after 8 days culture the cells were loaded with protoporphyrin, up to 1.5 micrograms 10(-7) cells. Photoirradiation of the cells for 2 min, accumulating endogenous porphyrins, induced cardinal deformations and cell disintegration in greater than 95% of the cells, as examined by scanning electron microscopy (SEM). The photodynamic destruction effects were dependent on cultivation time with 5-ALA. Flow cytometry analysis showed an immediate expansion of cell volume subsequent to irradiation, presumably a consequence of water influx. Transmission electron microscopy (TEM) of photosensitized cells after different time intervals of culture in 5-ALA medium, revealed initial damage to mitochondria and water influx into the nuclear envelope, after 2 days. After 3-4 days in culture the water influx phenomenon was pronounced, chromatin condensation took place and slight rupture of the outer membrane was detected. Cells photosensitized after 5-6 days of culture were completely disintegrated leaving a nuclear remnant and an enormously swollen nuclear envelope. The culture time dependence of the process, showed an interrelationship between the photodynamic effect and porphyrin accumulation sites in cellular compartments. The study presents a specific method for erythroleukaemic cell inactivation.

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.

Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria.

Partial deficiencies in enzymes activity of the heme biosynthesis pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients suffering from congenital erythropoietic porphyria and hereditary coproporphyria. Using a new fluorimetric method, we have assessed quantitatively porphyrin biosynthesis from added delta-aminolevulinic acid in cultured fibroblasts of two congenital erythropoietic porphyria patients and one homozygous case of hereditary corproporphyria. The results were compared with those of the patients' parents and those of normal controls. All the porphyrins synthesized remained within the cells of normal subjects and of patients with congenital erythropoietic porphyria; these porphyrins were mostly (95%) protoporphyrin. The fibroblasts of the patient with homozygous hereditary coproporphyria synthesized the same amount of porphyrins, but only 25% were found within the cells, whereas 75% were found in the medium. The porphyrins found within the cells were coproporphyrin (25%) and protoporphyrin (75%); in the medium, only coproporphyrin was identified.

Uroporphyrin- and coproporphyrin I-accumulating mutant of Escherichia coli K12.

A new type of haem-deficient mutant was isolated in Escherichia coli K12 by neomycin selection. The mutant was deficient in uroporphyrinogen III cosynthase activity as indicated by the accumulation of uroporphyrin I and coproporphyrin. The mapping of the corresponding hemD gene by P1-mediated transduction showed that the new gene was located between ilv and cya, at min 83 on the chromosomal map of Escherichia coli K12.

[Effect of ultrasound on porphyrin biosynthesis. I. Changes in the levels of proto-, copro- and uroporphyrins after a single exposure to ultrasound]. / Vliianie ul'trazvuka na zven'ia biosinteza porfirinov. I. Izmenenie urovnia proto-, kopro- i uroporfirinov posle odnokratnogo vozdeistviia ul'trazvuka.

Abdominal region of white rat males was exposed to ultrasound with the intensity of 0.2, 0.6 and 1.0 Wt/cm2. 1, 24 and 48 hours after sonication proto-, copro- and uroporphyrines were determined in erythrocytes and excrements, copro- and uroporphyrines in urea. Single ultrasonic exposure does not affect the biosynthesis of uro- and coproporphyrines in gialoplasm, however, it results in the change of their excretion with excrements and urea. Biosynthesis of protoporphyrine IX which is the final stage of haemoglobin biosynthesis is changed under the effect of ultrasonic energy.