APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.

Semen biochemistry of leprosy patients.

Studies have been made on the semen of three categories (borderline, borderline tuberculoid and lepromatous) of leprosy patients to evaluate the seminal biochemical constituents viz. fructose, glycerylphosphorylcholine and acid phosphatase besides the physical properties viz. volume, pH, liquefaction time, sperm density and sperm motility. In all categories of leprosy patients, seminal pH, liquefaction time and sperm density underwent significant decline. The decline in the seminal volume and sperm motility was significant only in borderline leprosy. It was observed that seminal glycerylphosphorylcholine (GPC) concentration and acid phosphatase activity declined in all categories of leprosy patients but GPC showed a significant decline only in borderline tuberculoid and acid phosphatase declined significantly only in borderline and lepromatous leprosy.

Nature of peritoneal macrophages from DCC immunized mice.

Peritoneal macrophages from mice immunized with the delipidified cell component (DCC) of Mycobacterium leprae showed changes in various parameters such as increased protein synthesis, levels of hydrolytic enzyme and augmented phagocytic ability indicating activation of the cells. Furthermore, the surface structure of the cells were quite different from that of the macrophages of normal mice. These observations indicate that the peritoneal macrophages have been activated to phagocytose and kill M. leprae better in the immunized mice. The ability to kill the pathogen by these cells was reported by us earlier.

Evidence for phagosome-lysosome fusion in Mycobacterium leprae-infected murine Schwann cells.

Murine Schwann cells were infected with viable armadillo-derived Mycobacterium leprae in vitro, and the lysosomal marker enzyme, acid phosphatase, was stained by the Gomori reaction. Electron microscopic analysis revealed that Schwann cells infected with M. leprae possess acid phosphatase and that lysosomes fuse with infected phagosomes.

Mycobacterium leprae surface components intervene in the early phagosome-lysosome fusion inhibition event.

Bone marrow-derived cultured macrophages were infected with Mycobacterium leprae. The bacteria were either used as freshly isolated organisms or incubated with M. leprae antiserum (1:5) for 30 min prior to phagocytosis. Immediately after inoculation (1 to 4 h) and at 1 to 8 days later, macrophages were stained for acid phosphatase activity to assess fusions between phagosomes and lysosomes. Inhibition of fusions was essentially apparent as an early event, which was partially reversed by antiserum treatment of the bacteria, suggesting a role for M. leprae immunogenic surface components in this early phenomenon. Later incubation times (1 to 8 days) did not show any considerable difference between antiserum-treated and nontreated bacteria. The formation of an electron-transparent zone around phagocytized bacteria and its role in phagosome-lysosome fusion was investigated, and a direct relationship could not be established.

N-acetyl-beta-glucosaminidase, beta-glucuronidase and acid phosphatase in Mycobacterium leprae.

N-Acetyl-beta-glucosaminidase, beta-glucuronidase and acid phosphatase activities were detected in cell-free extracts of Mycobacterium leprae (from armadillo liver). Extracts of bacteria which had been treated with 7-diazonaphthalene-1,3-disulphonic acid to inactivate surface enzymes retained 30-45% of the activity of the glycosidases and 15% of the activity of the acid phosphatase. When intact bacteria were treated with 1 M-NaOH, the corresponding activity in the extracts was 4--9% for the glycosidases and 7% for the acid phosphatase. Inhibition studies with lactones and the use of concanavalin A-agarose showed differences between the glycosidases in extracts of M. leprae and those of armadillo liver. Inhibition studies with vanadate using extracts from NaOH-treated bacteria and extracts of armadillo liver showed differences between the acid phosphatases. Enzymes removed from the surface of M. leprae could have been adsorbed to the surface from host tissue (i.e. lysosomal enzymes) or they could have been extracellular enzymes or associated with the bacterial membrane.

The cellular basis for extremity bone loss in leprosy.

Osteoclasts and osteolytic osteocytes have been observed in the majority of 60 samples of bone taken from five patients with lepromatous or tuberculoid leprosy. These results are interpreted to mean that bone loss in patients with leprosy is an acceleration of a normal cellular process and not the result of avascular necrosis. The acceleration of bone resorption could be due to local release of products from M. leprae or host cells, a hypothesis testable by organ culture methods. The presence of lymphocytes and mononuclear cells in bone samples in this and previous studies is discussed with respect to recent evidence of a role for lymphoid cells in bone resorption.

Electron microscopy of peroxidase and acid phosphatase in leprous and uninfected armadillo macrophages: a macrophage subpopulation contains peroxisomes and lacks bacilli.

Lepromatous tissue from armadillos inoculated 24--36 months earlier with Mycobacterium leprae was obtained for electron microscopic studies. Cytochemically stained lepromas revealed a subpopulation of macrophages containing peroxisomes. These peroxidase reactive macrophages were not infected with bacilli. Acid phosphatase was present in macrophages and many of these were infected with bacilli and contained vacuoles and lipid globules. Within the membrane-bound vacuoles, acid phosphatase surrounded bacilli. However, the reaction product ended abruptly at a 15--40 millimicron thick zone of low electron density surrounding intact bacilli. Acid phosphatase was more intensely reactive and localized less precisely in heavily infected and vacuolated macrophages than in lightly and non-infected cells. The effectiveness of this bacillary barrier and the numerous infected macrophages with substantial acid phosphatase argue against the ability of acid phosphatase to protect host cells from leprosy bacilli. Evidence suggests a protective action of peroxidase or the rapid turnover of macrophages within lepromas. Granular and membranous debris were commonly seen within vacuoles of infected macrophages. A portion of the debris was ultrastructurally similar to bacillary matrix and was nonreactive for peroxidase and acid phosphatase. Following homogenization and centrifugation, similar materials banded with bacilli above 60% sucrose. Another portion of the debris was ultrastructurally similar to host lysosomal matrix and was reactive for acid phosphatase. Results support the concept of dual host and parasitic origins of the debris found in phagolysosomes of infected macrophages. Transparent, oval Epon defects remained eccentric to the majority of intact bacilli in centrifuged fractions. Apparently, an intrinsic property of leprosy produced these Epon defects.

Macrophage function in leprosy.

The macrophage function in patients with leprosy was assessed by estimating histochemically the acid phosphatase activity in skin biopsies and by assessment of phagocytic and lytic capability of in vitro cultured macrophages derived from peripheral blood monocytes, challenged with live M. leprae. Acid phosphatase was demonstrated in skin biopsies of different groups of leprosy patients classified according to the Ridley and Jopling scale. The degree of acid phosphatase positivity was correlated with clinical spectrum, Bacterial and Morphologic Indices and treatment status. Peripheral blood monocytes from patients with leprosy, either tuberculoid or lepromatous, were cultured in monolayers and challenged with M. leprae. The phagocytosis and lysis of mycobacteria by macrophages was observed at different time intervals from the 1st to the 28th day. The morphology of the macrophages in different types of leprosy was also studied. The results suggest that macrophages from patients with either tuberculoid or lepromatous leprosy are not by themselves capable of lysing live M. leprae. Live M. leprae injected into the foot pad of Wistar strain of rats evoked similar responses on the tenth day, in normal and protein deficient animals.