ABSTRACT
BACKGROUND The insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation. OBJECTIVES To determine the occurrence of alternative splicing in chitinases of L. longipalpis. METHODS We sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA. FINDINGS We showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae. MAIN CONCLUSIONS We show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.
Subject(s)
Animals , Chitinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Digestive System/enzymology , Chitinases/physiology , Alternative Splicing/geneticsABSTRACT
Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14) and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.
Subject(s)
Humans , Leprosy , Mycobacterium leprae , Osteoblasts/enzymology , Schwann Cells/enzymology , Down-Regulation , Flow Cytometry , Gene Expression Regulation , Immunohistochemistry , Leprosy , Leprosy/pathology , PHEX Phosphate Regulating Neutral Endopeptidase , PHEX Phosphate Regulating Neutral Endopeptidase , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.
Subject(s)
Humans , Mycobacterium leprae/enzymology , Serine Endopeptidases/biosynthesis , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Bacterial/physiology , Molecular Sequence Data , Mycobacterium leprae/genetics , Spectroscopy, Fourier Transform InfraredABSTRACT
La relación entre el buen estado físicao, ejercicio regular y densidad mineral ósea (BMD) de columna lumbar y cuello de fémur, fueron estudiadas en 60 pacientes, entre 113 mujeres psotmenopáausicas de Cleveland Menopause Clinic, dirigida por el Dr. Utian. En cada sujeto se evaluó la masa ósea por densitometría, midiendo el contenido mineral óseo. Se incluyeron sólo las pacientes de tipo corporal normal. Se objservó una diferencia estadisticamente significativa, entre mujeres de vida activa y sedentaria, en relación a pacientes con y sin osteoporosis
Subject(s)
Humans , Female , Middle Aged , Adult , Bone Density , Exercise/physiology , Menopause , Osteoporosis/physiopathology , Osteoporosis/therapy , Risk Factors , SportsABSTRACT
Se estudió en 113 mujeres postmenopáausicas (PostM) el efecto de los componentes de una dieta normal, estas pacientes respondieeron a un cuestionario entre 600 mujeres, a quienes se le efectuó una densitometría(BMD) en Cleveland Menopause Clinic, dirigida por el Pro. Dr. Utian. Las mismas fueron divididas en dos grupos de acuerdo a la densidad mineral ósea (BMD) pacientes con ossteoporosis y pacientes sin osteoporosis o normales