Continued proteomic analysis of Mycobacterium leprae subcellular fractions.

Recently the sequence of the Mycobacterium leprae chromosome, the only known obligate intracellular mycobacterium, was completed. It has a dramatic reduction in functional genes, with a coding capacity of only 49.5%, the lowest one so far observed among bacterial genomes. The leprosy bacillus seems to preserve a minimal set of genes that allows its survival in the host. The identification of genes that are actually expressed by the bacterium is of high significance in the context of mycobacterial pathogenesis. In this current study, a proteomic approach was undertaken to identify the proteins present in the soluble/cytosol and membrane subcellular fractions obtained from armadillo derived M. leprae. Proteins from each fraction were separated by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry. A total of 147 protein spots were identified from 2-DE patterns and shown to comprise products of 44 different genes, twenty eight of them corresponding to new proteins. Additionally, two highly basic proteins (with pI >10.0) were isolated by heparin affinity chromatography and identified by N-terminal sequencing. This study constitutes the first application of proteomics to a host-derived Mycobacterium.

Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis.

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.

Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients.

We have reported previously that both urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) are statistically significant prognostic variables in patients with high-risk breast cancer (Grondahl-Hansen et al., Cancer Res., 53:2513-2521, 1993), and we recently described that the uPA receptor (uPAR) is a prognostic marker in postmenopausal, node-positive breast cancer patients (Grondahl-Hansen et al., Clin. Cancer Res., 1:1079-1087, 1995). The present retrospective study describes the prognostic impact of uPA, its receptor uPAR, and PAI-1 in breast cancer cytosol from 111 low-risk premenopausal patients and 184 low-risk postmenopausal patients with a median follow-up of 6.0 years (range, 3.8-14.9) and 7.4 (range, 3.7-14.0) years, respectively. uPA, uPAR, and PAI-1 levels were determined by sandwich enzyme-linked immunosorbent assays, and data were dichotomized using the median value as the cutoff for calculation of recurrence-free survival and overall survival. A correlation was found between the levels of each of the three molecules. In univariate analysis, high PAI-1 was significantly associated with short overall survival in postmenopausal patients [relative risk (RR), 2.3; 95% confidence interval (CI), 1.3-4.3; P = 0.005] and shorter recurrence-free survival in both premenopausal (RR, 2.5; 95% CI, 1.3-4.7; P = 0.004) and postmenopausal (RR, 1.8; 95% CI, 1.1-2.9; P = 0.02) patients. Neither uPA nor uPAR reached statistical significance in the univariate analyses. The prognostic value of uPA, uPAR, and PAI-1 was then compared with that of other established prognostic variables by multivariate analysis. PAI-1 was an independent prognostic variable for recurrence-free survival in premenopausal patients, with a RR of 2.6 (95% CI, 1.3-5.0). For recurrence-free survival (RR, 1.9; 95% CI, 1.1-3.5) and overall survival (RR, 2.6; 95% CI, 1.2-5.7) in postmenopausal patients, PAI-1 was the only independent variable left in this group of patients. Neither uPA nor uPAR reached significance in the multivariate analysis. These data, together with previously published data on the prognostic significance of components of the urokinase plasminogen activation system in breast cancer cytosols, strongly indicate that PAI-1 is a statistically significant and independent prognostic marker in both low- and high-risk breast cancer patients.

Defective pH regulation of acidic compartments in human breast cancer cells (MCF-7) is normalized in adriamycin-resistant cells (MCF-7adr).

Alkalinization of normally acidic intracellular compartments or acidification of a mildly alkaline cytoplasm by biochemical or genetic manipulation has been demonstrated to inhibit both endocytosis and secretion (Tartakoff, 1983a; Cosson et al., 1989; Mellman et al., 1986; Davoust et al., 1987; Cosson et al., 1989; van Deurs et al., 1989; Maxfield & Yamashiro, 1991; Hansen et al., 1993). These results provide the basis for the conclusion that the maintenance of pH gradients between acidic vesicular compartments and a mildly alkaline cytoplasm is an essential biochemical requirement for the correct functioning of the endocytotic and secretory machinery. Tumor cells have been shown to have an abnormally acidic cytoplasmic pH (Warburg, 1956; Simon & Schindler, 1994). Here we report that the intracellular vesicular compartments in tumor cells (MCF-7) derived from a human breast cancer fail to acidify. This failure results in a significant decrease in the pH gradient (0.9 pH unit) between the vesicular luminal compartments and the cytoplasm. These defects are correlated with a disruption in the organization and function of the trans-Golgi network (TGN) and the pericentriolar recycling compartment (PRC). In marked distinction, drug-resistant tumor cells (MCF-7adr) derived from the MCF-7 line that are resistant to the most widely employed chemotherapeutic drug, adriamycin, appear normal in both acidification and organization of the PRC and TGN. Treatment of drug-resistant MCF-7adr cells with nigericin and monensin, ionophores demonstrated to disrupt vesicular acidification (Tartakoff, 1983b), leads to a resensitization of these cells to adriamycin. Drug sensitivity is proposed to result from an acidification defect within vesicles of the recycling and secretory pathways. A functional consequence of this defect is the diminished capacity of cells to remove cytotoxic drugs from the cytoplasm by sequestration of protonated drugs within the vesicles, followed by drug secretion through the activity of the secretory and recycling pathways.

Suppress of the increase in free cytosolic calcium during the inhibition of T-cell activation by an autoantibody present in the serum of leprosy patients.

A serum factor, believed to be an IgG autoantibody, in certain patients with lepromatous leprosy inhibits the proliferation of mitogen-stimulated lymphocytes. To investigate which stage of the cell cycle was inhibited, we examined the effect of these sera on the kinetics of lymphocyte activation induced by several mitogenic agents: phytohaemagglutinin (PHA), the calcium ionophore A23187, the phorbol ester phorbol myristate acetate (PMA) and purified protein derivative of BCG (PPD). Seven out of 54 sera tested were found to inhibit PHA-stimulated proliferation. Inhibitory sera and to a lesser extent serum IgG from leprosy patients were capable of suppressing the increase in free cytosolic calcium normally observed immediately after PHA stimulation. Subsequent stages of the cell cycle, increase in cell size, the expression of the IL-2 receptor and increase in DNA were also suppressed. The inhibitory sera was not toxic and, if addition of the sera was delayed, would not inhibit lymphocytes that had already entered the cell cycle. Using mitogenic agents which act intracellularly, the normal early increase in cell size with A23187- and PMA-stimulated lymphocytes was not affected by inhibitory leprosy sera or serum IgG, but all subsequent steps in the cell cycle were suppressed; although the inhibition of proliferation in PMA-stimulated cultures was incomplete. The mechanism of action of the inhibitory sera and derived IgG, although acting through a cell surface antigen, appears to interfere with a fundamental process in activation since the effect was seen with all of the diverse stimuli examined in this study.