TY - JOUR A1 - Cull, Benjamin A1 - Lima Prado Godinho, Joseane A1 - Fernandes Rodrigues, Juliany Cola A1 - Frank, Benjamin A1 - Schurigt, Uta A1 - Williams, Roderick AM A1 - Coombs, Graham H A1 - Mottram, Jeremy C T1 - Glycosome turnover in Leishmania major is mediated by autophagy JF - Autophagy N2 - Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes. KW - ATG8 KW - Leishmania KW - TEM KW - glycosome KW - protozoan parasite KW - ATG KW - autophagy-related KW - GFP KW - green fluorescent protein KW - MVT KW - multivesicular tubule KW - RFP KW - red fluorescent protein KW - transmission electron microscopy KW - adaptation KW - autophagy KW - mC KW - mCherry KW - fluorescent protein Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150277 VL - 10 IS - 12 ER - TY - JOUR A1 - Abda, Ebrahim M. A1 - Krysciak, Dagmar A1 - Krohn-Molt, Ines A1 - Mamat, Uwe A1 - Schmeisser, Christel A1 - Förstner, Konrad U. A1 - Schaible, Ulrich E. A1 - Kohi, Thomas A. A1 - Nieman, Stefan A1 - Streit, Wolfgang R. T1 - Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and \(\beta\)-Lactamase Expression JF - Frontiers in Microbiology N2 - Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas rnaltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the Li and L2 beta-lactamases in response to beta-lactam treatment. Here we report that the patient isolate S. rnaltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bleu and bla(L2) were transcriptionally the most strongly upregulated genes. Promoter fusions of b/a(L1) and b/a(L2) genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla(L2) expressing cells as identified by RNA(seq) analysis. Overexpression of cornE in S. maltophilia K279a reduced the level of cells that were in a bla(L2)-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including b/a(L1), b/a(L2), and comE. KW - xanthomonas maltophilia KW - gram-negative bacteria KW - RNA-seq KW - pseudomas aeruginosa KW - antibiotic resistance KW - colony morphotypes KW - beta-lactamases KW - K279a KW - Stenotrophomonas maltophilia KW - phenotypic heterogeneity KW - persister cells KW - streptococcus pneumoniae KW - nosocomial pathogen KW - membrane vesicles KW - sinorhizobium fredii NGR234 KW - red fluorescent protein KW - escherichia coli Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136446 VL - 6 IS - 1373 ER -