Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes

Abstract

Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.

Figure 1. Isolation of hydrogenosomal fractions from T. vaginalis cells grown under iron-enriched (+Fe) and iron-depleted (−Fe) conditions.

(A) The fraction enriched in hydrogenosomes was further fractionated with an Optiprep density gradient. (B) Analysis of Optiprep fractions by Western blotting using an antibody against hydrogenosomal malic enzyme. (C–D) Transmission electron microscopy of fraction #7, which was chosen for proteomic analysis.

Figure 2. Proteins of the iron-sulfur cluster assembly machinery identified in this study.

Gene IDs marked with an asterisk denote proteins that were significantly regulated in response to iron availability.

Figure 3. Proteins of hydrogenosomal energy metabolism identified in this study.

Gene IDs marked with an asterisk denote proteins that were significantly regulated in response to iron availability.

Figure 4. Comparison between iron-regulated proteins determined with the proteomic approach and the expression of corresponding genes studied by DNA microarrays and comparative EST analysis .

Red square, significant upregulation under high iron; pink square, insignificant upregulation under high iron; green square; significant upregulation under low iron; light green square, insignificant upregulation under low iron; empty square, no change in the transcript level; black square, a gene that was not included in the analysis.