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1.
Proc Natl Acad Sci U S A ; 110(43): 17564-9, 2013 Oct 22.
Article in English | MEDLINE | ID: mdl-24101497

ABSTRACT

Metronidazole and other 5-nitroimidazoles (5-NI) are among the most effective antimicrobials available against many important anaerobic pathogens, but evolving resistance is threatening their long-term clinical utility. The common 5-NIs were developed decades ago, yet little 5-NI drug development has since taken place, leaving the true potential of this important drug class unexplored. Here we report on a unique approach to the modular synthesis of diversified 5-NIs for broad exploration of their antimicrobial potential. Many of the more than 650 synthesized compounds, carrying structurally diverse functional groups, have vastly improved activity against a range of microbes, including the pathogenic protozoa Giardia lamblia and Trichomonas vaginalis, and the bacterial pathogens Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis. Furthermore, they can overcome different forms of drug resistance, and are active and nontoxic in animal infection models. These findings provide impetus to the development of structurally diverse, next-generation 5-NI drugs as agents in the antimicrobial armamentarium, thus ensuring their future viability as primary therapeutic agents against many clinically important infections.


Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Nitroimidazoles/chemistry , Nitroimidazoles/pharmacology , Animals , Bacteroides fragilis/drug effects , Cell Survival/drug effects , Clostridioides difficile/drug effects , Combinatorial Chemistry Techniques , Giardia lamblia/drug effects , Giardiasis/drug therapy , Giardiasis/parasitology , HeLa Cells , Helicobacter pylori/drug effects , Humans , Mice , Mice, Inbred C57BL , Molecular Structure , Structure-Activity Relationship , Treatment Outcome , Trichomonas vaginalis/drug effects
2.
Br J Nutr ; 108(10): 1839-46, 2012 Nov 28.
Article in English | MEDLINE | ID: mdl-22264879

ABSTRACT

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.


Subject(s)
Bacterial Adhesion/drug effects , Entamoeba histolytica/drug effects , Entamoeba histolytica/physiology , Milk, Human/chemistry , Oligosaccharides/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/microbiology , Humans , Intestinal Mucosa/cytology , Lactose/chemistry , Oligosaccharides/chemistry
3.
Antimicrob Agents Chemother ; 55(10): 4643-51, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21825286

ABSTRACT

Infections with the diarrheagenic protozoan pathogen Giardia lamblia are most commonly treated with metronidazole (Mz). Treatment failures with Mz occur in 10 to 20% of cases and Mz resistance develops in the laboratory, yet clinically, Mz-resistant (Mz(r)) G. lamblia has rarely been isolated from patients. To understand why clinical Mz(r) isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mz(r) cell lines with stable, high-level resistance to Mz and significant cross-resistance to tinidazole, nitazoxanide, and furazolidone. Oral infection of suckling mice revealed that three of five Mz(r) cell lines could not establish infection, while two Mz(r) cell lines infected pups, albeit with reduced efficiencies. Failure to colonize resulted from a diminished capacity of the parasite to attach to the intestinal mucosa in vivo and to epithelial cells and plastic surfaces in vitro. The attachment defect was related to impaired glucose metabolism, since the noninfectious Mz(r) lines consumed less glucose, and glucose promoted ATP-independent parasite attachment in the parental lines. Thus, resistance of Giardia to Mz is accompanied by a glucose metabolism-related attachment defect that can interfere with colonization of the host. Because glucose-metabolizing pathways are important for activation of the prodrug Mz, it follows that a fitness trade-off exists between diminished Mz activation and reduced infectivity, which may explain the observed paucity of clinical Mz(r) isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.


Subject(s)
Antiprotozoal Agents/pharmacology , Drug Resistance , Giardia lamblia/drug effects , Giardiasis/parasitology , Metronidazole/pharmacology , Animals , Cell Line , Furazolidone/pharmacology , Giardia lamblia/metabolism , Giardia lamblia/physiology , Giardiasis/drug therapy , Glucose/metabolism , Mice , Mice, Inbred C57BL , Nitro Compounds , Thiazoles/pharmacology , Tinidazole/pharmacology
4.
J Antimicrob Chemother ; 65(3): 531-4, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20067984

ABSTRACT

OBJECTIVES: Attachment to the small intestinal mucosa is crucial for initiating and maintaining Giardia infection. We tested the effect of isoflavones on Giardia attachment. METHODS: We evaluated the effect of formononetin on trophozoite attachment to glass, to intestinal epithelial cell layers in vitro and to murine small intestinal explants, and on the intestinal load in mice. RESULTS: We found that the isoflavone formononetin inhibits both attachment and flagellar motility within minutes and reduces the trophozoite load of Giardia in mice within 1.5 h after treatment. CONCLUSIONS: The antigiardial activity of formononetin is at least partially due to its capacity to rapidly detach trophozoites.


Subject(s)
Antiprotozoal Agents/pharmacology , Cell Adhesion/drug effects , Giardia lamblia/drug effects , Isoflavones/pharmacology , Animals , Cell Line , Flagella/drug effects , Humans , In Vitro Techniques , Intestines/parasitology , Mice , Mice, Inbred C57BL , Trophozoites/drug effects
5.
PLoS Negl Trop Dis ; 14(4): e0008224, 2020 04.
Article in English | MEDLINE | ID: mdl-32302296

ABSTRACT

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.


Subject(s)
Antiparasitic Agents/pharmacology , Click Chemistry/methods , Drug Discovery/methods , Giardia lamblia/drug effects , Indazoles/pharmacology , Protozoan Proteins/metabolism , Animals , Antiparasitic Agents/chemical synthesis , Antiparasitic Agents/therapeutic use , Disease Models, Animal , Female , Giardiasis/drug therapy , Indazoles/chemical synthesis , Indazoles/therapeutic use , Intestine, Small/parasitology , Male , Mice, Inbred C57BL , Parasite Load , Protein Binding , Proteomics/methods
6.
Curr Opin Microbiol ; 10(6): 554-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17981075

ABSTRACT

To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoites attach to enterocytes that mature and die. The parasites must 'decide' whether to re-attach or differentiate into cysts that survive in the environment and re-activate when ingested. Other intestinal parasites face similar challenges. Study of these parasites is limited because they do not encyst in vitro. Giardia trophozoites were persuaded to encyst in vitro by mimicking physiological stimuli. Cysts are dormant, yet 'spring-loaded for action' to excyst upon ingestion. Giardial encystation has been studied from morphological, cell biological, biochemical, and molecular viewpoints. Yet important gaps remain and the mechanisms that co-ordinate responses to external signals remain enigmatic.


Subject(s)
Giardia lamblia/physiology , Animals , Cell Wall/metabolism , Gene Expression Regulation , Giardia lamblia/cytology , Giardia lamblia/genetics , Giardia lamblia/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism
7.
Int J Parasitol ; 38(3-4): 353-69, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17964578

ABSTRACT

Giardia lamblia is a major cause of diarrhoeal disease worldwide. Since it has no known toxin, the ability of trophozoites to colonise the human small intestine is required for its pathogenesis. Mitosis in this protozoan parasite is a unique challenge because its two equivalent nuclei and complex cytoskeleton must be duplicated and segregated accurately. Giardial mitosis is a complex and rapid event that is poorly understood at the cellular and molecular levels. Higher eukaryotes have one to three members of the highly conserved Ser/Thr aurora kinase (AK) family that regulate key aspects of mitosis and cytokinesis. Giardia has a single AK orthologue (gAK) with 61% similarity to human AK A. In addition to the conserved active site residues, activation loop and destruction-box motifs characteristic of AKs, gAK contains a unique insert near the active site region. We epitope-tagged gAK at its C-terminus and expressed it under its own promoter. During interphase, gAK localises exclusively to the nuclei, but is not phosphorylated as shown by lack of staining with an antibody specific to phosphorylated AK A (pAK). In contrast, during mitosis pAK localises to the basal bodies/centrosomes and co-localises with tubulin to the spindle. During specific stages of mitosis, giardial pAK also localised dynamically to cytoskeletal structures unique to Giardia: the paraflagellar dense rods of the anterior flagella and the median body, whose functions are unknown, as well as to the parent attachment disc. Two AK inhibitors significantly decreased giardial growth and increased the numbers of cells arrested in cytokinesis. These inhibitors appeared to increase microtubule nucleation and cell-ploidy. Our data show that gAK is phosphorylated in mitosis and suggest that it plays an important role in the Giardia cell cycle. The pleiotropic localisation of AK suggests that it may co-ordinate the reorganisation and segregation of tubulin-containing structures in mitosis. We believe this is the first report of a signalling protein regulating cell division in Giardia.


Subject(s)
Antigens, Protozoan/genetics , Giardia lamblia/enzymology , Mitosis/physiology , Protein Serine-Threonine Kinases/genetics , Animals , Antigens, Protozoan/analysis , Aurora Kinase A , Aurora Kinases , Base Sequence , Centrosome/enzymology , Diarrhea/parasitology , Enzyme Inhibitors/pharmacology , Gene Expression , Host-Parasite Interactions , Humans , Intestinal Diseases, Parasitic/immunology , Microtubules/enzymology , Molecular Sequence Data , Parasitology/methods , Protein Serine-Threonine Kinases/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment
8.
Mol Biochem Parasitol ; 152(1): 80-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17204341

ABSTRACT

The ability of Giardia lamblia to undergo two distinct differentiations in response to physiologic stimuli is central to its pathogenesis. The giardial cytoskeleton changes drastically during encystation and excystation. However, the signal transduction pathways mediating these transformations are poorly understood. We tested the hypothesis that PP2A, a highly conserved serine/threonine protein phosphatase, might be important in giardial differentiation. We found that in vegetatively growing trophozoites, gPP2A-C protein localizes to basal bodies/centrosomes, and to cytoskeletal structures unique to Giardia: the ventral disk, and the dense rods of the anterior, posterior-lateral, and caudal flagella. During encystation, gPP2A-C protein disappears from only the anterior flagellar dense rods. During excystation, gPP2A-C localizes to the cyst wall in excysting cysts but is not found in the wall of cysts with emerging excyzoites. Transcriptome and immunoblot analyses indicated that gPP2A-C mRNA and protein are upregulated in mature cysts and during the early stage of excystation that models passage through the host stomach. Stable expression of gPP2A-C antisense RNA did not affect vegetative growth, but strongly inhibited the formation of encystation secretory vesicles (ESV) and water-resistant cysts. Moreover, the few cysts that formed were highly defective in excystation. Thus, gPP2A-C localizes to universal cytoskeletal structures and to structures unique to Giardia. It is also important for encystation and excystation, crucial giardial transformations that entail entry into and exit from dormancy.


Subject(s)
Gene Expression Regulation, Developmental , Giardia lamblia/enzymology , Giardia lamblia/growth & development , Phosphoprotein Phosphatases/metabolism , Adaptation, Physiological , Amino Acid Sequence , Animals , Blotting, Western , Centrosome/chemistry , Cytoskeleton/chemistry , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Flagella/chemistry , Giardia lamblia/genetics , Immunohistochemistry , Microscopy, Fluorescence , Molecular Sequence Data , Morphogenesis/genetics , Phosphoprotein Phosphatases/biosynthesis , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 2 , Protozoan Proteins/analysis , Protozoan Proteins/genetics , RNA, Protozoan/analysis , RNA, Protozoan/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid
9.
J Cancer Res Clin Oncol ; 131(1): 49-59, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15480783

ABSTRACT

PURPOSE: We have previously demonstrated that conditioned medium from bacteria, some of which were isolated from the colon of cancer patients, stimulate cancer cell invasion in vitro through a 13-mer beta-casein-derived peptide. Since invasion signalling pathways are coordinated by the balance between protein kinases and phosphatases, we investigated the effect of conditioned medium from bacteria on the overall cellular tyrosine phosphorylation. METHODS: The tyrosine phosphorylation level of HCT-8/E11 human colon cancer cells treated with the pro-invasive conditioned medium of Listeria, prepared on top of collagen type I gels (CM(Coll) Listeria/TSB), were analysed by means of immunoprecipitation and Western blot, with specific anti-phosphotyrosine antibodies. RESULTS: We demonstrated that CM(Coll) Listeria/TSB increases the tyrosine phosphorylation level of ErbB2 and ErbB3, members of the epidermal growth factor receptor (EGFR) family, and the association between ErbB3 and the phosphatidylinositol 3-kinase (PI3K) regulatory subunit (p85alpha). CM(Coll) Listeria/TSB-stimulated ErbB3 tyrosine phosphorylation and cancer cell invasion were independent from EGFR expression and activity but dependent on ErbB2 activity. CONCLUSIONS: The interaction between Listeria and collagen type I produces, next to the 13-mer peptide, at least another pro-invasive factor that signals via ErbB2/ErbB3 heterodimers.


Subject(s)
Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Listeria monocytogenes/metabolism , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Signal Transduction , Tyrosine/metabolism , Blotting, Western , Cell Line, Tumor , Collagen Type I/metabolism , Colonic Neoplasms/microbiology , Humans , Immunoprecipitation , Listeriosis/metabolism , Neoplasm Invasiveness , Phosphorylation
10.
Int J Parasitol ; 34(5): 549-56, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15064119

ABSTRACT

Transfer of antigens frequently follows adhesion of protozoan parasites to host cells. We were interested in such transfer from the Entamoeba surface to enterocytes following adhesion of trophozoites. Therefore, cocultures of enterocytes in vitro and ex vivo with Entamoeba histolytica (strain HM-1:IMSS) or Entamoeba dispar (strain SAW760) trophozoites were processed for immunocytochemistry. The EH5 monoclonal antibody against amoebic proteophosphoglycans marked a dotted pattern on the apical side of enterocytes in in vitro cocultures with HM-1:IMSS and SAW760 trophozoites. Basolateral staining was present in cocultures following dysfunction of tight junctions, or when trophozoites made direct contact with the basolateral side of enterocytes in in vitro and ex vivo cocultures. Based on the molecular mass in Western blot, the transferred proteophosphoglycan was identified as a lipophosphopeptidoglycan. In conclusion, trophozoites transfer LPPG to the apical side of enterocytes following adhesion and prior to dysfunction of tight junctions.


Subject(s)
Entamoeba/physiology , Enterocytes/physiology , Glycosylphosphatidylinositols/physiology , Peptidoglycan/metabolism , Protozoan Proteins/physiology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Protozoan/immunology , Blotting, Western/methods , Caco-2 Cells , Cell Adhesion/physiology , Coculture Techniques , Colon/physiology , Entamoeba/immunology , Entamoeba histolytica/immunology , Entamoeba histolytica/physiology , Glycosylphosphatidylinositols/immunology , Humans , Immunohistochemistry/methods , Lectins/physiology , Membrane Proteins/immunology , Membrane Proteins/physiology , Peptidoglycan/immunology , Tight Junctions/physiology
11.
Int J Parasitol ; 34(7): 785-94, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15157761

ABSTRACT

Tight junctions and microvilli constitute an anti-invasive barrier at the luminal side of enteric cell layers. Both subcellular structures are disrupted following adhesion of Entamoeba histolytica trophozoites to enteric cell layers in vitro. It was our aim to analyse the molecular mechanism underlying this disruption. Therefore, we cocultured enteric T84 cell layers established on filter inserts with E. histolytica trophozoites and tested various modulators of enteric molecules, involved in the functional regulation of tight junctions, as well as inhibitors of trophozoite virulence factors on their capacity to maintain the transepithelial electrical resistance. Pretreatment of trophozoites with the proteinase inhibitor N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone prevented the decrease in transepithelial electrical resistance whereas none of the modulators used to pretreat enterocytes were successful. Moreover, zymography and Western blot analysis revealed that both N-Tosyl-Phenylalanine chloromethyl ketone and N-Tosyl-l-Lysine chloromethyl ketone inhibited E. histolytica cysteine proteinases and prevented proteolysis of tight junction molecules ZO-1 and ZO-2 and of villin, the major actin bundling molecule in microvilli. Immunocytochemistry with an antibody against ezrin, an actin-binding molecule in microvilli, and phase contrast microscopy demonstrated that pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone also prevented disturbance of microvilli and destruction of Caco-2 enteric cell layers in cocultures. Taken together, our results indicate that trophozoites use their proteinases to overcome microvilli and tight junction barriers during the invasion of enteric cell layers, that these phenomena could be prevented by pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone, and that such pretreatment disabled trophozoites to destroy enteric cell layers in vitro.


Subject(s)
Entamoeba histolytica/physiology , Enterocytes/parasitology , Protein Synthesis Inhibitors/pharmacology , Tight Junctions/parasitology , Tosyllysine Chloromethyl Ketone/pharmacology , Tosylphenylalanyl Chloromethyl Ketone/pharmacology , Animals , Cells, Cultured , Cysteine Endopeptidases/metabolism , Electric Impedance , Entamoeba histolytica/drug effects , Entamoeba histolytica/enzymology , Enterocytes/drug effects , Enterocytes/physiology , Microfilament Proteins/metabolism , Microvilli/drug effects , Microvilli/parasitology , Microvilli/physiology , Tight Junctions/drug effects , Tight Junctions/physiology
12.
Int J Parasitol ; 42(4): 411-9, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22429767

ABSTRACT

The NIMA-related serine/threonine kinases (Neks) function in the cell cycle and regulate ciliary and flagellar length. The Giardia lamblia genome encodes 198 Neks, of which 56 are predicted to be active. Here we believe that we report the first functional analysis of two G. lamblia Neks. The GlNek1 and GlNek2 kinase domains share 57% and 43% identity to the kinase domains of human Nek1 and Nek2, respectively. Both GlNeks are active in vitro, have dynamic relocalisation during the cell cycle, and are expressed throughout the life cycle, with GlNek1 being upregulated in cysts. Over-expression of inactive GlNek1 delays disassembly of the parental attachment disc and cytokinesis, whilst over-expression of either wild type GlNek1 or inactive mutant GlNek2 inhibits excystation.


Subject(s)
Giardia lamblia/enzymology , Giardia lamblia/physiology , Mitosis , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Cytosol/chemistry , Gene Expression Profiling , Giardia lamblia/growth & development , Molecular Sequence Data , Protozoan Proteins/metabolism , Sequence Homology, Amino Acid
13.
Mol Biochem Parasitol ; 184(2): 122-5, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22569588

ABSTRACT

The ability of Giardia to differentiate into cysts which survive in the environment and release the virulent trophozoites after ingestion in the small intestine is essential for transmission and disease. We examined the role of enolase, a glycolytic enzyme, in Giardia differentiation. The sequence of Giardia lamblia enolase (gEno) is most similar to enolases in Homo sapiens and Leishmania mexicana, and shows the conserved catalytic and metal-binding residues. We used an integration vector to stably express wild type and mutant gEno. In trophozoites, wild type gEno localized to the cell membrane, caudal flagella and cytosol. gEno is present on the wall of mature cysts, but not in encystation secretory vesicles (ESV). The expression of gEno with a deletion of residues G167-K169, or mutations H389Q/R390S significantly inhibited excystation while mutation of residue D257K had no effect. These results suggest a role for enolase in regulation of Giardia excystation.


Subject(s)
Giardia lamblia/enzymology , Phosphopyruvate Hydratase/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Catalytic Domain , Consensus Sequence , Giardia lamblia/cytology , Giardia lamblia/physiology , Molecular Sequence Data , Mutagenesis , Phosphopyruvate Hydratase/genetics , Protozoan Proteins/genetics , Sequence Deletion , Trophozoites/enzymology
14.
Genome Biol ; 12(7): R66, 2011 Jul 25.
Article in English | MEDLINE | ID: mdl-21787419

ABSTRACT

BACKGROUND: The major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest. RESULTS: To explore early kinase evolution and regulation of Giardia biology, we cataloged the kinomes of three sequenced strains. Comparison with published kinomes and those of the excavates Trichomonas vaginalis and Leishmania major shows that Giardia's 80 core kinases constitute the smallest known core kinome of any eukaryote that can be grown in pure culture, reflecting both its early origin and secondary gene loss. Kinase losses in DNA repair, mitochondrial function, transcription, splicing, and stress response reflect this reduced genome, while the presence of other kinases helps define the kinome of the last common eukaryotic ancestor. Immunofluorescence analysis shows abundant phospho-staining in trophozoites, with phosphotyrosine abundant in the nuclei and phosphothreonine and phosphoserine in distinct cytoskeletal organelles. The Nek kinase family has been massively expanded, accounting for 198 of the 278 protein kinases in Giardia. Most Neks are catalytically inactive, have very divergent sequences and undergo extensive duplication and loss between strains. Many Neks are highly induced during development. We localized four catalytically active Neks to distinct parts of the cytoskeleton and one inactive Nek to the cytoplasm. CONCLUSIONS: The reduced kinome of Giardia sheds new light on early kinase evolution, and its highly divergent sequences add to the definition of individual kinase families as well as offering specific drug targets. Giardia's massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton.


Subject(s)
Biological Evolution , Giardia lamblia/enzymology , Giardia lamblia/genetics , Phosphotransferases/genetics , Phosphotransferases/metabolism , Animals , Catalysis , Cell Cycle , DNA Repair , Histidine/metabolism , Phosphorylation , Phosphotransferases/classification , Phylogeny , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, Tertiary , Protein Transport , RNA Splicing , Signal Transduction , Transcription, Genetic , Tyrosine/metabolism
15.
Int J Parasitol ; 41(10): 1079-92, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21723868

ABSTRACT

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.


Subject(s)
Genome, Protozoan , Giardia lamblia/chemistry , Giardia lamblia/genetics , Organelles/chemistry , Organelles/genetics , Proteome/analysis , Protozoan Proteins/analysis , Computational Biology , Genes, Protozoan , Microscopy, Confocal
16.
J Biol Chem ; 278(25): 22650-6, 2003 Jun 20.
Article in English | MEDLINE | ID: mdl-12690119

ABSTRACT

Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.


Subject(s)
Carrier Proteins/metabolism , Cysteine Endopeptidases/metabolism , Entamoeba histolytica/enzymology , Intestinal Mucosa/parasitology , Microfilament Proteins/metabolism , Microvilli/parasitology , Animals , Cell Adhesion , Coculture Techniques , Colorectal Neoplasms , Entamoeba histolytica/physiology , Humans , Tumor Cells, Cultured , Urea/metabolism
17.
EMBO J ; 22(22): 6161-73, 2003 Nov 17.
Article in English | MEDLINE | ID: mdl-14609961

ABSTRACT

In colon cancer, enteric bacteria and dietary factors are major determinants of the microenvironment but their effect on cellular invasion is not known. We therefore incubated human HCT-8/E11 colon cancer cells with bacteria or bacterial conditioned medium on top of collagen type I gels. Listeria monocytogenes stimulate cellular invasion through the formation of a soluble motility-promoting factor, identified as a 13mer beta-casein-derived peptide (HKEMPFPKYPVEP). The peptide is formed through the combined action of Mpl, a Listeria thermolysin-like metalloprotease, and a collagen-associated trypsin-like serine protease. The 13mer peptide was also formed by tumour biopsies isolated from colon cancer patients and incubated with a beta-casein source. The pro- invasive 13mer peptide-signalling pathway implicates activation of Cdc42 and inactivation of RhoA, linked to each other through the serine/threonine p21- activated kinase 1. Since both changes are necessary but not sufficient, another pathway might branch upstream of Cdc42 at phosphatidylinositol 3-kinase. Delta opioid receptor (deltaOR) is a candidate receptor for the 13mer peptide since naloxone, an deltaOR antagonist, blocks both deltaOR serine phosphorylation and 13mer peptide-mediated invasion.


Subject(s)
Bacterial Proteins , Caseins/genetics , Cell Movement/physiology , Neoplasm Invasiveness , Neoplasms/metabolism , Peptides/metabolism , Collagen Type I/metabolism , Humans , Listeria monocytogenes/metabolism , Metalloendopeptidases/metabolism , Peptides/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, sigma/metabolism , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/metabolism
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