Tineke Lauwaet

The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology

BackgroundThe major human intestinal pathogen Giardia lamblia is a very early branching eukaryote with a minimal genome of broad evolutionary and biological interest.ResultsTo 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 Giardias 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.ConclusionsThe 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. Giardias massive Nek expansion may reflect its distinctive lifestyle, biphasic life cycle and complex cytoskeleton.

Human milk oligosaccharides reduce Entamoeba histolytica attachment and cytotoxicity in vitro

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infants 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.

Impaired parasite attachment as fitness cost of metronidazole resistance in Giardia lamblia

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 (Mzr) G. lamblia has rarely been isolated from patients. To understand why clinical Mzr isolates are rare, we questioned whether Mz resistance entails fitness costs to the parasite. Our studies employed several newly generated and established isogenic Mzr 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 Mzr cell lines could not establish infection, while two Mzr 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 Mzr 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 Mzr isolates of Giardia. However, the data also caution that some forms of Mz resistance do not markedly interfere with in vivo infectivity.

Mining the Giardia genome and proteome for conserved and unique basal body proteins.

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.

Expanded therapeutic potential in activity space of next-generation 5-nitroimidazole antimicrobials with broad structural diversity

Significance Drugs against disease-causing microbes are among the major achievements of modern medicine, but many microbes show a tenacious ability to develop resistance, so they are no longer killed by available drugs. We show here for an important class of these drugs, represented by the common drug metronidazole, that broad modifications of the basic drug structure can improve drug activities against several clinically important microbes and unexpectedly overcome different forms of resistance. Several of these new drugs cure infections in animal models and are safe in initial toxicity evaluations. These findings provide reasons to develop this class of drugs as human medicines in the ongoing fight against disease-causing microbes. 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.

Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin

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.

An atypical proprotein convertase in Giardia lamblia differentiation.

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Importance of enolase in Giardia lamblia differentiation

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.

Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation.

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.

Signaling Pathways in Giardia lamblia

Giardia trophozoites colonize the ever-changing small intestinal environment and must constantly react to external mucosal signals in order to “decide” whether to multiply and cause disease or to differentiate into cysts. Similarly, upon ingestion, cysts react to stimuli from the new host in order to excyst. Giardia is an excellent model to study signaling because its life cycle can be completed in vitro and genome analyses revealed a limited but broad selection of signaling proteins. Encystation is an entry into dormancy, while excystation is a rapid cellular awakening. Although the stimuli for encystation and excystation are known, understanding of the transduction of these important signals is incomplete. The localization of the various signaling proteins to universal or Giardia-specifi c structures and their redistribution in response to environmental signals will provide insights into their functions in the Giardia cell cycle and differentiation. However, research on signaling proteins and pathways in Giardia is hampered by the lack of specifi c antibodies, substrates, and inhibitors. The most striking fi nding is the very large number of Nek kinases in the Giardia genome. The Neks are promising targets for further studies and their function and regulation will likely disclose more insights into the regulation of Giardia motility, cell, and life cycle. Here, we summarize current published information on Giardia signaling in growth, encystation, and excystation.