Frances D. Gillin

Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardias requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardias genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Nitric Oxide Production by Human Intestinal Epithelial Cells and Competition for Arginine as Potential Determinants of Host Defense Against the Lumen-Dwelling Pathogen Giardia lamblia

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i.e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.

Central Importance of Immunoglobulin A in Host Defense against Giardia spp.

ABSTRACT The protozoan pathogen Giardia is an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controlling Giardia infection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls with Giardia muris or Giardia lamblia GS/M-83-H7. We found that IgA-deficient mice could not eradicate either G. muris or G. lamblia infection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clear G. muris infections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies cleared G. muris infection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to control G. muris infection, show that IgA-dependent host defenses are central for eradicating Giardia spp. Moreover, B-cell-dependent but IgA-independent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.

Chronic giardiasis: studies on drug sensitivity, toxin production, and host immune response.

UNLABELLED We investigated a patient (W.B.) with chronic symptomatic Giardiasis despite seven separate courses of either quinacrine or metronidazole who was cured by combined quinacrine and metronidazole. After isolating Giardia lamblia from W.B., we cultured the trophozoites to make the following observations. In vitro drug testing showed that (a) W.B.s organisms were not more drug resistant than three other isolates and that (b) W.B.s organisms were more sensitive to combined quinacrine and metronidazole than to either drug alone. Isolates of Giardia lamblia from W.B. and 3 other patients did not produce detectable enterotoxin in four different assays. W.B. had normal levels of circulating immunoglobulins, detectable intestinal immunoglobulin A, circulating immunoglobulin G anti-Giardia lamblia antibodies, and lymphocyte responsiveness to solubilized giardia lamblia. However, monocytes-macrophages from W.B. exhibited reduced killing for Giardia lamblia compared with normal subjects. CONCLUSIONS (a) The chronicity of our patients infection was not due to the organism having unique properties of drug resistance. (b) Combined quinacrine and metronidazole, which cured our patients chronic giardiasis, should be tried in patients in whom infection persists after single drug therapy. (c) The diarrhea in our patient was probably caused by a mechanism other than Giardia lamblia-induced secretion by currently recognized enterotoxins. (d) Reduced cellular cytotoxicity for Giardia lamblia may have contributed to the persistence of our patients infection and should be suspected in other patients with chronic giardiasis.

Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Giardia lamblia: The roles of bile, lactic acid, and pH in the completion of the life cycle in vitro

Large numbers (10(4) to greater than 10(5)/ml) of Type I water-resistant Giardia lamblia cysts were produced in vitro under conditions that are characteristic of the human intestinal lumen. We define Type I cyst morphology as oval shaped, smooth, and refractile, with cyst wall, axostyle, and median body visible in relief by Normarski differential interference contrast optics. Human and porcine bile induced higher levels of encystation than bovine bile at the alkaline pH (7.8) which occurs in the human lower small intestine. High-pressure liquid chromatography analysis showed that the porcine bile had a preponderance of hyocholate, rather than cholate, while bovine bile had less chenodeoxycholate and more deoxycholate than human bile. Lactic acid, a major product of bacterial metabolism in the human colon, further stimulated encystation. Growth of the preencystation culture without bile also increased subsequent encystation. More than 90% of Type I cysts produced with porcine bile plus lactic acid were viable as indicated by the uptake and retention of fluorescein diacetate and exclusion of propidium iodide. Biological activity of in vitro-derived water-resistant cysts was demonstrated by the observation that 1 to 9.5% excysted in vitro. The percentage of excystation was greatly decreased following encystation at pH 7.0 or by omission of bile or lactic acid. This is the first quantitative in vitro demonstration of the complete life cycle of G. lamblia from humans.

Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica.

Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent.

A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia

Giardia lamblia is an important human intestinal parasite that survives outside of the host by differentiation of trophozoites into infectious cysts. Transcriptional regulation is key for encystation gene expression, but the mechanisms are unknown. Giardia genome database searches identified a myb ‐like gene ( gmyb2 ) whose expression increased during encystation. Epitope‐tagged gMyb2 localized to both nuclei. DNA binding and mutation analysis showed that gMyb2 binds specifically to C(T/A)ACAG, a c‐Myb‐like target sequence in the promoters of encystation‐induced genes encoding gMyb2, three cyst wall proteins and G6PI‐B, a key enzyme in cyst wall polysaccharide biosynthesis. gMyb2 binding sites were not found in the upstream regions of 31 other giardial genes. Deletion of the putative gMyb2 binding site greatly reduced encystation‐specific promoter activity of g6pi‐b . Fusion of gMyb2 binding sites to the constitutive ran promoter or g6pi‐b promoter deletion lacking the gMyb2 binding site in‐duced encystation‐specific expression. gMyb2 may play an important role in transcriptional regulation of encystation genes, and may help co‐ordinate synthesis of cyst wall proteins and polysaccharide. gMyb2 is the first giardial transcription factor to be functionally identified and the first that is associated with upregulation of encystation genes. This work provides a model for study of differential gene regulation in early diverging eukaryotic organisms.

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.

Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5' mRNA processing.

Giardia lamblia must encyst to survive in the environment and subsequently infect new hosts. We investigated the expression of glucosamine‐6‐phosphate isomerase (Gln6PI), the first enzyme required for biosynthesis of N‐acetylgalactosamine, for the major cyst wall polysaccharide. We isolated two Gln6PI genes that encode proteins with large areas of identity, but distinctive central and terminal regions. Both recombinant enzymes have comparable kinetics. Interestingly, these genes have distinct patterns of expression. Gln6PI‐A has a conventional, short 5′ untranslated region (UTR), and is expressed at a low level during vegetative growth and encystation. The Gln6PI‐B gene has two transcripts — one is expressed constitutively and the second species is highly upregulated during encystation. The non‐regulated Gln6PI‐B transcript has the longest 5′‐UTR known for Giardia and is 5′ capped or blocked. In contrast, the Gln6PI‐B upregulated transcript has a short, non‐capped 5′‐UTR. A small promoter region (< 56 bp upstream from the start codon) is sufficient for the regulated expression of Gln6PI‐B. Gln6PI‐B also has an antisense overlapping transcript that is expressed constitutively. A shorter antisense transcript is detected during encystation. This is the first report of a developmentally regulated promoter in Giardia, as well as evidence for a potential role of 5′ RNA processing and antisense RNA in differential gene regulation.