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.

Antimicrobial polypeptides such as the defensins kill a wide range of organisms, including bacteria, fungi, viruses, and tumor cells. Because of the recent finding that intestinal defensins, also known as cryptdins, are synthesized by the Paneth cells of the small intestinal crypts and released into the lumen, we asked whether defensins and other small cationic antimicrobial peptides could kill the trophozoites of Giardia lamblia, which colonize the small intestine. Four mouse cryptdins, two neutrophil defensins (HNP-1 [human] and NP-2 [rabbit]), and the unique tryptophan-rich bovine neutrophil polypeptide indolicidin each had some antigiardial activity against trophozoites in vitro. Cryptdins 2 and 3, indolicidin, and NP-2 each reduced viability by more than 3 log units in 2 h, and killing by all peptides was dose and time dependent. Exposure of trophozoites to peptides frequently resulted in cell aggregation and dramatic changes in morphology. The mechanism of binding and lysis appeared to involve charge interactions, since 150 mM NaCl as well as millimolar levels of Ca2' and Mg2' inhibited killing by most of the peptides. Our studies show that G. lamblia is sensitive to defensins and indolicidin and suggest that these small polypeptides could play a role in nonimmune host defenses.

SummaryEncystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome-like vacuoles. As short-range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation-excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.

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Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca 2؉ levels (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acidtetra(acetyloxymethyl) ester, a Ca 2؉ channel blocker, Ca 2؉ ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca 2؉ levels, and peak Ca 2؉ responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca 2؉ storage compartment in cysts. The ability to sequester ions in membranebounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca 2؉ storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence.

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Expression of TSA 417, the predominant cysteine-rich variable surface protein of Giardia lamblia WB clone C6 trophozoites, did not change during encystation in vitro. However, in vitro excystation of cysts derived in vitro or in vivo consistently produced TSA 417 nonexpressing trophozoite populations, suggesting that completion of the life cycle leads to antigenic switching.