Antigenic variation and the murine immune response to Giardia lamblia

The protozoan parasite Giardia lamblia is an important causative agent of acute or chronic diarrhoea in humans and various animals. During infection, the parasite survives the hosts reactions by undergoing continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). The VSPs form a unique family of cysteine-rich proteins that are extremely heterogeneous in size. The relevance of antigenic variation for the survival in the host has been most successfully studied by performing experimental infections in a combined mother/offspring mouse system and by using the G. lamblia clone GS/M-83-H7 (human isolate) as model parasite. In-vivo antigenic variation of G. lamblia clone GS/M-83-H7 is characterised by a diversification of the intestinal parasite population into a complex mixture of different variant antigen types. It could be shown that maternally transferred lactogenic anti-VSP IgA antibodies exhibit cytotoxic activity on the Giardia variant-specific trophozoites in suckling mice, and thus express a modulatory function on the proliferative parasite population characteristics. Complementarily, in-vitro as well as in-vivo experiments in adult animals indicated that non-immunological factors such as intestinal proteases may interfere into the process of antigen variation in that they favour proliferation of those variant antigen-type populations which resist the hostile physiological conditions within the intestine. These observations suggest that an interplay between immunological and physiological factors, rather than one of these two factor alone, modulates antigenic diversification of a G. lamblia population within an experimental murine host and thus influences the survival rate and strategy of the parasite.     

Antigenic variation in Giardia lamblia

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.     

Antigenic variation in Giardia lamblia

Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.     

Antigenic variation of Giardia lamblia in experimental human infections

To determine if Giardia surface Ag vary in human infections volunteers were inoculated enterally with trophozoites of uncloned GS/M-85 and in later experiments with two clones derived from GS/M. The surface Ag of trophozoites reisolated from 6/6 volunteers differed from the inoculum. To determine if the surface Ag of trophozoites derived from clones would also change, volunteers were inoculated with two clones, B6 or H7. B6 possesses a 200-kDa surface Ag recognized by mAb 3F6 and H7 has a 72-kDa surface Ag recognized by mAb G10/4. One of thirteen B6 and four of four H7-inoculated volunteers became infected. Analysis of Giardia obtained on day 22 from the intestines of the four H7-infected volunteers and cultures derived from these trophozoites revealed loss of the initial major surface Ag as determined by surface IFA using mAb, surface radiolabeling and loss of cytotoxicity to mAb, and Western blots. Loss of the 72-kDa Ag began after day 14 and was practically complete by day 22. The 200-kDa surface Ag was almost totally absent from the surface of Giardia isolated from the single B6-infected volunteer. Serum surface-reactive antibodies, as measured by IFA and cytotoxicity to H7 and the day 22 isolates, showed high levels of antibodies to H7, primarily to the 72-kDa surface Ag, but negligible or low levels of late-appearing antibodies to the day 22 isolates. These studies document antigenic variation of Giardia in human infections and show that humoral responses are in part isolate-specific.     

Surface antigenic variation in Giardia lamblia

Giardia lamblia, a common intestinal dwelling protozoan and a cause of diarrhoea in humans and animals world-wide, undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) are a family of related, highly unusual proteins that cover the entire surface of the parasite. VSPs are cysteine-rich proteins containing many CXXC motifs, one or two GGCY motifs, a conserved hydrophobic tail and a Zn finger motif. The biological role(s) of VSPs is unclear. As VSPs are resistant to the effects of intestinal proteases, they likely allow the organism to survive in the protease-rich small intestine. Although immune escape is commonly mentioned as the reason antigenic variation occurs, VSP expression changes in vivo even in the absence of an adaptive immune system suggesting the biological role of antigenic variation is more complex. The molecular mechanisms involved in antigenic variation are not known but appear to differ from those known to occur in other protozoa.     

Surface antigen variability and variation in Giardia lamblia

Recent studies show that Giardia isolates are heterogeneous but fall into at least three groups as determined by a number of complementary techniques. Giardia undergoes surface antigenic variation, both in vitro, and in humans and other animal model infections. Many of the characteristics of antigenic variation and the proteins involved, called variant-specific surface proteins (VSPs), are unique. The sequences of five VSPs reveal a family of cysteine-rich proteins. Here Theodore Nash reviews the relationship between antigenic variation and Giardia heterogeneity.     

Chromosome-size variation in Giardia lamblia: the role of rDNA repeats.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.     

Synonymous codon usage variation among Giardia lamblia genes and isolates.

The pattern of codon usage in the amitochondriate diplomonad Giardia lamblia has been investigated. Very extensive heterogeneity was evident among a sample of 65 genes. A discrete group of genes featured unusual codon usage due to the amino acid composition of their products: these variant surface proteins (VSPs) are unusually rich in Cys and, to a lesser extent, Gly and Thr. Among the remaining 50 genes, correspondence analysis revealed a single major source of variation in synonymous codon usage. This trend was related to the extent of use of a particular subset of 21 codons which are inferred to be those which are optimal for translation; at one end of this trend were genes expected to be expressed at low levels with near random codon usage, while at the other extreme were genes expressed at high levels in which these optimal codons are used almost exclusively. These optimal codons all end in C or G so G + C content at silent sites varies enormously among genes, from values around 40%, expected to reflect the background level of the genome, up to nearly 100%. Although VSP genes are occasionally extremely highly expressed, they do not, in general, have high frequencies of optimal codons, presumably because their high expression is only intermittent. These results indicate that natural selection has been very effective in shaping codon usage in G. lamblia. These analyses focused on sequences from strains placed within G. lamblia "assemblage A"; a few sequences from other strains revealed extensive divergence at silent sites, including some divergence in the pattern of codon usage.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Antigenic conservation and variation in Giardia cysts from various vertebrate hosts.

Monoclonal antibodies produced against Giardia muris cysts reacted in indirect immunofluorescence with homologous cysts and cysts from a Giardia-infected wild Norway rat but did not cross-react with Giardia lamblia cysts of human, dog, or beaver sources. Another monoclonal antibody raised against Giardia simoni cysts from the Norway rat reacted with homologous cysts (rat) and cross-reacted with cysts from a cow. The demonstration of antigenic differences at the cyst surfaces of Giardia organisms of animal and human origin suggests that it is possible to identify the animal source of Giardia cysts according to their pattern of reactivity with monoclonal antibodies. Such identification would have a useful application in affirming the possible zoonotic transmission of animal source Giardia species to humans.     

Gut-associated immune effector responses in immunocompetent and immunocomprimised mice with Giardia lamblia

Abstract Significantly higher Giardia lamblia trophozoites load in the intestine of infected mice accompanied pronounced influx of suppressor/cytotoxic T cells (Lyt 2.2⁺), T cells (Thy 1.2⁺) and significant reduction in IgA-containing cells in the gut during the establishment and peak phases of infection. The induction of helper/inducer T cells (Lyt 1.1⁺) and significant enhancement of IgA-containing cells in gut resulted in the decline of the trophozoite loads. However, the prior treatment of animals with dexamethasone alone resulted in significant reduction in helper/inducer T cells (Lyt 1.1⁺) and the IgA-containing cells in the gut; the percents of suppressor/cytotoxic T cells (Lyt 2.2⁺) and IgM-containing cells remained unaltered. Although the G. lamblia infection in such animals further significantly increased the influx of suppressor/cytotoxic T cells, the late response of helper/inducer T cells and IgA-containing cells was abrogated during the decline phase of infection. The significant reduction in the trophozoite load — despite immuno-suppressive therapy — appeared to be due to unaltered IgM response in such animals which probably took over the function of IgA in defense against G. lamblia . The data of the investigation thus suggested a role of helper/inducer T cells and antibodies producing cells in gut as important effector cells resulting in the termination of primary G. lamblia infection.     

Unusually low levels of genetic variation among Giardia lamblia isolates

Giardia lamblia, an intestinal pathogen of mammals, including humans, is a significant cause of diarrheal disease around the world. Additionally, the parasite is found on a lineage which separated early from the main branch in eukaryotic evolution. The extent of genetic diversity among G. lamblia isolates is insufficiently understood, but this knowledge is a prerequisite to better understand the role of parasite variation in disease etiology and to examine the evolution of mechanisms of genetic exchange among eukaryotes. Intraisolate genetic variation in G. lamblia has never been estimated, and previous studies on interisolate genetic variation have included a limited sample of loci. Here we report a population genetics study of intra- and interisolate genetic diversity based on six coding and four noncoding regions from nine G. lamblia isolates. Our results indicate exceedingly low levels of genetic variation in two out of three G. lamblia groups that infect humans; this variation is sufficient to allow identification of isolate-specific markers. Low genetic diversity at both coding and noncoding regions, with an overall bias towards synonymous substitutions, was discovered. Surprisingly, we found a dichotomous haplotype structure in the third, more variable G. lamblia group, represented by a haplotype shared with one of the homogenous groups and an additional group-specific haplotype. We propose that the distinct patterns of genetic-variation distribution among lineages are a consequence of the presence of genetic exchange. More broadly, our findings have implications for the regulation of gene expression, as well as the mode of reproduction in the parasite.     

A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.     

Codon Usage in Giardia lamblia

ABSTRACT A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonomous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei ( r = 0.434) and Plasmodium falciparum ( r =–0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Chromosome rearrangements in Giardia lamblia

Recent studies have shown that the genome of Giardia lamblia is plastic. Clinical isolates exhibit extensive karyotypic heterogeneity and chromosome rearrangements occur frequently, in vitro. In this review, Sylvie Le Blancq looks at genome organization and the impact of DNA rearrangement events.     

The immune response to Giardia

The flagellate Giardia duodenalis has been considered for many years to be a commensal living in the lumen of the small intestine of its host. It is only 25 years ago that it was accepted that Giardia is a significant pathogen of humans. Knowledge that Giardia can elicit an immune response that would probably contribute to the onset or absence of symptoms is not much older. The use of animal models to study the disease in the laboratory, together with the production of the whole life cycle in a test tube, have contributed greatly to our present knowledge of the immune responses to Giardia and of antigens that are specific to the trophozoite or cyst stages. In this review, Gaétan Faubert focuses on studies published since the last review in Parasitology Today in 1988, and examines the roles played by the humoral and cell-mediated immune responses in the control of the infection. It also covers the immunodiagnostic assays that have been recently developed on the basis of advances in our knowledge of the antigens of Giardia.