Previous studies determined that arginine deiminase plays a role in the energy metabolism in Giardia. In the present work, we show an additional gADI action, which affects or controls essential biological activities of the organism. There are four major findings. First, we establish that gADI acts as a peptidyl-arginine deiminase in addition to its known function as a metabolic enzyme. Secondly, gADI influences important processes in Giardia. Specifically, by way of its ability to citrullinate VSPs gADI alters VSP biology, the response to cytotoxic antibodies, and antigenic variation. Moreover, gADI appears to play an important, if not controlling, role in encystation. Lastly, because arginine deiminase is sumoylated, it is likely that this modification alters its the activity, degradation and/or localization.
The evidence that VSPs are citrullinated by gADI is compelling. gADI binds specifically to the CRGKA tail and localizes close to the VSPs found on the plasma membrane of the trophozoite. Using specific antibodies to modify citrulline, VSPs were shown to be citrullinated specifically, at the arginine residue (R) of the CRGKA cytoplasmic tail. Also, purified gADI citrullinates the arginine in the conserved tail in vitro, verifying gADI's PAD activity. This modification has profound effects on the biology of the parasite.
The most commonly cited biological role of antigenic variation in pathogenic microorganisms is immunological escape, in which the host antibodies produced against a dominant antigen destroy those organisms bearing it, resulting in the organisms being replaced by ones that possess a variant form of the antigen. However, the function of antigenic variation among organisms differs and, in some cases, its analysis is complex. In Giardia
, antibodies directed against VSPs, including VSP-specific surface reacting mAbs (Nash and Aggarwal, 1986
), immune lactogenic IgA (Stager et al., 1998
), and serum from infected humans (Nash et al., 1990a
; Nash et al., 1990b
) or animals, either inhibit growth or kill recognized trophozoites (Hemphill et al., 1996
; Stager and Muller, 1997
), thus allowing the repopulation of trophozoites expressing other VSPs. The in vitro
effects range from little or no inhibition of growth to cytotoxicity, and the effects appear to depend on the concentration (direct correlation), affinity, and even perhaps the nature of the antibody and target epitopes (Hemphill et al., 1996
; Nash and Aggarwal, 1986
; Stager and Muller, 1997
). In contrast, monovalent F(ab') of the same antibodies exhibited none of these cytological effects (Hemphill et al., 1996
; Nash and Aggarwal, 1986
), although the VSP switching was not analyzed in this case.
Our results support the findings that exposure of trophozoites to a high level of specific-VSP Abs results in cell death and the emergence of trophozoites expressing an antigenically different VSP. Most significantly, this event is strongly linked to deimination (citrullination) of the cytoplasmic tail of VSPs, since mutation of the amino acid R led to survival of targeted trophozoites and failure to switch. Conversely, we have now shown that an increase in the VSP citrullination by over-expression of gADI in the presence of the specific Ab causes deregulation of VSP switching, probably due to an amplification of a signal transduction event (Touz et al., 2005
and the present work). Therefore, it is now clear that post-translational modifications are important for the control of the antigenic variation in Giardia
. These studies are consistent with the hypothesis that both immunological and biological factors act in concert to select which VSPs are expressed in a particular host.
The functional significance of gADI in cell survival appears not to be restricted to its role in energy production and antigenic variation. We found that the subcellular localization of gADI is cytoplasmic and significantly close to the plasma membrane but it is up-regulated and translocated to the nuclei when the trophozoites are induced to encyst. At least two possibilities arise from this event: 1) as an arginine deiminase, gADI may be sequestered from the cytoplasm to enter in a “stand-by” process during encystation, because the requirement of energy during this process is lower than that needed during active growth; 2) as a PAD, it may be directed into the nuclei for histone modification and transcription regulation (Wang et al., 2004
). However, the results presented in this work suggest that the second possibility is more likely. Post-translational histone modifications, such as phosphorylation, acetylation, methylation, and citrullination, regulate a broad range of DNA and chromatin-templated nuclear events, including transcription (Jenuwein and Allis, 2001
; Wang et al., 2004
). Because acetylation was shown to be a Giardia
histone post-translational modification (Kulakova et al., 2006
), we propose that gADI modifies histones by citrullination, with this posttranslational modification being involved in the down-regulation of the encystation process. This hypothesis is based on the fact that translocation of the functionally over-expressed gADI to the nuclei avoids cyst formation. We also suggest that gADI causes the cwps
genes to turn off as an essential requirement in order to successfully complete the encystation. This effect takes place early on in gADI-transgenic cells, where the high over-expression of gADI together with its nuclear translocation avoids CWP expression and cyst formation. Nevertheless, we can not exclude the possibility that gADI is also involved in the control of its major surface antigen when its life cycle is completed, as was demonstrated by Svard et al. (Svard et al., 1998
). Differentiation-associated switching of surface antigens could be one reason for the common occurrence of repeated infections (Gilman et al., 1988
), due to the down-regulation of the major VSP expressed during encystation and the appearance of new VSPs after excystation.
gADI possesses two predicted nuclear localization signals (PQRRREQ and RRGIVMGQFQAPQRRRE) (SignalIP program) (Le Panse et al., 1997
), suggesting that these motifs are involved in the translocation of this protein to the nuclei during encystation. There is, however, no evidence of the participation of these motifs in the nuclear protein translocation of any protein in this parasite. Thus, further analyses are needed to show how this parasite utilizes the nuclear signaling motifs that are highly conserved during evolution. Another possibility is that gADI is translocated to the nuclei by means of SUMO protein binding. Recently, it was shown that conjugation of SUMO-1 could lead to protein stabilization and protection from degradation, but an increasing body of evidence implicates sumoylation in the targeting of certain proteins to the cell nucleus and to the subnuclear structures (Dohmen, 2004
). The underlying mechanisms, however, are still largely unknown. In Giardia
, the molecular biology of enzymes of the SUMO system has never been addressed. In fact, only three putative proteins involved in sumoylation have been posted in the Giardia
DB: SUMO-1 (or Sentrin, GGD 7760), the dimmer of the SUMO-activating enzyme E1 (Uba2, GGD 6288), and the SUMO ligase (GGD 17386). Our results indicate that gADI is a sumoylated protein based on “in silico”
data, Western blotting, and immunoprecipitation using anti-SUMO mAb. This protein modification may be essential in the nuclear translocation of gADI in order to accomplish its function during encystation, since the 85 kDa-gADI was mainly located in the nuclear subfractions. Further research into the possibility that gADI function could be regulated by sumoylation may provide insight into the biology of this important intestinal parasite and also contribute to the understanding of the evolution of protein modification in eukaryotic cells.
Taking earlier studies and this work together, we have been able to unravel the multiple functions of arginine deiminase in the survival of Giardia
(). One of these concerns the ability to obtain energy by using free-arginine as the preferential fuel under anaerobic conditions. During the proliferative stages of growth, gADI converts arginine into citrulline, with ATP production occurring at the final enzymatic step of the ADH pathway (Schofield et al., 1992
) (). In addition to this function, it was reported that Giardia
uses gADI as a competitor to NOS from the host cell for the free-arginine, thereby reducing the production of NO and the host defense mechanism against microbial infection (Eckmann et al., 2000
). Supporting this finding, it was confirmed that gADI is released from Giardia
to the extracellular space as a 66 kDa protein when the trophozoites are in contact with human colon epithelial cells (Ringqvist et al., 2008
) (). Also, during growth, gADI acts as a peptidyl-arginine deiminase on the cytoplasmic tail of VSPs, probably by functioning as a regulator of antigenic variation (). During encystation, gADI is in the nuclei and cysts formation is reduced (). This translocation event is most likely associated with gADI sumoylation, but the possibility of the participation of gADI nuclear localization signals can not been discarted.
Schematic representation of gADI functions during growth and encystation
Changes in the environment (anaerobiosis, presence of Abs, presence of the host cell, or depletion of cholesterol) define which functions are essential for gADI to be performed at one point in time. The underlying mechanisms, however, are still largely unknown. We believe that the analysis of the protein modifications associated with differentiation, along with the mediators of these activities reported here, represent an important contribution in our understanding of the control of gene expression in parasitic protozoa.