Background The parasitic protozoa belonging to  Leishmania (L.) donovani  complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene,  Ldccys2  from  Leishmania (L.) chagasi . Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of  Leishmania (L.) chagasi  within the U937 macrophage cells.
Background
The parasitic protozoa belonging to  Leishmania (L.) donovani  complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene,  Ldccys2  from  Leishmania (L.) chagasi . Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of  Leishmania (L.) chagasi  within the U937 macrophage cells.Leishmania (L.) donovaniLdccys2Leishmania (L.) chagasiLeishmania (L.) chagasiResults The amastigote specific Ldccys2 genes of  L. (L.) chagasi  and  L. (L.) donovani  have identical gene organization, as determined by southern blots. In  vivo  expression analyses by Northern blots showed that  Ldccys2  is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from  Ldccys2  heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate  Ldcccys2  gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in  Leishmania  infection and pathogenesis.
Results
The amastigote specific Ldccys2 genes of  L. (L.) chagasi  and  L. (L.) donovani  have identical gene organization, as determined by southern blots. In  vivo  expression analyses by Northern blots showed that  Ldccys2  is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from  Ldccys2  heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate  Ldcccys2  gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in  Leishmania  infection and pathogenesis.L. (L.) chagasiL. (L.) donovanivivoLdccys2Ldccys2Ldcccys2LeishmaniaConclusions The study shows that  Ldccys2  is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of  L. (L.) chagasi  amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in  L. (L.) chagasi , especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.
Conclusions
The study shows that  Ldccys2  is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of  L. (L.) chagasi  amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in  L. (L.) chagasi , especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.Ldccys2L. (L.) chagasiL. (L.) chagasi

Background Leishmania  are the etiological agents of a variety of disease manifestations, collectively termed as leishmaniasis. Visceral leishmaniasis caused by  Leishmania (L.) donovani  and  Leishmania (L.) chagasi  is a serious health problem in many tropical and subtropical countries [ 1 - 3 ]. During the digenetic life cycles of  Leishmania , it alternates between gut of sand fly vector as an extra cellular promastigote and in the acidic phagolysosome of macrophage as an intracellular amastigote. However, the most intriguing question is the capacity of  Leishmania  to withstand the hydrolytic conditions of the macrophages, the mechanism of which is still unclear. Thus, identifying the genes expressed specifically in the amastigote stage of the parasites and elucidating their biological function is very important as it would provide new insights into the role of these gene products in the intracellular life cycle of the parasites. Further, this will also help in designing specific drugs and identifying vaccine candidates. Cysteine proteases play an important role in the infection, replication, development and metabolism of protozoan parasites [ 4 , 5 ]. They have been implicated in the invasion of human erythrocyte by  Plasmodium falciparum  [ 6 ] and considered as virulence factors in the pathogenesis of  Entamoeba histolytica  [ 7 ]. Cysteine protease activity is necessary for the survival of  Leishmania (L.) mexicana  [ 8 , 9 ] and related protozoan,  Trypanosoma cruzi , within the macrophages,  in vitro  [ 10 ]. Knockout studies in  L. (L.) mexicana  have shown that cysteine proteases not only are virulence factors but also act as modulators of host immune responses [ 11 , 12 ]. Thus, cysteine proteases have become a potential target for chemotherapy and a candidate for vaccine development. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of  T. cruzi ,  P. falciparum  and  L. (L.) major  [ 13 - 15 ]. Immunization with the hybrid protein vaccine, consisting of  L. (L.) major  cysteine proteases CPB and CPA, partially protected against leishmaniasis [ 16 ]. So far, functionally well characterized cysteine proteases are from the New World species of  Lesihamania  causing the cutaneous forms of leishmaniasis. The members of  L .(L.) donovani  complex also possess multiple classes of cysteine proteases, which are developmentally regulated [ 17 , 18 ] and are not functionally well characterized. Therefore, there is a need to study the function of these proteases and their role in visceral leishmaniasis. Studies aimed at defining the function of protozoan parasite components have often used gene disruption approach by homologous recombination. Recently an alternative antisense RNA method was followed that may easily and rapidly answer the complex biological questions. Anti sense RNA approach has been used to study the functions of certain gene products in  Entamoeba  and  Leishmania , to elucidate the functions of cysteine protease [ 19 , 20 ], A2 protein [ 21 ] and gp63 [ 22 ]. Previously, we have isolated and characterized two distinct cysteine protease cDNA clones  Ldccys1  and  Ldccys2  from promastigote and amastigote specific cDNA libararies of  L. (L.) chagasi  [ 17 ].  Ldccys1 , a member of multi gene family was characterized both in  L. (L.) chagasi  ( Ldccys1 ) and  L. (L.) donovani  ( Lddcys1 ) parasites [ 18 ]. In the present study, we have characterized the functional role of amastigote specific cysteine protease gene ( Ldccys2 ) of  L. (L.) chagasi . We have generated  Ldccys2  heterozygous knockout mutants of  L. (L.) chagasi  by homologous recombination. As an alternative approach, antisense mRNA expression was also employed. Results obtained from both, gene disruption and antisense mRNA expression shows that Ldccys2 plays an important role in the survival of amastigotes within the U937 macrophages.
Background
Leishmania  are the etiological agents of a variety of disease manifestations, collectively termed as leishmaniasis. Visceral leishmaniasis caused by  Leishmania (L.) donovani  and  Leishmania (L.) chagasi  is a serious health problem in many tropical and subtropical countries [ 1 - 3 ]. During the digenetic life cycles of  Leishmania , it alternates between gut of sand fly vector as an extra cellular promastigote and in the acidic phagolysosome of macrophage as an intracellular amastigote. However, the most intriguing question is the capacity of  Leishmania  to withstand the hydrolytic conditions of the macrophages, the mechanism of which is still unclear. Thus, identifying the genes expressed specifically in the amastigote stage of the parasites and elucidating their biological function is very important as it would provide new insights into the role of these gene products in the intracellular life cycle of the parasites. Further, this will also help in designing specific drugs and identifying vaccine candidates.LeishmaniaLeishmania (L.) donovaniLeishmania (L.) chagasiLeishmaniaLeishmaniaCysteine proteases play an important role in the infection, replication, development and metabolism of protozoan parasites [ 4 , 5 ]. They have been implicated in the invasion of human erythrocyte by  Plasmodium falciparum  [ 6 ] and considered as virulence factors in the pathogenesis of  Entamoeba histolytica  [ 7 ]. Cysteine protease activity is necessary for the survival of  Leishmania (L.) mexicana  [ 8 , 9 ] and related protozoan,  Trypanosoma cruzi , within the macrophages,  in vitro  [ 10 ]. Knockout studies in  L. (L.) mexicana  have shown that cysteine proteases not only are virulence factors but also act as modulators of host immune responses [ 11 , 12 ]. Thus, cysteine proteases have become a potential target for chemotherapy and a candidate for vaccine development. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of  T. cruzi ,  P. falciparum  and  L. (L.) major  [ 13 - 15 ]. Immunization with the hybrid protein vaccine, consisting of  L. (L.) major  cysteine proteases CPB and CPA, partially protected against leishmaniasis [ 16 ]. So far, functionally well characterized cysteine proteases are from the New World species of  Lesihamania  causing the cutaneous forms of leishmaniasis. The members of  L .(L.) donovani  complex also possess multiple classes of cysteine proteases, which are developmentally regulated [ 17 , 18 ] and are not functionally well characterized. Therefore, there is a need to study the function of these proteases and their role in visceral leishmaniasis.Plasmodium falciparumEntamoeba histolyticaLeishmania (L.) mexicanaTrypanosoma cruziin vitroL. (L.) mexicanaT. cruziP. falciparumL. (L.) majorL. (L.) majorLesihamaniaL .(L.) donovaniStudies aimed at defining the function of protozoan parasite components have often used gene disruption approach by homologous recombination. Recently an alternative antisense RNA method was followed that may easily and rapidly answer the complex biological questions. Anti sense RNA approach has been used to study the functions of certain gene products in  Entamoeba  and  Leishmania , to elucidate the functions of cysteine protease [ 19 , 20 ], A2 protein [ 21 ] and gp63 [ 22 ]. Previously, we have isolated and characterized two distinct cysteine protease cDNA clones  Ldccys1  and  Ldccys2  from promastigote and amastigote specific cDNA libararies of  L. (L.) chagasi  [ 17 ].  Ldccys1 , a member of multi gene family was characterized both in  L. (L.) chagasi  ( Ldccys1 ) and  L. (L.) donovani  ( Lddcys1 ) parasites [ 18 ]. In the present study, we have characterized the functional role of amastigote specific cysteine protease gene ( Ldccys2 ) of  L. (L.) chagasi . We have generated  Ldccys2  heterozygous knockout mutants of  L. (L.) chagasi  by homologous recombination. As an alternative approach, antisense mRNA expression was also employed. Results obtained from both, gene disruption and antisense mRNA expression shows that Ldccys2 plays an important role in the survival of amastigotes within the U937 macrophages.EntamoebaLeishmaniaLdccys1Ldccys2L. (L.) chagasiLdccys1L. (L.) chagasiLdccys1L. (L.) donovaniLddcys1Ldccys2L. (L.) chagasiLdccys2L. (L.) chagasiResults Ldccys2 is a single copy gene From our earlier studies it is known that  Ldccys2  is a single copy gene in  L. (L.) chagasi . In order to compare the genomic organization of  Ldccys2  gene in  L. (L.) donovani and L. (L.) chagasi , the members of  L. (L.) donovani  complex, Southern analysis of genomic DNA was performed. Genomic DNA was digested with different restriction enzymes as shown in Figure  1A  and probed with  Ldccys2  coding region as a probe. Identical hybridizing bands were present in both  L. (L.) chagasi  and  L. (L.) donovani  (Figure  1A ). PCR amplification and sequencing of  Ldccys2  coding region from  L. (L.) donovani  genomic DNA showed identical nucleotide sequences, indicating that  L. (L.) chagasi  and  L. (L.) donovani  had identical  Ldccys2  cysteine protease genes. Ldccys2 gene is differentially expressed To study the expression of  Ldccys2  cysteine protease genes, Northern blot analysis was performed using  L. (L.) chagasi  total RNA from log and stationary phase promastigotes and amastigotes. The 3'UTR region of  Ldccys2  used as a probe, hybridized to a 2.5 kb transcript in amastigote (Figure  1 BI) unlike the cysteine protease,  Ldccys1 , which is predominantly expressed in promastigote stages of the parasite (Figure  1  BII). The α- tubulin  gene expression was used as an internal control (Figure  1  BIII).  Ldccys2  showed similar pattern of expression in  L. (L.) donovani  (data not shown). It is important to note that this result is in contrast to the earlier report (17), wherein  Ldccys2  was identified as promastigote specific cysteine protease gene and  Ldccys1  was thought to be amastigote specific. We have confirmed the expression pattern and rectified this error in the following publication (18). To further confirm the amastigote expression of Ldccys2, Western blot analysis was carried out using extracts from both promastigotes and U937 infected amastigotes of  L. (L.) chagasi . The anti-Ldccys2 antibody detected a 30 kDa band in amastigotes alone (Figure  1C , lane 3). However, a single 46 kDa band seen in log and stationary phase promastigotes (Figure  1C , lanes 1 and 2) represents promastigote specific cysteine protease Ldccys1, due to the cross-reactivity of the antibody. Taken together, the above results show that Ldccys2 is expressed only in the amastigote stage of  L. (L.) chagasi . Recombinant Ldccys2 expressed in insect cells is functionally active In order to determine the cysteine protease activity encoded by  Ldccys2 , the predicted coding region was cloned into pIE1/153A.jhe (6hep) vector and transfected into High Five insect cells (invitrogen). The supernatant was harvested and analyzed for the presence of recombinant protein by Western blot analysis using JHE and Ldccys2 antibodies. Western blot analyses with α-JHE antibody (Figure  2A , lane3) detected a band of 96 kDa, corresponding to the fusion protein. The lower molecular mass bands are due to the processed products as a result of autocatalytic activity. Anti- Ldccys2 antibody however, could not detect the recombinant fusion protein (Figure  2B  lane 3). The processed products were detected instead, which may be because of inaccessibility of the epitope in the fusion protein. Both the antibodies did not give any background with the negative control (cell extract, Figure  2B  and  2C , lane1). Protease activity determined by gelatin SDS-PAGE, showed a band of approximately 30 kDa (Figure  2C , lane 3), confirming the protease activity. Cysteine protease activity was completely inhibited by a specific inhibitor, E-64 at 20 μM concentration (data not shown). Ldccys2 single allele gene replacement by homologous recombination In order to investigate the function  Ldccys2  gene, we have performed gene replacement by disrupting a 500 bp coding region of the gene with  hyg  and  dhfr-ts  cassette as shown in figure  3A . Following transfection, hygromycin B resistant parasites ( Ldccys2KO , heterozygous knockout mutants) were selected for further analysis. The  Pst I restriction enzyme digested genomic DNA from wild type  L. (L.) chagasi  and  Ldccys2 KO  were subjected to Southern analysis and results are presented in figure  3B . The disrupted  Ldccys2  allele had an additional  Pst I site that is present within the  hyg  gene. As shown in the figure  3B , 5' and 3'probes (probes a and b) hybridized to a single 3.5 kb band with wild type genomic DNA. In  Ldccys2KO , probe-a hybridized to two bands (3.5 kb and 2.8 kb) and probe-b hybridized to 3.5 kb and 3.0 kb bands. As expected, one of the bands corresponds to the wild type allele whereas the other band corresponded to the disrupted allele. The  hyg  specific probe-c resulted in no hybridizing band with  Pst I digested wild type genomic DNA, while in  Ldccys2KO , two bands (2.8 kb and 3.0 kb) corresponding to the disrupted allele were detected (Figure  3B , probe c). These results clearly indicate that one of the alleles of the  Ldccys2  gene has been replaced by hygromycin gene. Similarly, a second cassette using neomycin gene was used in order to replace the second allele of  Ldccys2  gene. Despite numerous attempts, we were unable to obtain a null mutant of  Ldccys2 . We then checked for any events of rearrangements or change in ploidy levels in the heterozygous mutants. No such detectable events were detected (data not shown). Ldccys2 single allele knock-out mutants had reduced mRNA and protein levels In order to compare the expression of  Ldccys2  gene in wild type and heterozygous knockout mutants ( Ldccys2 KO ), amastigote RNA was isolated from U937 cells infected with wild type as well as  Ldccys2KO  mutant  L. (L.) chagasi  promastigotes. Northern blot analysis with Ldccys2 coding region probe detected a single transcript of 2.5 kb from wild type as well as  Ldccys2 KO  amastigotes (Figure  4A , panel 1). However, the transcript level of  Ldccys2  in the  Ldccys2KO  amastigotes was reduced by 64% compared to that in the wild type  L. (L.) chagasi . There was no change in the level of α-tubulin expression either in the  Ldccys2KO  or in the wild type amastigotes (Figure  4A , panel 2). The effect of  Ldccys2  single allele disruption at protein level was studied using western blot analysis. Equal amounts of lysates from wild type and  Ldccys2KO L. (L.) chagasi  amastigotes were analyzed using antiserum raised against Ldccys2 protein. Wild type  L. (L.) chagasi  amastigotes showed a major band of 30 kDa (Figure  4B , panel 1), while  Ldccys2KO  amastigotes showed an identical sized band, but there was a reduction in amounts by 45%, based on the quantitation of bands. The antibody did not pick up any signal from the negative control (U937 cells). Figure  4B , panel 2 indicates the equal loading of proteins. These results once again, confirm the deletion of one of the alleles of  Ldccys2 . Ldccys2 heterozygous knockout mutants exhibit reduced infectivity and intra-macrophage survival of amastigotes In order to study the effect of Ldccys2 heterozygous knockout mutants on parasite growth, a growth curve analysis was performed. No significant differences were observed between the wildtype parasites and the  Ldccys2KO  (data not shown). Next, we performed an infection assay with differentiated U937 macrophage cells using with wild type and  Ldccys2KO L. (L.) chagasi  promastigotes. Interestingly, twelve hours after infection, the number of amastigotes in  Ldccys2KO  infected macrophages were reduced by half as compared to that of wild type infected macrophage cells. This trend remained same at all the time intervals tested (24, 36 and 48 hours post infection), indicating that the intracellular amastigotes were not able to multiply efficiently (Figure  4C ). The percentage of infected macrophages in the wildtype and  Ldccys2KO  were similar at 12, 24, 36 and 48 hours post infection (Figure  4D ), however, there was a clear decrease in the initial infection in case of  Ldccys2KO , as compared to the wildtype parasites. The above data suggests that Ldccys2 heterozygous knockout has resulted in reduced levels of Ldccys2 expression and furthermore, has decreased the ability of the amastigotes to infect the macrophages and survive inside the macrophage cells. Taken together, these results suggest that Ldccys2 gene has a significant role in the infection and intramacrophage survival of amastigotes. Antisense mRNA inhibition of Ldccys2 resulted in reduced macrophage infection and poor amastigote survival within the macrophages We were unable to generate a complete knock out mutant of  Ldccys2  and the heterozygous knockout mutants only partially inhibited the gene expression. Therefore, we used an alternative approach of antisense mRNA inhibition to silence the  Ldccys2  gene and to confirm the conclusions derived from  Ldccys2  heterozygous knockout mutants studies. Plasmid constructs containing the  Ldccys2  antisense and sense sequences were transfected into  L. (L.) chagasi  promastigotes. The presence of plasmids in these transfectants was confirmed by Southern analysis with  Ldccys2  probes (data not shown). Northern blot analyses of amastigotes expressing different plasmid constructs were carried out using specific sense and antisense probes. As expected,  Ldccys2  antisense mRNA was expressed only in amastigotes with antisense constructs (Figure  5A , panel 1), and these amastigotes showed a 74% decrease in the  Ldccys2  mRNA levels (Figure  5A , panel 2). The amastigotes containing either the sense plasmid or the control plasmid did not show any change in the levels of  Ldccys2  mRNA (Figure  5A , panel 2). The stained agarose gel indicates the equal amounts of RNA used in the Northern blots (Figure  5A , panel 3). These results clearly demonstrate that the episomal expression of  Ldccys2  antisense mRNA reduces the levels of endogenous  Ldccys2  mRNA. We next performed Western blot analysis of transfectants expressing sense and antisense  Ldccys2  mRNA using α-Ldccys2 antibody. Amastigotes expressing antisense mRNA showed a significant reduction (by 76%) in the levels of Ldccys2, however, the expression of sense mRNA did not increase the levels of Ldccys2 compared to amastigotes containing the control plasmid (Figure  5B , panel1). Equal loading of the protein is depicted by the Coomassie stained gel (Figure  5B , panel 2). The results further confirm that  Ldccys2  antisense mRNA expression does inhibit the endogenous levels of Ldccys2. Survival of  L. (L.) chagasi  amastigotes within the macrophages was assessed  in vitro , using U937 cells. Results from three individual experiments are pooled and the collective data is presented in figure  5C . Results showed that the amastigotes carrying either the control plasmid (P6.5) or expressing  Ldccys2  sense mRNA did not have any effect on their capacity to infect and survive inside the macrophages. The number of amastigotes per macrophage cell remained the same at initial stages of infection (data not shown). However, amastigotes expressing  Ldccys2  antisense mRNA showed significant reduction (by almost 50%) in terms of the initial infection and intra macrophage survival starting from 12 hours post infection and remained consistent up to 48 hours after infection (Figure  5D ). The percentage of total infected macrophages is shown in figure  5D . Consistent with the results from the heterozygous knockout mutants, the  Ldccys2  antisense transfectants had reduced levels of infection when compared to that of the wildtype and control plasmids. These results further underline the importance of Ldccys2 in infection of macrophages and subsequent survival of amastigotes within the macrophage cells. Taken together, these results indicate that the amastigote specific cysteine protease, Ldccys2 plays an important role in intramacrophage survival of  L. (L.) chagasi  amastigotes.
Results
Ldccys2 is a single copy gene From our earlier studies it is known that  Ldccys2  is a single copy gene in  L. (L.) chagasi . In order to compare the genomic organization of  Ldccys2  gene in  L. (L.) donovani and L. (L.) chagasi , the members of  L. (L.) donovani  complex, Southern analysis of genomic DNA was performed. Genomic DNA was digested with different restriction enzymes as shown in Figure  1A  and probed with  Ldccys2  coding region as a probe. Identical hybridizing bands were present in both  L. (L.) chagasi  and  L. (L.) donovani  (Figure  1A ). PCR amplification and sequencing of  Ldccys2  coding region from  L. (L.) donovani  genomic DNA showed identical nucleotide sequences, indicating that  L. (L.) chagasi  and  L. (L.) donovani  had identical  Ldccys2  cysteine protease genes.
Ldccys2 is a single copy gene
From our earlier studies it is known that  Ldccys2  is a single copy gene in  L. (L.) chagasi . In order to compare the genomic organization of  Ldccys2  gene in  L. (L.) donovani and L. (L.) chagasi , the members of  L. (L.) donovani  complex, Southern analysis of genomic DNA was performed. Genomic DNA was digested with different restriction enzymes as shown in Figure  1A  and probed with  Ldccys2  coding region as a probe. Identical hybridizing bands were present in both  L. (L.) chagasi  and  L. (L.) donovani  (Figure  1A ). PCR amplification and sequencing of  Ldccys2  coding region from  L. (L.) donovani  genomic DNA showed identical nucleotide sequences, indicating that  L. (L.) chagasi  and  L. (L.) donovani  had identical  Ldccys2  cysteine protease genes.Ldccys2L. (L.) chagasiLdccys2L. (L.) donovani and L. (L.) chagasiL. (L.) donovaniLdccys2L. (L.) chagasiL. (L.) donovaniLdccys2L. (L.) donovaniL. (L.) chagasiL. (L.) donovaniLdccys2Ldccys2 gene is differentially expressed To study the expression of  Ldccys2  cysteine protease genes, Northern blot analysis was performed using  L. (L.) chagasi  total RNA from log and stationary phase promastigotes and amastigotes. The 3'UTR region of  Ldccys2  used as a probe, hybridized to a 2.5 kb transcript in amastigote (Figure  1 BI) unlike the cysteine protease,  Ldccys1 , which is predominantly expressed in promastigote stages of the parasite (Figure  1  BII). The α- tubulin  gene expression was used as an internal control (Figure  1  BIII).  Ldccys2  showed similar pattern of expression in  L. (L.) donovani  (data not shown). It is important to note that this result is in contrast to the earlier report (17), wherein  Ldccys2  was identified as promastigote specific cysteine protease gene and  Ldccys1  was thought to be amastigote specific. We have confirmed the expression pattern and rectified this error in the following publication (18). To further confirm the amastigote expression of Ldccys2, Western blot analysis was carried out using extracts from both promastigotes and U937 infected amastigotes of  L. (L.) chagasi . The anti-Ldccys2 antibody detected a 30 kDa band in amastigotes alone (Figure  1C , lane 3). However, a single 46 kDa band seen in log and stationary phase promastigotes (Figure  1C , lanes 1 and 2) represents promastigote specific cysteine protease Ldccys1, due to the cross-reactivity of the antibody. Taken together, the above results show that Ldccys2 is expressed only in the amastigote stage of  L. (L.) chagasi .
Ldccys2 gene is differentially expressed
To study the expression of  Ldccys2  cysteine protease genes, Northern blot analysis was performed using  L. (L.) chagasi  total RNA from log and stationary phase promastigotes and amastigotes. The 3'UTR region of  Ldccys2  used as a probe, hybridized to a 2.5 kb transcript in amastigote (Figure  1 BI) unlike the cysteine protease,  Ldccys1 , which is predominantly expressed in promastigote stages of the parasite (Figure  1  BII). The α- tubulin  gene expression was used as an internal control (Figure  1  BIII).  Ldccys2  showed similar pattern of expression in  L. (L.) donovani  (data not shown). It is important to note that this result is in contrast to the earlier report (17), wherein  Ldccys2  was identified as promastigote specific cysteine protease gene and  Ldccys1  was thought to be amastigote specific. We have confirmed the expression pattern and rectified this error in the following publication (18).Ldccys2L. (L.) chagasiLdccys2Ldccys1tubulinLdccys2L. (L.) donovaniLdccys2Ldccys1To further confirm the amastigote expression of Ldccys2, Western blot analysis was carried out using extracts from both promastigotes and U937 infected amastigotes of  L. (L.) chagasi . The anti-Ldccys2 antibody detected a 30 kDa band in amastigotes alone (Figure  1C , lane 3).L. (L.) chagasiHowever, a single 46 kDa band seen in log and stationary phase promastigotes (Figure  1C , lanes 1 and 2) represents promastigote specific cysteine protease Ldccys1, due to the cross-reactivity of the antibody. Taken together, the above results show that Ldccys2 is expressed only in the amastigote stage of  L. (L.) chagasi .L. (L.) chagasiRecombinant Ldccys2 expressed in insect cells is functionally active In order to determine the cysteine protease activity encoded by  Ldccys2 , the predicted coding region was cloned into pIE1/153A.jhe (6hep) vector and transfected into High Five insect cells (invitrogen). The supernatant was harvested and analyzed for the presence of recombinant protein by Western blot analysis using JHE and Ldccys2 antibodies. Western blot analyses with α-JHE antibody (Figure  2A , lane3) detected a band of 96 kDa, corresponding to the fusion protein. The lower molecular mass bands are due to the processed products as a result of autocatalytic activity. Anti- Ldccys2 antibody however, could not detect the recombinant fusion protein (Figure  2B  lane 3). The processed products were detected instead, which may be because of inaccessibility of the epitope in the fusion protein. Both the antibodies did not give any background with the negative control (cell extract, Figure  2B  and  2C , lane1). Protease activity determined by gelatin SDS-PAGE, showed a band of approximately 30 kDa (Figure  2C , lane 3), confirming the protease activity. Cysteine protease activity was completely inhibited by a specific inhibitor, E-64 at 20 μM concentration (data not shown).
Recombinant Ldccys2 expressed in insect cells is functionally active
In order to determine the cysteine protease activity encoded by  Ldccys2 , the predicted coding region was cloned into pIE1/153A.jhe (6hep) vector and transfected into High Five insect cells (invitrogen). The supernatant was harvested and analyzed for the presence of recombinant protein by Western blot analysis using JHE and Ldccys2 antibodies. Western blot analyses with α-JHE antibody (Figure  2A , lane3) detected a band of 96 kDa, corresponding to the fusion protein. The lower molecular mass bands are due to the processed products as a result of autocatalytic activity. Anti- Ldccys2 antibody however, could not detect the recombinant fusion protein (Figure  2B  lane 3). The processed products were detected instead, which may be because of inaccessibility of the epitope in the fusion protein. Both the antibodies did not give any background with the negative control (cell extract, Figure  2B  and  2C , lane1). Protease activity determined by gelatin SDS-PAGE, showed a band of approximately 30 kDa (Figure  2C , lane 3), confirming the protease activity. Cysteine protease activity was completely inhibited by a specific inhibitor, E-64 at 20 μM concentration (data not shown).Ldccys2Ldccys2 single allele gene replacement by homologous recombination In order to investigate the function  Ldccys2  gene, we have performed gene replacement by disrupting a 500 bp coding region of the gene with  hyg  and  dhfr-ts  cassette as shown in figure  3A . Following transfection, hygromycin B resistant parasites ( Ldccys2KO , heterozygous knockout mutants) were selected for further analysis. The  Pst I restriction enzyme digested genomic DNA from wild type  L. (L.) chagasi  and  Ldccys2 KO  were subjected to Southern analysis and results are presented in figure  3B . The disrupted  Ldccys2  allele had an additional  Pst I site that is present within the  hyg  gene. As shown in the figure  3B , 5' and 3'probes (probes a and b) hybridized to a single 3.5 kb band with wild type genomic DNA. In  Ldccys2KO , probe-a hybridized to two bands (3.5 kb and 2.8 kb) and probe-b hybridized to 3.5 kb and 3.0 kb bands. As expected, one of the bands corresponds to the wild type allele whereas the other band corresponded to the disrupted allele. The  hyg  specific probe-c resulted in no hybridizing band with  Pst I digested wild type genomic DNA, while in  Ldccys2KO , two bands (2.8 kb and 3.0 kb) corresponding to the disrupted allele were detected (Figure  3B , probe c). These results clearly indicate that one of the alleles of the  Ldccys2  gene has been replaced by hygromycin gene. Similarly, a second cassette using neomycin gene was used in order to replace the second allele of  Ldccys2  gene. Despite numerous attempts, we were unable to obtain a null mutant of  Ldccys2 . We then checked for any events of rearrangements or change in ploidy levels in the heterozygous mutants. No such detectable events were detected (data not shown).
Ldccys2 single allele gene replacement by homologous recombination
In order to investigate the function  Ldccys2  gene, we have performed gene replacement by disrupting a 500 bp coding region of the gene with  hyg  and  dhfr-ts  cassette as shown in figure  3A . Following transfection, hygromycin B resistant parasites ( Ldccys2KO , heterozygous knockout mutants) were selected for further analysis. The  Pst I restriction enzyme digested genomic DNA from wild type  L. (L.) chagasi  and  Ldccys2 KO  were subjected to Southern analysis and results are presented in figure  3B . The disrupted  Ldccys2  allele had an additional  Pst I site that is present within the  hyg  gene. As shown in the figure  3B , 5' and 3'probes (probes a and b) hybridized to a single 3.5 kb band with wild type genomic DNA. In  Ldccys2KO , probe-a hybridized to two bands (3.5 kb and 2.8 kb) and probe-b hybridized to 3.5 kb and 3.0 kb bands. As expected, one of the bands corresponds to the wild type allele whereas the other band corresponded to the disrupted allele. The  hyg  specific probe-c resulted in no hybridizing band with  Pst I digested wild type genomic DNA, while in  Ldccys2KO , two bands (2.8 kb and 3.0 kb) corresponding to the disrupted allele were detected (Figure  3B , probe c). These results clearly indicate that one of the alleles of the  Ldccys2  gene has been replaced by hygromycin gene. Similarly, a second cassette using neomycin gene was used in order to replace the second allele of  Ldccys2  gene. Despite numerous attempts, we were unable to obtain a null mutant of  Ldccys2 . We then checked for any events of rearrangements or change in ploidy levels in the heterozygous mutants. No such detectable events were detected (data not shown).Ldccys2hygdhfr-tsLdccys2KOPstL. (L.) chagasiLdccys2 KOLdccys2PsthygLdccys2KOhygPstLdccys2KOLdccys2Ldccys2Ldccys2Ldccys2 single allele knock-out mutants had reduced mRNA and protein levels In order to compare the expression of  Ldccys2  gene in wild type and heterozygous knockout mutants ( Ldccys2 KO ), amastigote RNA was isolated from U937 cells infected with wild type as well as  Ldccys2KO  mutant  L. (L.) chagasi  promastigotes. Northern blot analysis with Ldccys2 coding region probe detected a single transcript of 2.5 kb from wild type as well as  Ldccys2 KO  amastigotes (Figure  4A , panel 1). However, the transcript level of  Ldccys2  in the  Ldccys2KO  amastigotes was reduced by 64% compared to that in the wild type  L. (L.) chagasi . There was no change in the level of α-tubulin expression either in the  Ldccys2KO  or in the wild type amastigotes (Figure  4A , panel 2). The effect of  Ldccys2  single allele disruption at protein level was studied using western blot analysis. Equal amounts of lysates from wild type and  Ldccys2KO L. (L.) chagasi  amastigotes were analyzed using antiserum raised against Ldccys2 protein. Wild type  L. (L.) chagasi  amastigotes showed a major band of 30 kDa (Figure  4B , panel 1), while  Ldccys2KO  amastigotes showed an identical sized band, but there was a reduction in amounts by 45%, based on the quantitation of bands. The antibody did not pick up any signal from the negative control (U937 cells). Figure  4B , panel 2 indicates the equal loading of proteins. These results once again, confirm the deletion of one of the alleles of  Ldccys2 .
Ldccys2 single allele knock-out mutants had reduced mRNA and protein levels
In order to compare the expression of  Ldccys2  gene in wild type and heterozygous knockout mutants ( Ldccys2 KO ), amastigote RNA was isolated from U937 cells infected with wild type as well as  Ldccys2KO  mutant  L. (L.) chagasi  promastigotes. Northern blot analysis with Ldccys2 coding region probe detected a single transcript of 2.5 kb from wild type as well as  Ldccys2 KO  amastigotes (Figure  4A , panel 1). However, the transcript level of  Ldccys2  in the  Ldccys2KO  amastigotes was reduced by 64% compared to that in the wild type  L. (L.) chagasi . There was no change in the level of α-tubulin expression either in the  Ldccys2KO  or in the wild type amastigotes (Figure  4A , panel 2).Ldccys2Ldccys2 KOLdccys2KOL. (L.) chagasiLdccys2 KOLdccys2Ldccys2KOL. (L.) chagasiLdccys2KOThe effect of  Ldccys2  single allele disruption at protein level was studied using western blot analysis. Equal amounts of lysates from wild type and  Ldccys2KO L. (L.) chagasi  amastigotes were analyzed using antiserum raised against Ldccys2 protein. Wild type  L. (L.) chagasi  amastigotes showed a major band of 30 kDa (Figure  4B , panel 1), while  Ldccys2KO  amastigotes showed an identical sized band, but there was a reduction in amounts by 45%, based on the quantitation of bands. The antibody did not pick up any signal from the negative control (U937 cells). Figure  4B , panel 2 indicates the equal loading of proteins. These results once again, confirm the deletion of one of the alleles of  Ldccys2 .Ldccys2Ldccys2KO L. (L.) chagasiL. (L.) chagasiLdccys2KOLdccys2Ldccys2 heterozygous knockout mutants exhibit reduced infectivity and intra-macrophage survival of amastigotes In order to study the effect of Ldccys2 heterozygous knockout mutants on parasite growth, a growth curve analysis was performed. No significant differences were observed between the wildtype parasites and the  Ldccys2KO  (data not shown). Next, we performed an infection assay with differentiated U937 macrophage cells using with wild type and  Ldccys2KO L. (L.) chagasi  promastigotes. Interestingly, twelve hours after infection, the number of amastigotes in  Ldccys2KO  infected macrophages were reduced by half as compared to that of wild type infected macrophage cells. This trend remained same at all the time intervals tested (24, 36 and 48 hours post infection), indicating that the intracellular amastigotes were not able to multiply efficiently (Figure  4C ). The percentage of infected macrophages in the wildtype and  Ldccys2KO  were similar at 12, 24, 36 and 48 hours post infection (Figure  4D ), however, there was a clear decrease in the initial infection in case of  Ldccys2KO , as compared to the wildtype parasites. The above data suggests that Ldccys2 heterozygous knockout has resulted in reduced levels of Ldccys2 expression and furthermore, has decreased the ability of the amastigotes to infect the macrophages and survive inside the macrophage cells. Taken together, these results suggest that Ldccys2 gene has a significant role in the infection and intramacrophage survival of amastigotes.
Ldccys2 heterozygous knockout mutants exhibit reduced infectivity and intra-macrophage survival of amastigotes
In order to study the effect of Ldccys2 heterozygous knockout mutants on parasite growth, a growth curve analysis was performed. No significant differences were observed between the wildtype parasites and the  Ldccys2KO  (data not shown). Next, we performed an infection assay with differentiated U937 macrophage cells using with wild type and  Ldccys2KO L. (L.) chagasi  promastigotes. Interestingly, twelve hours after infection, the number of amastigotes in  Ldccys2KO  infected macrophages were reduced by half as compared to that of wild type infected macrophage cells. This trend remained same at all the time intervals tested (24, 36 and 48 hours post infection), indicating that the intracellular amastigotes were not able to multiply efficiently (Figure  4C ). The percentage of infected macrophages in the wildtype and  Ldccys2KO  were similar at 12, 24, 36 and 48 hours post infection (Figure  4D ), however, there was a clear decrease in the initial infection in case of  Ldccys2KO , as compared to the wildtype parasites. The above data suggests that Ldccys2 heterozygous knockout has resulted in reduced levels of Ldccys2 expression and furthermore, has decreased the ability of the amastigotes to infect the macrophages and survive inside the macrophage cells. Taken together, these results suggest that Ldccys2 gene has a significant role in the infection and intramacrophage survival of amastigotes.Ldccys2KOLdccys2KO L. (L.) chagasiLdccys2KOLdccys2KOLdccys2KOAntisense mRNA inhibition of Ldccys2 resulted in reduced macrophage infection and poor amastigote survival within the macrophages We were unable to generate a complete knock out mutant of  Ldccys2  and the heterozygous knockout mutants only partially inhibited the gene expression. Therefore, we used an alternative approach of antisense mRNA inhibition to silence the  Ldccys2  gene and to confirm the conclusions derived from  Ldccys2  heterozygous knockout mutants studies. Plasmid constructs containing the  Ldccys2  antisense and sense sequences were transfected into  L. (L.) chagasi  promastigotes. The presence of plasmids in these transfectants was confirmed by Southern analysis with  Ldccys2  probes (data not shown). Northern blot analyses of amastigotes expressing different plasmid constructs were carried out using specific sense and antisense probes. As expected,  Ldccys2  antisense mRNA was expressed only in amastigotes with antisense constructs (Figure  5A , panel 1), and these amastigotes showed a 74% decrease in the  Ldccys2  mRNA levels (Figure  5A , panel 2). The amastigotes containing either the sense plasmid or the control plasmid did not show any change in the levels of  Ldccys2  mRNA (Figure  5A , panel 2). The stained agarose gel indicates the equal amounts of RNA used in the Northern blots (Figure  5A , panel 3). These results clearly demonstrate that the episomal expression of  Ldccys2  antisense mRNA reduces the levels of endogenous  Ldccys2  mRNA. We next performed Western blot analysis of transfectants expressing sense and antisense  Ldccys2  mRNA using α-Ldccys2 antibody. Amastigotes expressing antisense mRNA showed a significant reduction (by 76%) in the levels of Ldccys2, however, the expression of sense mRNA did not increase the levels of Ldccys2 compared to amastigotes containing the control plasmid (Figure  5B , panel1). Equal loading of the protein is depicted by the Coomassie stained gel (Figure  5B , panel 2). The results further confirm that  Ldccys2  antisense mRNA expression does inhibit the endogenous levels of Ldccys2. Survival of  L. (L.) chagasi  amastigotes within the macrophages was assessed  in vitro , using U937 cells. Results from three individual experiments are pooled and the collective data is presented in figure  5C . Results showed that the amastigotes carrying either the control plasmid (P6.5) or expressing  Ldccys2  sense mRNA did not have any effect on their capacity to infect and survive inside the macrophages. The number of amastigotes per macrophage cell remained the same at initial stages of infection (data not shown). However, amastigotes expressing  Ldccys2  antisense mRNA showed significant reduction (by almost 50%) in terms of the initial infection and intra macrophage survival starting from 12 hours post infection and remained consistent up to 48 hours after infection (Figure  5D ). The percentage of total infected macrophages is shown in figure  5D . Consistent with the results from the heterozygous knockout mutants, the  Ldccys2  antisense transfectants had reduced levels of infection when compared to that of the wildtype and control plasmids. These results further underline the importance of Ldccys2 in infection of macrophages and subsequent survival of amastigotes within the macrophage cells. Taken together, these results indicate that the amastigote specific cysteine protease, Ldccys2 plays an important role in intramacrophage survival of  L. (L.) chagasi  amastigotes.
Antisense mRNA inhibition of Ldccys2 resulted in reduced macrophage infection and poor amastigote survival within the macrophages
We were unable to generate a complete knock out mutant of  Ldccys2  and the heterozygous knockout mutants only partially inhibited the gene expression. Therefore, we used an alternative approach of antisense mRNA inhibition to silence the  Ldccys2  gene and to confirm the conclusions derived from  Ldccys2  heterozygous knockout mutants studies. Plasmid constructs containing the  Ldccys2  antisense and sense sequences were transfected into  L. (L.) chagasi  promastigotes. The presence of plasmids in these transfectants was confirmed by Southern analysis with  Ldccys2  probes (data not shown). Northern blot analyses of amastigotes expressing different plasmid constructs were carried out using specific sense and antisense probes. As expected,  Ldccys2  antisense mRNA was expressed only in amastigotes with antisense constructs (Figure  5A , panel 1), and these amastigotes showed a 74% decrease in the  Ldccys2  mRNA levels (Figure  5A , panel 2). The amastigotes containing either the sense plasmid or the control plasmid did not show any change in the levels of  Ldccys2  mRNA (Figure  5A , panel 2). The stained agarose gel indicates the equal amounts of RNA used in the Northern blots (Figure  5A , panel 3). These results clearly demonstrate that the episomal expression of  Ldccys2  antisense mRNA reduces the levels of endogenous  Ldccys2  mRNA.Ldccys2Ldccys2Ldccys2Ldccys2L. (L.) chagasiLdccys2Ldccys2Ldccys2Ldccys2Ldccys2Ldccys2We next performed Western blot analysis of transfectants expressing sense and antisense  Ldccys2  mRNA using α-Ldccys2 antibody. Amastigotes expressing antisense mRNA showed a significant reduction (by 76%) in the levels of Ldccys2, however, the expression of sense mRNA did not increase the levels of Ldccys2 compared to amastigotes containing the control plasmid (Figure  5B , panel1). Equal loading of the protein is depicted by the Coomassie stained gel (Figure  5B , panel 2). The results further confirm that  Ldccys2  antisense mRNA expression does inhibit the endogenous levels of Ldccys2.Ldccys2Ldccys2Survival of  L. (L.) chagasi  amastigotes within the macrophages was assessed  in vitro , using U937 cells. Results from three individual experiments are pooled and the collective data is presented in figure  5C . Results showed that the amastigotes carrying either the control plasmid (P6.5) or expressing  Ldccys2  sense mRNA did not have any effect on their capacity to infect and survive inside the macrophages. The number of amastigotes per macrophage cell remained the same at initial stages of infection (data not shown). However, amastigotes expressing  Ldccys2  antisense mRNA showed significant reduction (by almost 50%) in terms of the initial infection and intra macrophage survival starting from 12 hours post infection and remained consistent up to 48 hours after infection (Figure  5D ). The percentage of total infected macrophages is shown in figure  5D . Consistent with the results from the heterozygous knockout mutants, the  Ldccys2  antisense transfectants had reduced levels of infection when compared to that of the wildtype and control plasmids. These results further underline the importance of Ldccys2 in infection of macrophages and subsequent survival of amastigotes within the macrophage cells. Taken together, these results indicate that the amastigote specific cysteine protease, Ldccys2 plays an important role in intramacrophage survival of  L. (L.) chagasi  amastigotes.L. (L.) chagasiin vitroLdccys2Ldccys2Ldccys2L. (L.) chagasiDiscussion In this study, we have shown that amastigote cysteine protease, Ldccys2 necessary for macrophage infection and for survival of  L. (L.) chagasi  amastigotes inside the macrophage cells using two different approaches, gene disruption and antisense mRNA expression.  Leishmania (L.) chagasi  cysteine protease,  Ldccys2  is a single copy gene [ 18 ] and is expressed only in amastigote stage of the parasite.  L. (L.) donovani  also possesses an identical gene. Expression studies performed demonstrates that  Ldccys2  is expressed only in amastigotes of  L. (L.) chagasi  and  L. (L.) donovani . A similar single copy gene in  L. (L.) mexicana  ( Lmcpa)  has a significant sequence identity (80%) to  Ldccys2 .  Lmcpa  gene of  L. (L.) mexicana  which is the closest homolog of  Ldccys2  is differentially expressed in amastigotes [ 23 ], whereas the  Ldccys2  gene of  L. (L.) chagasi  is expressed specifically in the amastigote stage. In addition, the major amastigote specific cysteine proteinase gene of  L. (L.) mexicana ,  cpb2.8  is a multicopy gene, located within the  lmcpb  cluster [ 24 ] and there is no significant sequence identity (34%) between  Ldccys2  and  lmcpb2.8 . Therefore, gene organization and expression of amastigote specific cysteine proteases in  L. (L.) mexicana  and  L. (L.) donovani  complex are not identical. Interestingly,  Llacys1 , an amastigote specific cysteine protease from  L. (L.) amazonensis  was identified recently, which has high homology (88%) with  Ldccys2  and like  Ldccys2 , is only expressed in amastigote stages [ 25 ]. In order to confirm the protease activity of amastigote cysteine protease, full-length  Ldccys2  was expressed as JHE-fusion protein in an insect cell expression system. The recombinant protein hydrolysed gelatin in activity gels. Recently, functions of many genes have been established in  Leishmania  by following gene disruption approach [ 26 , 27 ]. To study the biological role of Ldccys2 cysteine protease, we have followed targeted gene disruption using  hyg  gene as the selectable marker. Genomic Southern analyses of the hygromycin B resistant clones using specific probes confirmed the replacement of 500 bp region within the ORF of the  Ldccys2  by  hyg/dhfr-ts . Northern and Western blot analyses  Ldccys2KO  amastigotes showed a clear decrease in the endogenous levels of Ldccys2 compared to that of wild type  L. (L.) chagasi  amastigotes (Figure  4 ), demonstrating the disruption of one of the  Ldccys2  alleles. Despite our repeated effort using different selectable marker genes, we failed to create a null mutant of  Ldccys2  in  L. (L.) chagasi . Since gene amplification and increase in ploidy level has been reported for  Leishmania  species [ 28 ], we analyzed for changes in ploidy level and or gene amplification and found no such events occurring in these heterozygous knockout mutants. This suggests that  Ldccys2  is an important gene necessary for the initiation of macrophage infection and survival of amastigotes within the macrophages. Therefore we used the  Ldccys2  heterozygous knockout parasites for further functional studies. The U937 macrophage cells infected with  Ldccys2  heterozygous knockout mutants showed a significant decrease in the initial infection and survival within the macrophage cells, compared to wild type (Figure  4 ). This trend remained consistent from 12 hours up to 48 hours after infection. The decrease in initiation of infection and subsequent survival of amastigotes may explain the role of Ldccys2 in amastigote metabolism and its survival inside the macrophages [ 4 ]. Consistent with our results, null mutants of the  lmcpb  cluster in  L. (L.) mexicana  exhibited reduction in macrophage infectivity and survival [ 23 ]. However, lack of  Ldccys2  null mutant prevented us from a detailed study on the role of Ldccys2 in survival and pathogenesis, using animal models. The lack of  Ldccys2  null mutants prompted us to use an alternative approach of antisense mRNA inhibtion in order to confirm the results obtained from  Ldccys2  heterozygous knockout studies. Using antisense RNA technique it has become possible to investigate the contribution of cysteine proteases to virulence mechanism of  E. histolytica  [ 19 , 20 ] and A2- protein in the survival of  L. (L.) donovani  amastigotes in macrophages [ 21 ]. Furthermore, episomal expression of sense and anti sense mRNA of  L. (L.) amazonensis  gp63using  Leishmania - specific P6.5 vector provided evidence for a role both in binding of macrophages and the intracellular survival and replication [ 22 ]. Not surprisingly,  L. (L.) chagasi  amastigotes expressing  Ldccys2  antisense mRNA has clearly down regulated the endogenous  Ldccys2  mRNA levels (Figure  5 ). However, a small amount of  Ldccys2  mRNA is still present in antisense transfectants, which may be due to the incomplete suppression, which is inherent to this approach. Infection of U937 macrophage cells with transfectants expressing  Ldccys2  antisense mRNA showed a significant decrease in amastigote survival inside the macrophage cells, as compared to amastigotes expressing either sense  Ldccys2  mRNA or the control plasmid (Figure  5 ). This is in agreement with the results obtained from  Ldccys2  heterozygous knockout mutants, reaffirming the role of Ldccys2 in initiation of intracellular infection, and subsequent survival and multiplication of amastigotes. However, the actual effect of antisense mRNA expression may be greater, since reversal of antisense effects is expected from the release of the selective pressure necessary to avoid drug toxicity to macrophages [ 21 ]. This is especially true in the assay for intracellular survival and replication, since it requires prolonged period of incubation. The transient nature of the antisense suppression in the absence of selection prevented us from taking up animal studies that also require prolonged infection with these transfectants. The out come of this study shows that  Ldccys2  is expressed only in amastigote stage of the parasite. Both the heterozygous knockout mutants and antisense mRNA expression shows that Ldccys2 is important for infection of macrophages and survival of  L. (L.) chagasi  amastigotes inside the macrophage cells. Based on the data obtained in this study, there is a clear justification for targeting the  Ldccys2  gene in  L (L.). chagasi  and carrying out further studies in animal models. Currently, we are working towards generating null mutants of  Ldccys2  in  L. (L.) chagasi  and to further understand the role of this protein in the pathogenesis of  L. (L.) chagasi  parasites.
Discussion
In this study, we have shown that amastigote cysteine protease, Ldccys2 necessary for macrophage infection and for survival of  L. (L.) chagasi  amastigotes inside the macrophage cells using two different approaches, gene disruption and antisense mRNA expression.  Leishmania (L.) chagasi  cysteine protease,  Ldccys2  is a single copy gene [ 18 ] and is expressed only in amastigote stage of the parasite.  L. (L.) donovani  also possesses an identical gene. Expression studies performed demonstrates that  Ldccys2  is expressed only in amastigotes of  L. (L.) chagasi  and  L. (L.) donovani . A similar single copy gene in  L. (L.) mexicana  ( Lmcpa)  has a significant sequence identity (80%) to  Ldccys2 .  Lmcpa  gene of  L. (L.) mexicana  which is the closest homolog of  Ldccys2  is differentially expressed in amastigotes [ 23 ], whereas the  Ldccys2  gene of  L. (L.) chagasi  is expressed specifically in the amastigote stage. In addition, the major amastigote specific cysteine proteinase gene of  L. (L.) mexicana ,  cpb2.8  is a multicopy gene, located within the  lmcpb  cluster [ 24 ] and there is no significant sequence identity (34%) between  Ldccys2  and  lmcpb2.8 . Therefore, gene organization and expression of amastigote specific cysteine proteases in  L. (L.) mexicana  and  L. (L.) donovani  complex are not identical. Interestingly,  Llacys1 , an amastigote specific cysteine protease from  L. (L.) amazonensis  was identified recently, which has high homology (88%) with  Ldccys2  and like  Ldccys2 , is only expressed in amastigote stages [ 25 ]. In order to confirm the protease activity of amastigote cysteine protease, full-length  Ldccys2  was expressed as JHE-fusion protein in an insect cell expression system. The recombinant protein hydrolysed gelatin in activity gels.L. (L.) chagasiLeishmania (L.) chagasiLdccys2L. (L.) donovaniLdccys2L. (L.) chagasiL. (L.) donovaniL. (L.) mexicanaLmcpa)Ldccys2LmcpaL. (L.) mexicanaLdccys2Ldccys2L. (L.) chagasiL. (L.) mexicanacpb2.8lmcpbLdccys2lmcpb2.8L. (L.) mexicanaL. (L.) donovaniLlacys1L. (L.) amazonensisLdccys2Ldccys2Ldccys2Recently, functions of many genes have been established in  Leishmania  by following gene disruption approach [ 26 , 27 ]. To study the biological role of Ldccys2 cysteine protease, we have followed targeted gene disruption using  hyg  gene as the selectable marker. Genomic Southern analyses of the hygromycin B resistant clones using specific probes confirmed the replacement of 500 bp region within the ORF of the  Ldccys2  by  hyg/dhfr-ts . Northern and Western blot analyses  Ldccys2KO  amastigotes showed a clear decrease in the endogenous levels of Ldccys2 compared to that of wild type  L. (L.) chagasi  amastigotes (Figure  4 ), demonstrating the disruption of one of the  Ldccys2  alleles. Despite our repeated effort using different selectable marker genes, we failed to create a null mutant of  Ldccys2  in  L. (L.) chagasi . Since gene amplification and increase in ploidy level has been reported for  Leishmania  species [ 28 ], we analyzed for changes in ploidy level and or gene amplification and found no such events occurring in these heterozygous knockout mutants. This suggests that  Ldccys2  is an important gene necessary for the initiation of macrophage infection and survival of amastigotes within the macrophages. Therefore we used the  Ldccys2  heterozygous knockout parasites for further functional studies. The U937 macrophage cells infected with  Ldccys2  heterozygous knockout mutants showed a significant decrease in the initial infection and survival within the macrophage cells, compared to wild type (Figure  4 ). This trend remained consistent from 12 hours up to 48 hours after infection. The decrease in initiation of infection and subsequent survival of amastigotes may explain the role of Ldccys2 in amastigote metabolism and its survival inside the macrophages [ 4 ]. Consistent with our results, null mutants of the  lmcpb  cluster in  L. (L.) mexicana  exhibited reduction in macrophage infectivity and survival [ 23 ]. However, lack of  Ldccys2  null mutant prevented us from a detailed study on the role of Ldccys2 in survival and pathogenesis, using animal models.LeishmaniahygLdccys2hyg/dhfr-tsLdccys2KOL. (L.) chagasiLdccys2Ldccys2L. (L.) chagasiLeishmaniaLdccys2Ldccys2Ldccys2lmcpbL. (L.) mexicanaLdccys2The lack of  Ldccys2  null mutants prompted us to use an alternative approach of antisense mRNA inhibtion in order to confirm the results obtained from  Ldccys2  heterozygous knockout studies. Using antisense RNA technique it has become possible to investigate the contribution of cysteine proteases to virulence mechanism of  E. histolytica  [ 19 , 20 ] and A2- protein in the survival of  L. (L.) donovani  amastigotes in macrophages [ 21 ]. Furthermore, episomal expression of sense and anti sense mRNA of  L. (L.) amazonensis  gp63using  Leishmania - specific P6.5 vector provided evidence for a role both in binding of macrophages and the intracellular survival and replication [ 22 ]. Not surprisingly,  L. (L.) chagasi  amastigotes expressing  Ldccys2  antisense mRNA has clearly down regulated the endogenous  Ldccys2  mRNA levels (Figure  5 ). However, a small amount of  Ldccys2  mRNA is still present in antisense transfectants, which may be due to the incomplete suppression, which is inherent to this approach. Infection of U937 macrophage cells with transfectants expressing  Ldccys2  antisense mRNA showed a significant decrease in amastigote survival inside the macrophage cells, as compared to amastigotes expressing either sense  Ldccys2  mRNA or the control plasmid (Figure  5 ). This is in agreement with the results obtained from  Ldccys2  heterozygous knockout mutants, reaffirming the role of Ldccys2 in initiation of intracellular infection, and subsequent survival and multiplication of amastigotes. However, the actual effect of antisense mRNA expression may be greater, since reversal of antisense effects is expected from the release of the selective pressure necessary to avoid drug toxicity to macrophages [ 21 ]. This is especially true in the assay for intracellular survival and replication, since it requires prolonged period of incubation. The transient nature of the antisense suppression in the absence of selection prevented us from taking up animal studies that also require prolonged infection with these transfectants.Ldccys2Ldccys2E. histolyticaL. (L.) donovaniL. (L.) amazonensisLeishmaniaL. (L.) chagasiLdccys2Ldccys2Ldccys2Ldccys2Ldccys2Ldccys2The out come of this study shows that  Ldccys2  is expressed only in amastigote stage of the parasite. Both the heterozygous knockout mutants and antisense mRNA expression shows that Ldccys2 is important for infection of macrophages and survival of  L. (L.) chagasi  amastigotes inside the macrophage cells. Based on the data obtained in this study, there is a clear justification for targeting the  Ldccys2  gene in  L (L.). chagasi  and carrying out further studies in animal models. Currently, we are working towards generating null mutants of  Ldccys2  in  L. (L.) chagasi  and to further understand the role of this protein in the pathogenesis of  L. (L.) chagasi  parasites.Ldccys2L. (L.) chagasiLdccys2L (L.). chagasiLdccys2L. (L.) chagasiL. (L.) chagasiMethods Parasite culture L. (L.) chagasi  strain MHOM/BR/74/PP75 promastigotes were grown in HOMEM, pH 7.4 (Gibco BRL) supplemented with 10% (v/v) heat inactivated calf serum, at 26°C. Promastigote cultures were seeded at 1 × 10 6 parasites/ml and harvested in logarithmic or stationary growth phase, as defined by cell concentration.  L. (L.) chagasi  promastigotes were transformed to axenic amastigotes using the standardized protocol as described earlier [ 29 ]. All the transfectants were periodically converted to amastigotes by infecting U937 macrophage cells in order to ensure the property of virulence. RNA isolation and northern hybridization Total RNA was isolated using trizol (GibcoBRL) as recommended by the manufacturer. Promastigote RNA was obtained from logarithmic and stationary growth phase parasites of  L. (L.) chagasi . To isolate amastigote RNA, human macrophage cell line (U937, ATCC) was infected with promastigote parasites, which were subsequently converted into intracellular amastigotes [ 29 ]. RNA (10 μg/lane) was separated on 1.2% (w/v) formaldehyde agarose gels and transferred onto Hybond N+ membrane (Amersham Pharmacia). Standard procedures were followed to perform Southern and Northern blot hybridizations. Using Quick Prime Labeling Kit (Amersham Pharmacia Biotech), probes representing specific region of  Ldccys2  cDNA clone was labeled with [α 32 P] dCTP. The sense and antisense probes were generated by 5'end-labeling the specific sense and antisense oligonuecleotide primers with γ- 32 P ATP, using T4 polynucleotide kinase according to the standard protocol. Polyclonal antibody production against Ldccys2 Direct DNA immunization [ 30 ] was used to generate antibody against Ldccys2. Briefly, the pro-mature region of the gene was PCR amplified from the  Ldccys2  cDNA clone using specific 5' (5'CAGACAGGATCCG CGGCCGCCATGGACGACTTCATTGCC 3') and 3' primers (5' GGCCGCGGATCCGCG GCCGCCTATGAGGTGTTGGAGTCGTC 3') and the PCR product was sub-cloned into  BamH I site of pcDNA 3.1 (Invitrogen). After confirmation by sequencing the plasmid constructs were amplified in  E. coli  and DNA was isolated using endotoxin free maxi prep kit (Qiagen). 50 μg of plasmid DNA in 100 μl of saline was injected into the hind limb quadriceps muscles of BALB/c mice. All the mice were boosted after 14 days and tested for antibody response seven days after the boost. Mice with positive response were boosted one more time and after a week sera was collected and stored at -20°C for further use. Western blot analysis Denaturing gel electrophoresis was carried out according to the standard protocols. Following SDS-PAGE, proteins were transferred onto to Hybond P membrane (Amersham Pharmacia Biotech) using semi-dry transblot apparatus (BioRad). Membranes were blocked for 2 hours with 5% (w/v) skim milk solution and probed overnight at 4°C with 1:200 dilution of anti-Ldccys2 antiserum. Membranes were washed three times with phosphate buffered saline containing 0.1% (v/v) Tween and incubated with 1:5000 dilution of anti-mouse horseradish peroxidase linked secondary antibody (Molecular Probes). Enhanced chemiluminescent kit (Amersham Pharmacia Biotech) was used for detection. Expression of cysteine protease genes In order to express  Ldccys2  cysteine protease gene, the coding region was amplified by PCR. The digested PCR product was cloned in-frame into unique NotI site of pIE1/153A.jhe (6hep) vector [ 31 ] and confirmed by sequencing. The fusion of the Cysteine proteases to the c-terminus of Juvenile hormone esterase (JHE) directs the secretion into the culture supernatant of transfected insect cells. Culture of insect cells and procurement of samples containing recombinant protein from transfected insect cells are described earlier [ 32 ]. Briefly, High Five insect cells (Invitrogen) were seeded into6-well culture plates (35 mm diameter) at a density of 5 × 10 5  cells/ml (2 ml/well) and transfected for 5 h with 0.5 ml of transfection solution containing 30 μg/ml lipofectin (Life Technologies) and 6 μg/ml total plasmid DNA in basal IPL-41 medium. Then the transfection solution was removed, the cells were rinsed with basal media and 2 ml IPL-41 + 10% FBS was added and incubated for sixty hours. The supernatant was then harvested and stored at -20°C for analysis. Enzyme activity was analyzed on gelatin SDS-PAGE containing 0.2% gelatin and 8% (w/v) acrylamide as described previously [ 17 ]. For inhibition studies, gels were pre-incubated with 20 μM E-64 (Sigma), a Cysteine protease specific inhibitor. Gels were stained with Coomassie Brilliant Blue for half hour and destained with 30% Methanol for 2 hours, to visualize the bands. Bands were photographed using Diamed apparatus (Bio-Rad). Ldccys2 gene disruption by homologous recombination Gene replacement vector,  pXLdccys2-HygKO  was designed to replace 500 bp mature region of  Ldccys2  coding region and constructed as outlined below (Figure  2b ). The sense primer  Ldccys2 Hind III (5' GCTCTCAAGCTTGCTCACGCATCCGCCGC-3') and the antisense primer  Ldccys2  Xho I (5'GAAGGCCTCGAGCGAGCCGCACATTCCCTG-3') were used to amplify a 500 bp region from  Ldccys2  cDNA and cloned between  Hind III –  Xho I sites in pX63-Hyg (kindly provided by Dr. S. M. Beverley) for the 5' end homologous recombination. A 450 bp 3' end homologous recombination fragment was amplified using the sense primer  Ldccys2 Sma I (5'CCGGAGCCCGGGCCCACGGCGCTTGTGCAG-3') and antisense primer  Ldccys2 Bgl II (5' ACTGTCAGATCTGCTGTGCGCCAGATCGCG-3') and cloned between  Sma I and  Bgl II sites of  pX63-Hyg . The plasmid construct  pXLdccys2-HygKO  was confirmed by sequencing and digested with  Hind III and  Bgl II. The resulting 3.8 kb linear fragment containing hygromycin phospotransferase/dihydrofolate reductase-thymidylate synthase ( hyg/dhfr-ts ) gene sequence and the homologous recombination fragment was purified and used for transfection. Episomal expression of Ldccys2 specific sense and antisense mRNA The  Ldccys2  cDNA clone was isolated from an amastigote specific cDNA library of  L. (L.) chagasi .  Ldccys2  was earlier reported as promastigote specific cDNA clone [ 17 ]. However, it was further verified to be amastigote specific gene (18). The coding region (1 kb) of  Ldccys2  was inserted at  BamH I site of  Leishmania  specific vector P6.5 [ 22 ], a kind gift from Dr. K.P.Chang. Cloning was done in both sense and antisense orientations with reference to that of N-acetylglucosamine 1-phosphate transferase  (nagt) , which was used as the selective marker for tunicamycin resistance. Plasmid constructs were confirmed by sequencing. P6.5 plasmid and the sense and antisense plasmid constructs were amplified in  E. coli  and isolated with a DNA maxi prep kit (Qiagen) and the purified DNA samples were used for transfection. DNA transfection All the transfections were carried out using  L. (L.) chagasi  promastigotes. Briefly, 4 × 10 7  log phase promastigotes were pelleted, washed twice with phosphate buffered saline and resuspended in 0.4 ml of electroporation buffer in a 0.2 cm cuvette. Twenty-five microgram of P6.5 plasmid DNA constructs was used for episomal expression, whereas for homologous recombination 5 μg of linearized DNA fragment was transfected. DNA was electroporated using BioRad Gene Pulser Unit at 0.45 kV, capacitance of 25 μF and resistance of 250 ohms. Following electroporation, cells were resuspended in drug-free media for 24 hours. Parasites with P6.5 plasmid DNA constructs were selected for tunicamycin resistance at 5, 10 and 20 μg/ml tunicamycin (Sigma). Parasites transfected with DNA fragment for homologous recombination were selected for hygromycin B (GibcoBRL) resistance at 100 μg/ml. In vitro assay for intra macrophage survival The U937 macrophage cells were infected with  L. (L.) chagasi  stationary promastigotes at a host to parasite ratio of 1:10 [ 33 ]. Briefly, U937 suspension cells (10  6  cells/ml) in 10 ml RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mM L-glutamine and 50 μg gentamycin (Gibco BRL) were allowed to grow at 37°C for 3 days in 10% (v/v) CO 2 . The cells were then induced to adhere with 7.5 ng/ml of phorbol myristate acetate (PMA) and allowed to recover for 72 hours. The macrophage cells were then infected with stationary phase promastigotes and allowed to infect for 6 hours. The unbound parasites were washed away with RPMI medium and the infected macrophage cells were fed with fresh RPMI medium and left for 4 days. At every 12 hours interval up to 72 hours, cells were scraped and Diff-Quick stained slides were prepared for microscopic counting,
Methods
Parasite culture L. (L.) chagasi  strain MHOM/BR/74/PP75 promastigotes were grown in HOMEM, pH 7.4 (Gibco BRL) supplemented with 10% (v/v) heat inactivated calf serum, at 26°C. Promastigote cultures were seeded at 1 × 10 6 parasites/ml and harvested in logarithmic or stationary growth phase, as defined by cell concentration.  L. (L.) chagasi  promastigotes were transformed to axenic amastigotes using the standardized protocol as described earlier [ 29 ]. All the transfectants were periodically converted to amastigotes by infecting U937 macrophage cells in order to ensure the property of virulence.
Parasite culture
L. (L.) chagasi  strain MHOM/BR/74/PP75 promastigotes were grown in HOMEM, pH 7.4 (Gibco BRL) supplemented with 10% (v/v) heat inactivated calf serum, at 26°C. Promastigote cultures were seeded at 1 × 10 6 parasites/ml and harvested in logarithmic or stationary growth phase, as defined by cell concentration.  L. (L.) chagasi  promastigotes were transformed to axenic amastigotes using the standardized protocol as described earlier [ 29 ]. All the transfectants were periodically converted to amastigotes by infecting U937 macrophage cells in order to ensure the property of virulence.L. (L.) chagasiL. (L.) chagasiRNA isolation and northern hybridization Total RNA was isolated using trizol (GibcoBRL) as recommended by the manufacturer. Promastigote RNA was obtained from logarithmic and stationary growth phase parasites of  L. (L.) chagasi . To isolate amastigote RNA, human macrophage cell line (U937, ATCC) was infected with promastigote parasites, which were subsequently converted into intracellular amastigotes [ 29 ]. RNA (10 μg/lane) was separated on 1.2% (w/v) formaldehyde agarose gels and transferred onto Hybond N+ membrane (Amersham Pharmacia). Standard procedures were followed to perform Southern and Northern blot hybridizations. Using Quick Prime Labeling Kit (Amersham Pharmacia Biotech), probes representing specific region of  Ldccys2  cDNA clone was labeled with [α 32 P] dCTP. The sense and antisense probes were generated by 5'end-labeling the specific sense and antisense oligonuecleotide primers with γ- 32 P ATP, using T4 polynucleotide kinase according to the standard protocol.
RNA isolation and northern hybridization
Total RNA was isolated using trizol (GibcoBRL) as recommended by the manufacturer. Promastigote RNA was obtained from logarithmic and stationary growth phase parasites of  L. (L.) chagasi . To isolate amastigote RNA, human macrophage cell line (U937, ATCC) was infected with promastigote parasites, which were subsequently converted into intracellular amastigotes [ 29 ]. RNA (10 μg/lane) was separated on 1.2% (w/v) formaldehyde agarose gels and transferred onto Hybond N+ membrane (Amersham Pharmacia). Standard procedures were followed to perform Southern and Northern blot hybridizations. Using Quick Prime Labeling Kit (Amersham Pharmacia Biotech), probes representing specific region of  Ldccys2  cDNA clone was labeled with [α 32 P] dCTP. The sense and antisense probes were generated by 5'end-labeling the specific sense and antisense oligonuecleotide primers with γ- 32 P ATP, using T4 polynucleotide kinase according to the standard protocol.L. (L.) chagasiLdccys2Polyclonal antibody production against Ldccys2 Direct DNA immunization [ 30 ] was used to generate antibody against Ldccys2. Briefly, the pro-mature region of the gene was PCR amplified from the  Ldccys2  cDNA clone using specific 5' (5'CAGACAGGATCCG CGGCCGCCATGGACGACTTCATTGCC 3') and 3' primers (5' GGCCGCGGATCCGCG GCCGCCTATGAGGTGTTGGAGTCGTC 3') and the PCR product was sub-cloned into  BamH I site of pcDNA 3.1 (Invitrogen). After confirmation by sequencing the plasmid constructs were amplified in  E. coli  and DNA was isolated using endotoxin free maxi prep kit (Qiagen). 50 μg of plasmid DNA in 100 μl of saline was injected into the hind limb quadriceps muscles of BALB/c mice. All the mice were boosted after 14 days and tested for antibody response seven days after the boost. Mice with positive response were boosted one more time and after a week sera was collected and stored at -20°C for further use.
Polyclonal antibody production against Ldccys2
Direct DNA immunization [ 30 ] was used to generate antibody against Ldccys2. Briefly, the pro-mature region of the gene was PCR amplified from the  Ldccys2  cDNA clone using specific 5' (5'CAGACAGGATCCG CGGCCGCCATGGACGACTTCATTGCC 3') and 3' primers (5' GGCCGCGGATCCGCG GCCGCCTATGAGGTGTTGGAGTCGTC 3') and the PCR product was sub-cloned into  BamH I site of pcDNA 3.1 (Invitrogen). After confirmation by sequencing the plasmid constructs were amplified in  E. coli  and DNA was isolated using endotoxin free maxi prep kit (Qiagen). 50 μg of plasmid DNA in 100 μl of saline was injected into the hind limb quadriceps muscles of BALB/c mice. All the mice were boosted after 14 days and tested for antibody response seven days after the boost. Mice with positive response were boosted one more time and after a week sera was collected and stored at -20°C for further use.Ldccys2BamHE. coliWestern blot analysis Denaturing gel electrophoresis was carried out according to the standard protocols. Following SDS-PAGE, proteins were transferred onto to Hybond P membrane (Amersham Pharmacia Biotech) using semi-dry transblot apparatus (BioRad). Membranes were blocked for 2 hours with 5% (w/v) skim milk solution and probed overnight at 4°C with 1:200 dilution of anti-Ldccys2 antiserum. Membranes were washed three times with phosphate buffered saline containing 0.1% (v/v) Tween and incubated with 1:5000 dilution of anti-mouse horseradish peroxidase linked secondary antibody (Molecular Probes). Enhanced chemiluminescent kit (Amersham Pharmacia Biotech) was used for detection.
Western blot analysis
Denaturing gel electrophoresis was carried out according to the standard protocols. Following SDS-PAGE, proteins were transferred onto to Hybond P membrane (Amersham Pharmacia Biotech) using semi-dry transblot apparatus (BioRad). Membranes were blocked for 2 hours with 5% (w/v) skim milk solution and probed overnight at 4°C with 1:200 dilution of anti-Ldccys2 antiserum. Membranes were washed three times with phosphate buffered saline containing 0.1% (v/v) Tween and incubated with 1:5000 dilution of anti-mouse horseradish peroxidase linked secondary antibody (Molecular Probes). Enhanced chemiluminescent kit (Amersham Pharmacia Biotech) was used for detection.Expression of cysteine protease genes In order to express  Ldccys2  cysteine protease gene, the coding region was amplified by PCR. The digested PCR product was cloned in-frame into unique NotI site of pIE1/153A.jhe (6hep) vector [ 31 ] and confirmed by sequencing. The fusion of the Cysteine proteases to the c-terminus of Juvenile hormone esterase (JHE) directs the secretion into the culture supernatant of transfected insect cells. Culture of insect cells and procurement of samples containing recombinant protein from transfected insect cells are described earlier [ 32 ]. Briefly, High Five insect cells (Invitrogen) were seeded into6-well culture plates (35 mm diameter) at a density of 5 × 10 5  cells/ml (2 ml/well) and transfected for 5 h with 0.5 ml of transfection solution containing 30 μg/ml lipofectin (Life Technologies) and 6 μg/ml total plasmid DNA in basal IPL-41 medium. Then the transfection solution was removed, the cells were rinsed with basal media and 2 ml IPL-41 + 10% FBS was added and incubated for sixty hours. The supernatant was then harvested and stored at -20°C for analysis. Enzyme activity was analyzed on gelatin SDS-PAGE containing 0.2% gelatin and 8% (w/v) acrylamide as described previously [ 17 ]. For inhibition studies, gels were pre-incubated with 20 μM E-64 (Sigma), a Cysteine protease specific inhibitor. Gels were stained with Coomassie Brilliant Blue for half hour and destained with 30% Methanol for 2 hours, to visualize the bands. Bands were photographed using Diamed apparatus (Bio-Rad).
Expression of cysteine protease genes
In order to express  Ldccys2  cysteine protease gene, the coding region was amplified by PCR. The digested PCR product was cloned in-frame into unique NotI site of pIE1/153A.jhe (6hep) vector [ 31 ] and confirmed by sequencing. The fusion of the Cysteine proteases to the c-terminus of Juvenile hormone esterase (JHE) directs the secretion into the culture supernatant of transfected insect cells. Culture of insect cells and procurement of samples containing recombinant protein from transfected insect cells are described earlier [ 32 ]. Briefly, High Five insect cells (Invitrogen) were seeded into6-well culture plates (35 mm diameter) at a density of 5 × 10 5  cells/ml (2 ml/well) and transfected for 5 h with 0.5 ml of transfection solution containing 30 μg/ml lipofectin (Life Technologies) and 6 μg/ml total plasmid DNA in basal IPL-41 medium. Then the transfection solution was removed, the cells were rinsed with basal media and 2 ml IPL-41 + 10% FBS was added and incubated for sixty hours. The supernatant was then harvested and stored at -20°C for analysis. Enzyme activity was analyzed on gelatin SDS-PAGE containing 0.2% gelatin and 8% (w/v) acrylamide as described previously [ 17 ]. For inhibition studies, gels were pre-incubated with 20 μM E-64 (Sigma), a Cysteine protease specific inhibitor. Gels were stained with Coomassie Brilliant Blue for half hour and destained with 30% Methanol for 2 hours, to visualize the bands. Bands were photographed using Diamed apparatus (Bio-Rad).Ldccys2Ldccys2 gene disruption by homologous recombination Gene replacement vector,  pXLdccys2-HygKO  was designed to replace 500 bp mature region of  Ldccys2  coding region and constructed as outlined below (Figure  2b ). The sense primer  Ldccys2 Hind III (5' GCTCTCAAGCTTGCTCACGCATCCGCCGC-3') and the antisense primer  Ldccys2  Xho I (5'GAAGGCCTCGAGCGAGCCGCACATTCCCTG-3') were used to amplify a 500 bp region from  Ldccys2  cDNA and cloned between  Hind III –  Xho I sites in pX63-Hyg (kindly provided by Dr. S. M. Beverley) for the 5' end homologous recombination. A 450 bp 3' end homologous recombination fragment was amplified using the sense primer  Ldccys2 Sma I (5'CCGGAGCCCGGGCCCACGGCGCTTGTGCAG-3') and antisense primer  Ldccys2 Bgl II (5' ACTGTCAGATCTGCTGTGCGCCAGATCGCG-3') and cloned between  Sma I and  Bgl II sites of  pX63-Hyg . The plasmid construct  pXLdccys2-HygKO  was confirmed by sequencing and digested with  Hind III and  Bgl II. The resulting 3.8 kb linear fragment containing hygromycin phospotransferase/dihydrofolate reductase-thymidylate synthase ( hyg/dhfr-ts ) gene sequence and the homologous recombination fragment was purified and used for transfection.
Ldccys2 gene disruption by homologous recombination
Gene replacement vector,  pXLdccys2-HygKO  was designed to replace 500 bp mature region of  Ldccys2  coding region and constructed as outlined below (Figure  2b ). The sense primer  Ldccys2 Hind III (5' GCTCTCAAGCTTGCTCACGCATCCGCCGC-3') and the antisense primer  Ldccys2  Xho I (5'GAAGGCCTCGAGCGAGCCGCACATTCCCTG-3') were used to amplify a 500 bp region from  Ldccys2  cDNA and cloned between  Hind III –  Xho I sites in pX63-Hyg (kindly provided by Dr. S. M. Beverley) for the 5' end homologous recombination. A 450 bp 3' end homologous recombination fragment was amplified using the sense primer  Ldccys2 Sma I (5'CCGGAGCCCGGGCCCACGGCGCTTGTGCAG-3') and antisense primer  Ldccys2 Bgl II (5' ACTGTCAGATCTGCTGTGCGCCAGATCGCG-3') and cloned between  Sma I and  Bgl II sites of  pX63-Hyg . The plasmid construct  pXLdccys2-HygKO  was confirmed by sequencing and digested with  Hind III and  Bgl II. The resulting 3.8 kb linear fragment containing hygromycin phospotransferase/dihydrofolate reductase-thymidylate synthase ( hyg/dhfr-ts ) gene sequence and the homologous recombination fragment was purified and used for transfection.pXLdccys2-HygKOLdccys2Ldccys2 HindLdccys2Ldccys2HindXhoLdccys2 SmaLdccys2 BglSmaBglpX63-HygpXLdccys2-HygKOHindBglhyg/dhfr-tsEpisomal expression of Ldccys2 specific sense and antisense mRNA The  Ldccys2  cDNA clone was isolated from an amastigote specific cDNA library of  L. (L.) chagasi .  Ldccys2  was earlier reported as promastigote specific cDNA clone [ 17 ]. However, it was further verified to be amastigote specific gene (18). The coding region (1 kb) of  Ldccys2  was inserted at  BamH I site of  Leishmania  specific vector P6.5 [ 22 ], a kind gift from Dr. K.P.Chang. Cloning was done in both sense and antisense orientations with reference to that of N-acetylglucosamine 1-phosphate transferase  (nagt) , which was used as the selective marker for tunicamycin resistance. Plasmid constructs were confirmed by sequencing. P6.5 plasmid and the sense and antisense plasmid constructs were amplified in  E. coli  and isolated with a DNA maxi prep kit (Qiagen) and the purified DNA samples were used for transfection.
Episomal expression of Ldccys2 specific sense and antisense mRNA
The  Ldccys2  cDNA clone was isolated from an amastigote specific cDNA library of  L. (L.) chagasi .  Ldccys2  was earlier reported as promastigote specific cDNA clone [ 17 ]. However, it was further verified to be amastigote specific gene (18). The coding region (1 kb) of  Ldccys2  was inserted at  BamH I site of  Leishmania  specific vector P6.5 [ 22 ], a kind gift from Dr. K.P.Chang. Cloning was done in both sense and antisense orientations with reference to that of N-acetylglucosamine 1-phosphate transferase  (nagt) , which was used as the selective marker for tunicamycin resistance. Plasmid constructs were confirmed by sequencing. P6.5 plasmid and the sense and antisense plasmid constructs were amplified in  E. coli  and isolated with a DNA maxi prep kit (Qiagen) and the purified DNA samples were used for transfection.Ldccys2L. (L.) chagasiLdccys2Ldccys2BamHLeishmania(nagt)E. coliDNA transfection All the transfections were carried out using  L. (L.) chagasi  promastigotes. Briefly, 4 × 10 7  log phase promastigotes were pelleted, washed twice with phosphate buffered saline and resuspended in 0.4 ml of electroporation buffer in a 0.2 cm cuvette. Twenty-five microgram of P6.5 plasmid DNA constructs was used for episomal expression, whereas for homologous recombination 5 μg of linearized DNA fragment was transfected. DNA was electroporated using BioRad Gene Pulser Unit at 0.45 kV, capacitance of 25 μF and resistance of 250 ohms. Following electroporation, cells were resuspended in drug-free media for 24 hours. Parasites with P6.5 plasmid DNA constructs were selected for tunicamycin resistance at 5, 10 and 20 μg/ml tunicamycin (Sigma). Parasites transfected with DNA fragment for homologous recombination were selected for hygromycin B (GibcoBRL) resistance at 100 μg/ml.
DNA transfection
All the transfections were carried out using  L. (L.) chagasi  promastigotes. Briefly, 4 × 10 7  log phase promastigotes were pelleted, washed twice with phosphate buffered saline and resuspended in 0.4 ml of electroporation buffer in a 0.2 cm cuvette. Twenty-five microgram of P6.5 plasmid DNA constructs was used for episomal expression, whereas for homologous recombination 5 μg of linearized DNA fragment was transfected. DNA was electroporated using BioRad Gene Pulser Unit at 0.45 kV, capacitance of 25 μF and resistance of 250 ohms. Following electroporation, cells were resuspended in drug-free media for 24 hours. Parasites with P6.5 plasmid DNA constructs were selected for tunicamycin resistance at 5, 10 and 20 μg/ml tunicamycin (Sigma). Parasites transfected with DNA fragment for homologous recombination were selected for hygromycin B (GibcoBRL) resistance at 100 μg/ml.L. (L.) chagasiIn vitro assay for intra macrophage survival The U937 macrophage cells were infected with  L. (L.) chagasi  stationary promastigotes at a host to parasite ratio of 1:10 [ 33 ]. Briefly, U937 suspension cells (10  6  cells/ml) in 10 ml RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mM L-glutamine and 50 μg gentamycin (Gibco BRL) were allowed to grow at 37°C for 3 days in 10% (v/v) CO 2 . The cells were then induced to adhere with 7.5 ng/ml of phorbol myristate acetate (PMA) and allowed to recover for 72 hours. The macrophage cells were then infected with stationary phase promastigotes and allowed to infect for 6 hours. The unbound parasites were washed away with RPMI medium and the infected macrophage cells were fed with fresh RPMI medium and left for 4 days. At every 12 hours interval up to 72 hours, cells were scraped and Diff-Quick stained slides were prepared for microscopic counting,
In vitro assay for intra macrophage survival
The U937 macrophage cells were infected with  L. (L.) chagasi  stationary promastigotes at a host to parasite ratio of 1:10 [ 33 ]. Briefly, U937 suspension cells (10  6  cells/ml) in 10 ml RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mM L-glutamine and 50 μg gentamycin (Gibco BRL) were allowed to grow at 37°C for 3 days in 10% (v/v) CO 2 . The cells were then induced to adhere with 7.5 ng/ml of phorbol myristate acetate (PMA) and allowed to recover for 72 hours. The macrophage cells were then infected with stationary phase promastigotes and allowed to infect for 6 hours. The unbound parasites were washed away with RPMI medium and the infected macrophage cells were fed with fresh RPMI medium and left for 4 days. At every 12 hours interval up to 72 hours, cells were scraped and Diff-Quick stained slides were prepared for microscopic counting,L. (L.) chagasiAbbreviations Hyg, hygromycin; JHE, juvenile hormone esterase; kb, kilobase; KO, knock-out; ORF, open reading frame; UTR, untranslated region
Abbreviations
Hyg, hygromycin; JHE, juvenile hormone esterase; kb, kilobase; KO, knock-out; ORF, open reading frame; UTR, untranslated regionAuthors' contributions VM carried out all the plasmid constructs for gene disruption and antisense mRNA inhibiton studies and performed the Southern and Northern blot analyses shown in Figures  1 ,  3 ,  4  & 6; wrote parts of the manuscript. ASK performed all the transfections, western blots and macrophage assays shown in Figures  1 ,  2 ,  4 ,  5 , 6 and 7; wrote parts of the manuscript. LG supervised the experiments and gave laboratory support, and critically read the manuscript. All authors read and approved the final manuscript.
Authors' contributions
VM carried out all the plasmid constructs for gene disruption and antisense mRNA inhibiton studies and performed the Southern and Northern blot analyses shown in Figures  1 ,  3 ,  4  & 6; wrote parts of the manuscript. ASK performed all the transfections, western blots and macrophage assays shown in Figures  1 ,  2 ,  4 ,  5 , 6 and 7; wrote parts of the manuscript. LG supervised the experiments and gave laboratory support, and critically read the manuscript. All authors read and approved the final manuscript.


The astounding diversity of life—different body shapes and sizes, physiologies, and behaviors—stems from the accumulation of genetic changes through the process we call evolution. But catching a glimpse into the process of evolution at the gene level is difficult, mostly because significant changes to the plant and animal species of today happened a long time ago. Nevertheless, biologists are keen to understand exactly how evolution progresses. For example, how many genes must be altered before noticeable shifts in appearance can be seen? Is evolution the result of changes in many genes with small additive effects, or of just a few mutations that exert a strong influence?To tackle these questions, Pamela Colosimo and colleagues turned to threespine stickleback fish, a longtime favorite model system of evolutionary biologists because of its relatively youthful evolutionary history. At the end of the last ice age 10,000 years ago, when glaciers all over the Northern Hemisphere began to melt, small populations of these originally marine-dwelling fish became trapped in newly formed lakes. There, isolated stickleback colonies adapted to new ecological conditions—different predators, food availability, water chemistry, and temperature—and now look distinctly different from their marine ancestors. One of the most obvious changes in appearance is in their body armor—they come in three distinct types, or “morphs.” Marine sticklebacks are covered from head to tail with rows of tightly packed boney plates (a complete morph), while those found in freshwater lakes have fewer body plates (a partial morph) or almost none at all (a low morph). Colosimo and colleagues found that a single region of the genome is largely responsible for the dramatic changes in plate morph, and that this is true for two widely separated populations of independently evolving freshwater sticklebacks.To uncover the genomic regions that affect armor, Colosimo's team crossed fully armored marine sticklebacks from Japan with deep-water, or benthic, low morph fish from Paxton Lake in British Columbia, Canada. They then “mapped” the full genome of second generation offspring using 160 known genetic markers, or loci, as guideposts for distinct regions of the genome—loci that are inherited along with differences in the overall type of plating, and individual plate number and size.The team found that one such locus explained 75% of the variation in plate morphs. Offspring that carried two alleles—versions of the gene—from their marine grandparents, genotype  AA,  were almost always fully plated. Those that inherited two copies of the allele from their benthic progenitors,  aa,  were mostly low morphs with very little plating. And  Aa  heterozygous fish (with one allele from each population) had mostly full or partial plates. Colosimo and colleagues also found three other regions in the genome that significantly affected the number and size of plates. These modifiers had an additive effect—the more benthic alleles inherited, the fewer and smaller the plates; more marine alleles caused a trend toward greater armor.AA,aa,AaBut is this genetic architecture the same for every independently evolving population of lake-bound sticklebacks in North America? Or did the geographically isolated freshwater groups loose their plates through mutations in different genes? Colosimo and colleagues mapped the genome of a population of sticklebacks from Friant, California, which is 800 miles away from Paxton Lake, and found that the same major locus seemed to be controlling plate morph there as well. Crossing a low morph from Friant with a low morph from Paxton yielded only offspring with very little armor. Further, some of the modifiers uncovered in the Paxton fish were also acting on the Friant sticklebacks. So, though these two populations of fish have been separated for 10,000 years, loss of armor in both groups probably stemmed from changes in the same genetic pathway.Without knowing the precise sequence of these genes, it is impossible to tell exactly how and when the alleles that reduce armor arose. Small numbers of individuals with genes causing less plating could have been present in ancestral populations of marine sticklebacks when they were originally locked in newly formed lakes. Alternatively, reduced armor could have arisen independently in different lakes following isolation if, for example, some genes that control armor are predisposed to mutation, or certain armor-related mutations are more advantageous than others. But however it happened, this study clearly shows that dramatic morphological evolution can result from a small number of genetic changes. Further study of this classic system should provide a detailed picture of the genes involved, and of the molecular events that underlie morphological changes in natural populations evolving in new environments.