A post-transcriptional respiratome regulon in trypanosomes.

Post-transcriptional regulons coordinate the expression of groups of genes in eukaryotic cells, yet relatively few have been characterized. Parasitic trypanosomatids are particularly good models for studies on such mechanisms because they exhibit almost exclusive polycistronic, and unregulated, transcription. Here, we identify the Trypanosoma brucei ZC3H39/40 RNA-binding proteins as regulators of the respiratome; the mitochondrial electron transport chain (complexes I-IV) and the FoF1-ATP synthase (complex V). A high-throughput RNAi screen initially implicated both ZC3H proteins in variant surface glycoprotein (VSG) gene silencing. This link was confirmed and both proteins were shown to form a cytoplasmic ZC3H39/40 complex. Transcriptome and mRNA-interactome analyses indicated that the impact on VSG silencing was indirect, while the ZC3H39/40 complex specifically bound and stabilized transcripts encoding respiratome-complexes. Quantitative proteomic analyses revealed specific positive control of >20 components from complexes I, II and V. Our findings establish a link between the mitochondrial respiratome and VSG gene silencing in bloodstream form T. brucei. They also reveal a major respiratome regulon controlled by the conserved trypanosomatid ZC3H39/40 RNA-binding proteins.

Oral infection of mice and host cell invasion by Trypanosoma cruzi strains from Mexico.

Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.

Genome organization and DNA accessibility control antigenic variation in trypanosomes.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies.

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection.

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation.

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

FACT plays a major role in histone dynamics affecting VSG expression site control in Trypanosoma brucei.

Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei. We show that the T. brucei FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing. We see an inverse correlation between transcription and condensed chromatin, whereby FACT knockdown results in ES derepression and more open chromatin around silent ES promoters. Derepressed ESs show increased sensitivity to micrococcal nuclease (MNase) digestion, and a decrease in histones at silent ES promoters but not telomeres. In contrast, FACT knockdown results in more histones at the active ES, correlated with transcription shut-down. ES promoters are derepressed in cells stalled at the G2/M cell cycle stage after knockdown of FACT, but not in G2/M cells stalled after knockdown of cyclin 6. This argues that the observed ES derepression is a direct consequence of histone chaperone activity by FACT at the G2/M cell cycle stage which could affect transcription elongation, rather than an indirect consequence of a cell cycle checkpoint. These experiments highlight the role of the FACT complex in cell cycle-specific chromatin remodelling within VSG ESs.

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity.

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.

Life and times: synthesis, trafficking, and evolution of VSG.

Evasion of the acquired immune response in African trypanosomes is principally mediated by antigenic variation, the sequential expression of distinct variant surface glycoproteins (VSGs) at extremely high density on the cell surface. Sequence diversity between VSGs facilitates escape of a subpopulation of trypanosomes from antibody-mediated killing. Significant advances have increased understanding of the mechanisms underpinning synthesis and maintenance of the VSG coat. In this review, we discuss the biosynthesis, trafficking, and turnover of VSG, emphasising those unusual mechanisms that act to maintain coat integrity and to protect against immunological attack. We also highlight new findings that suggest the presence of unique or highly divergent proteins that may offer therapeutic opportunities, as well as considering aspects of VSG biology that remain to be fully explored.

Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis.

BACKGROUND: The transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation. METHODS: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression. RESULTS: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. In intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region. CONCLUSIONS: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.

Histone H1 plays a role in heterochromatin formation and VSG expression site silencing in Trypanosoma brucei.

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6-8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Regulatory elements in the 3' untranslated region of the GP82 glycoprotein are responsible for its stage-specific expression in Trypanosoma cruzi metacyclic trypomastigotes.

Gene expression in Trypanosoma cruzi is regulated at the post-transcriptional level and cis-acting elements present in the 3' untranslated region (3'UTR) play an important role by interacting with regulatory proteins. Previous studies demonstrated that the GP82 surface glycoprotein, which is involved in host cell invasion, is up-regulated in the infective metacyclic trypomastigote form, and that GP82 mRNA half-life is longer in this form compared to the non-infective epimastigote form. Here, we demonstrate that the 3'UTR of the GP82 transcript is involved in this developmental regulation, promoting higher expression of the green fluorescent protein (GFP) reporter in metacyclic trypomastigotes than in epimastigotes. A series of stepwise deletions in the 3'UTR was created and results suggest that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.

Developmental regulation and extracellular release of a VSG expression-site-associated gene product from Trypanosoma brucei bloodstream forms.

Trypanosomes evade host immunity by exchanging variant surface glycoprotein (VSG) coats. VSG genes are transcribed from telomeric expression sites, which contain a diverse family of expression-site-associated genes (ESAGs). We have discovered that the mRNAs for one ESAG family, ESAG9, are strongly developmentally regulated, being enriched in stumpy forms, a life-cycle stage in the mammalian bloodstream that is important for the maintenance of chronic parasite infections and for tsetse transmission. ESAG9 gene sequences are highly diverse in the genome and encode proteins with weak similarity to the massively diverse MASP proteins in Trypanosoma cruzi. We demonstrate that ESAG9 proteins are modified by N-glycosylation and can be shed to the external milieu, this being dependent upon coexpression with at least one other family member. The expression profile and extracellular release of ESAG9 proteins represents a novel and unexpected aspect of the transmission biology of trypanosomes in their mammalian host. We suggest that these molecules might interact with the external environment, with possible implications for infection chronicity or parasite transmission.

Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes.

African trypanosomes evade the host immune response through antigenic variation, which is achieved by periodically expressing different variant surface glycoproteins (VSGs). VSG expression is monoallelic such that only one of approximately 15 telomeric VSG expression sites (ESs) is transcribed at a time. Epigenetic regulation is involved in VSG control but our understanding of the mechanisms involved remains incomplete. Histone deacetylases are potential drug targets for diseases caused by protozoan parasites. Here, using recombinant expression we show that the essential Trypanosoma brucei deacetylases, DAC1 (class I) and DAC3 (class II) display histone deacetylase activity. Both DAC1 and DAC3 are nuclear proteins in the bloodstream stage parasite, while only DAC3 remains concentrated in the nucleus in insect-stage cells. Consistent with developmentally regulated localization, DAC1 antagonizes SIR2rp1-dependent telomeric silencing only in the bloodstream form, indicating a conserved role in the control of silent chromatin domains. In contrast, DAC3 is specifically required for silencing at VSG ES promoters in both bloodstream and insect-stage cells. We conclude that DAC1 and DAC3 play distinct roles in subtelomeric gene silencing and that DAC3 represents the first readily druggable target linked to VSG ES control in the African trypanosome.

Chaperone requirements for biosynthesis of the trypanosome variant surface glycoprotein.

BACKGROUND: Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG) represents approximately 90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as approximately 2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation. CONCLUSIONS/SIGNIFICANCE: The retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only approximately 30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.

Trypanosoma brucei: trypanosome-specific endoplasmic reticulum proteins involved in variant surface glycoprotein expression.

In Trypanosoma brucei the GPI-anchored variant surface glycoprotein (VSG) represents approximately 90% of cell surface protein and a major proportion of endoplasmic reticulum (ER) biosynthetic output. We identified four trypanosomatid-specific genes encoding candidate ER-resident proteins; all were required for normal proliferation. For Tb11.01.2640 and Tb11.01.8120, an increase in VSG abundance was found on silencing, while the protein products localized to the ER; we designated these ERAP32 and ERAP18 for ER-associated protein of 32kDa and 18kDa. Silencing ERAP32 or ERAP18 did not alter expression levels of ISG65 or ISG75, the major surface trans-membrane domain proteins. Surface biotinylation or immunoflorescence did not identify intracellular VSG accumulation, while FACS and fluorescence microscopy indicated that the cells were not increased in size, arguing for increased VSG density on the cell surface. Therefore, ERAP32 and ERAP18 are trypanosome-specific ER-localized proteins with a major role in VSG protein export and, contrary to current paradigms, VSG is not saturated on the cell surface.

Processing and presentation of variant surface glycoprotein molecules to T cells in African trypanosomiasis.

Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.

Posttranscriptional mechanisms involved in the control of expression of the stage-specific GP82 surface glycoprotein in Trypanosoma cruzi.

Trypanosoma cruzi metacyclic trypomastigotes express the developmentally regulated GP82 glycoprotein, which is implicated in host cell invasion. Although GP82 mRNA and protein are not present and the mRNAs barely detectable in epimastigotes, nuclear run-on analysis showed that it is transcribed in both stages. This result indicates that accumulation of transcripts in metacyclic forms is not due to increased transcription of the GP82 gene. To investigate whether mRNA stability may be responsible for the differences in the steady-state levels of this mRNA, parasites were treated with actinomycin D or cycloheximide. When treated with actinomycin D, the half-lives estimated for GP82 transcripts were about 6h in metacyclic trypomastigotes and 0.5h in epimastigotes. In the presence of cycloheximide, the levels of GP82 mRNA decayed slightly after 8h in metacyclic trypomastigotes, whereas in epimastigotes the levels of this mRNA increased. This effect suggests a stabilizing mechanism acting in metacyclic trypomastigotes and a destabilizing mechanism in epimastigotes which could be mediated by an element present in the 3'-UTR of the transcripts. Consistent with this finding, northern blot analysis showed that GP82 mRNAs were mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes.

Designing sleeping sickness control.

Control of African trypanosomiasis caused by the protozoan parasite Trypanosoma brucei is an important issue in medicine, veterinary medicine, and agricultural economy. Because vaccine development is unlikely, development of safer and more effective chemotherapeutics is critical. The biosynthetic pathway of glycosylphosphatidylinositol (GPI), which acts as membrane anchors of coat proteins, variant surface glycoproteins, and transferrin receptors, is a validated target of drug development. An article in this issue reports the first chemically synthesized inhibitor of the third mannosyltransferase from the GPI pathway, stimulating further investigation toward practical and useful compounds.

Expression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus.

Trypanosoma cruzi, the parasite causing Chagas' disease, relies on triatomines for its transmission. T. cruzi metacyclic trypomastigotes express GP82 and GP90, which are developmentally regulated surface proteins that have been implicated in host cell invasion. We used quantitative RT-PCR to quantify GP90 and GP82 mRNA levels expressed by T. cruzi in the digestive tract of experimentally infected Rhodnius prolixus at different times post infection. Translation of these transcripts was assessed by immunofluorescence using specific monoclonal antibodies against GP90 and GP82. We found that although GP82 and GP90 proteins were not detected in epimastigote cells by immunofluorescence, transcripts were present at lower levels. Increased levels of GP90 and GP82 transcripts and the appearance of these proteins on the parasite surface were accompanied by morphological differentiation from epimastigotes into metacyclic forms. Our data suggest that during in vivo metacyclogenesis there is a coordinated mechanism that links stabilization of GP90 and GP82 mRNAs with their translation.

A function for a specific zinc metalloprotease of African trypanosomes.

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains approximately 30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B (-/-) trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC (-/-) cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B (-/-) PLC (-/-) trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.