Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.
The interplay among host innate immunity and resistance mechanisms in African trypanosomes has a major impact on the host range of these tsetse-fly transmitted parasites, defining their ability to cause disease in humans. A genome-scale RNAi screen identified a highly restricted set of four genes that sensitise trypanosomes to human serum: those encoding the haptoglobin-haemoglobin receptor, a predicted trans-membrane channel, a lysosomal membrane-protein and the cysteine peptidase inhibitor. An analysis of the cysteine peptidases revealed cathepsin-L as the protease regulated by the inhibitor – and with the capacity to render the parasite resistant to lysis by human serum. These findings emphasise the importance of parasite factors for the delivery and stability of host toxins. They also shed light on the control of proteolysis by parasites and potential unanticipated consequences of therapies that target the parasite proteases.
Citation: Alsford S, Currier RB, Guerra-Assunção JA, Clark TG, Horn D (2014) Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei. PLoS Pathog 10(5): e1004130. doi:10.1371/journal.ppat.1004130
Editor: Stephen M. Beverley, Washington University School of Medicine, United States of America
Received: October 16, 2013; Accepted: April 2, 2014; Published: May 15, 2014
Copyright: © 2014 Alsford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a Wellcome Trust Institutional Strategic Support Fund (LSHTM) fellowship (www.wellcome.ac.uk/) and the Medical Research Council/Department for International Development (project grant MR/K011987/1, www.mrc.ac.uk) to SA. JAG is supported by a Wellcome Trust grant (096249/Z/11/B). TGC receives support from a Medical Research Council UK programme grant (MR/J005398/1). DH is funded by a Wellcome Trust Senior Investigator Award (100320/Z/12/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The African trypanosomes are flagellated protozoan parasites comprising several species of the genus Trypanosoma, which cause devastating diseases in humans and livestock. One key feature that distinguishes members of this group is their sensitivity to innate trypanolytic factors (TLFs) found in human serum. T. b. brucei and related species cause nagana in livestock but these parasites are rapidly lysed by human TLFs , . T. b. gambiense and T. b. rhodesiense, on the other hand, although sharing >99% genome sequence identity with T. b. brucei , have evolved distinct mechanisms to escape lysis by human serum; these are the causative agents of human African trypanosomiasis (HAT), also known as sleeping sickness, in Western and Eastern Africa, respectively. T. b. gambiense is responsible for 97% of reported cases of HAT .
There are two classes of TLF found in normal human serum, TLF-1, which is a component of high density lipoprotein , , and TLF-2, which is an apolipoprotein-A1/IgM complex , ; the active lytic component in both TLFs is apolipoprotein-L1 (APOL1) . Both TLFs also contain haptoglobin-related protein, which, in the case of TLF-1, mediates binding to the T. b. brucei haptoglobin-haemoglobin receptor (HpHbR) and uptake into the cell , . Following uptake, APOL1 is inserted into endosomal and lysosomal membranes, where Bcl-2-like pore-formation is thought to be responsible for osmotic swelling and lysis , .
Human TLF resistance mechanisms of T. b. gambiense and T. b rhodesiense have now been described, and these involve reduced TLF binding/uptake, APOL1 sequestration, or reduced APOL1 toxicity, possibly due to membrane stiffening. Reduced TLF binding/uptake operates in T. b. gambiense due to reduced expression of HpHbR and/or mutations in HpHbR –. Endosomal sequestration of APOL1 operates in T. b. rhodesiense due to the expression of a serum resistance-associated protein (SRA) related to a glycosyl-phoshatidylinositol membrane-anchored variant surface glycoprotein (VSG) , . Expression of a VSG-related protein also confers TLF-resistance to T. b. gambiense , , but in this case the VSG-like T. b. gambiense-specific glycoprotein or TgsGP may protect cells from APOL1 by stiffening endosomal membranes rather than through direct interaction with, or sequestration of, APOL1 .
The lysosomal membrane protein, p67  and inhibitor of cysteine peptidase (ICP)  have also been shown to contribute to human TLF susceptibility using loss-of-function approaches in T. b. brucei. While HpHbR plays a role in TLF binding/uptake, the mechanism by which p67 contributes to human serum sensitivity in T. b. brucei remains unknown. Depletion of p67 causes lysosomal dysfunction, but does not increase lysosomal pH ; acidification has been proposed to be important for the insertion of APOL1 into membranes and the resulting lytic activity , , . The role of the individual cysteine peptidases, the targets of ICP, has not previously been investigated, although T. b. brucei and T. b. gambiense cells exposed to a cysteine peptidase inhibitor display increased accumulation of TLF-1  and APOL1 , strongly suggesting that a cysteine peptidase contributes to the destruction of APOL1. Cysteine peptidase inhibition by ICP likely similarly increases APOLI accumulation, explaining increased human serum resistance following ICP knockdown . Thus, gain-of-function, through the expression of modified VSGs, or loss of TLF-receptor function, have contributed to the emergence of human-infective African trypanosomes. However, other undiscovered resistance mechanisms are thought to operate in these parasites ; expression of TgsGP does not confer human serum resistance to T. b. brucei , and the main route of entry for TLF-2 in T. b. brucei is thought to be independent of HpHbR , .
We sought to confirm those factors known to render T. b. brucei susceptible to lysis by human serum and to screen for additional factors. A genome-scale RNA interference library screen for increased resistance to human serum identified all three known genes and only one additional gene, encoding a novel putative trans-membrane channel, with high-confidence. This library was previously shown to yield read-outs representing approximately 5-fold genome coverage, or more than 99% of the >7,000 non-redundant protein coding sequences in the T. b. brucei genome , and an approach related to the one described here was used to identify efficacy determinants for all five current anti-HAT drugs . We next explored the unexplained role of the cysteine peptidase inhibitor in this process, and show that ICP impacts human serum resistance by specifically modulating the activity of the lysosomal cysteine peptidase, cathepsin-L (CATL).
A genome-scale screen for genes controlling human serum sensitivity in T. b. brucei
Natural hosts for bloodstream form (BSF) T. b. brucei include bovids, and these parasites are typically propagated in a culture medium containing 10% bovine serum. In this culture environment, the half maximal effective growth-inhibitory concentration (EC50) of normal human serum (NHS) against cultured BSF T. b. brucei was less than 0.00025% (Figure 1A), revealing the exquisite sensitivity of these parasites to lytic factors in NHS. To identify T. b. brucei factors that contribute to the trypanolytic activity of NHS, we selected a multi-genome coverage BSF T. b. brucei RNAi library in 0.0005% NHS (see Figure 1B). Using this loss-of-function approach, knockdown of factors that normally contribute to human serum sensitivity will generate cells with increased resistance to this toxin. Under RNAi-inducing conditions, population growth was severely curtailed for six days in the presence of NHS; the human serum was added to the growth medium 24 h after inducing RNAi with tetracycline (Figure 1C). A population that displayed tetracycline-dependent tolerance of this concentration of NHS emerged thereafter (Figure 1D), and was harvested for DNA extraction and RNA interference target sequencing (RIT-seq) two days later.
(A) T. b. brucei (MITat 1.2; 2T1) have an EC50 of less than 0.00025% normal human serum (NHS) when incubated for 72 hours at 37°C. (B) Schematic showing selection of NHS-resistant parasites from the RNAi library. (C) A population resistant to NHS was selected. RNAi was induced in 1 µg/ml tetracycline (Tet) for 24 hours prior to selection initiated at day 0; red arrows indicate culture dilution and addition of fresh NHS and tetracycline at 0.0005% and 1 µg/ml, respectively. (D) The NHS resistance phenotype of the selected library was tetracycline-inducible; error bars represent standard deviation.
Using a modified RIT-seq  methodology (see Materials and Methods), we generated and mapped individual sequence reads representing the human serum-enriched RNAi target fragments, about 0.5 million reads in total. Approximately 24% of these reads incorporated a 14-bp RNAi construct signature found at the junction with each gene-specific RNAi target fragment, and this allowed us to focus on only ‘high-confidence hits’: genes identified in the screen by more than 99 reads per kilobase per CDS (Figure 2A), with more than 99 reads containing the RNAi construct signature, and at least two independent RNAi target fragments (Table 1). We previously applied similarly stringent criteria to define the key efficacy determinants of the anti-HAT drugs .
(A) Genome-wide NHS RIT-seq profile representing 7,433 non-redundant protein-coding sequences, showing those represented by more than 100 reads per kbp; three known human serum sensitivity determinants, HpHbR (Tb927.6.440), p67 (Tb927.5.1810/1830) and ICP (Tb927.8.6450), and the only other high-confidence and novel ‘hit’ (Tb927.8.5240) are highlighted in red. (B) Profiles of mapped RIT-seq reads for the four high-confidence hits identified in (A); boxes represent protein coding sequences; total reads (red) and tagged reads (blue; containing the 14-bp RNAi construct signature sequence) mapped to each region are shown in the top right corner of each panel; see Table 1 for further details.
As detailed above, three T. b. brucei genes have been shown to play a role in trypanolysis by NHS. Remarkably, we identified only four high-confidence hits in our screen. The presence of the three known genes within this set (Figure 2A), haptoglobin-haemoglobin receptor (HpHbR) , inhibitor of cysteine peptidase (ICP) , and the lysosomal membrane protein, p67 , provides excellent validation for the RNAi-screening approach. The schematic in Figure 2B shows the four high-confidence loci identified in the screen with mapped sequence reads. The novel high-confidence hit (Tb927.8.5240) encodes a ‘conserved hypothetical’ protein (Figure 2A and Table 1). Although it is likely membrane-associated, as it contains 11 putative trans-membrane domains, we have been unable to establish its sub-cellular localisation by C-terminal epitope tagging (data not shown). Specific stem-loop RNAi depletion of Tb927.8.5240 in three independent cell lines, confirmed by quantitative reverse transcriptase PCR (Figure 3A; Text S1), had no significant effect on bloodstream-form population growth over seven days (Figure 3B), but resulted in a 2.3-fold average increase in NHS EC50, demonstrating its contribution to NHS-sensitivity (Figure 3C, D).
(A) Tb927.8.5240 depletion, as demonstrated by reverse transcriptase qPCR (see also Tables S1 and S2 in Text S1), has no significant effect on parasite population growth (B); bars represent standard deviation from three independent strains. (C, D) RNAi against Tb927.8.5240 increases NHS EC50; bars represent standard error (C) or standard deviation (D); p values derived from extra sum-of-squares F-test (C) or paired t-test (D).
The additional genes highlighted in blue in Figure 2A are listed in Table 1. These failed to fulfil the stringent criteria for further analysis detailed above. The only exception being Tb927.8.6870 whose knockdown has previously been shown to lead to a significant gain of fitness ; we subsequently confirmed that loss of this protein did not influence sensitivity to NHS (data not shown).
Cathepsins fail to resist human serum trypanolytic activity in the presence of ICP
ICPs are conserved in protozoal and bacterial pathogens . The T. b. brucei and T. b. gambiense ICP genes are almost identical and are predicted to encode proteins of 13.5 kDa. They are thought to block cysteine peptidase activity by occupying the substrate-binding cleft , and to play a role in regulating parasite infectivity and VSG coat exchange during differentiation . African trypanosomes express two cathepsins, CATB and CATL , which are highly conserved between T. b. brucei and T. b. gambiense, and at least one (CATL) localises to the lysosome . We used chemical and genetic approaches to explore the potential roles of T. b. brucei CATB and CATL in resisting lysis by human serum.
Initially, we tested the dual CATB/L inhibitor, FMK024, in combination with NHS against T. b. brucei. Isobologram and EC50 analyses revealed that this inhibitor fails to synergise with NHS in cell-killing assays (Figure 4A, B). Indeed, the addition of increasing amounts of FMK024 causes little change in parasite sensitivity to NHS (Figure 4B), suggesting that the inhibitory function of endogenous ICP may be modulated as a consequence of changes in protease activity elicited by exogenous inhibitor. FMK024 applied at 10 or 20 µM (62.5 and 125-fold higher than the highest concentration used here) has been shown to cause lysosomal accumulation of TLF in T. b. gambiense  and of APOL1 in SRA-expressing T. b. brucei , respectively. It should be noted, however, that such high concentrations of FMK024 would likely lead to total inhibition of lysosomal cathepsin activity, and it is unlikely that ICP modulation would have any impact. Indeed, FMK024 treatment is lethal at these concentrations in our EC50 assays, independent of NHS exposure (see below).
(A) FMK024 fails to synergise with NHS against wild-type T. b. brucei; isobologram analysis showing 50% fractional inhibitory concentrations (FIC); dashed line indicates the expected output for no interaction. (B) Individual NHS EC50 curves generated in the presence of 5 to 160 nM FMK024; bars represent standard error.
We next used RNAi to knockdown CATB or CATL individually in T. b. brucei. Specific protein depletion was confirmed by western blot, and subsequent analyses revealed that only CATL activity appears to be particularly important for robust growth (Figure 5A, B). Previous findings suggested that CATB but not CATL was essential for growth , ; however, our results are consistent with the recent chemical and genetic validation of CATL as a more appropriate drug target , . Although we used a sub-lethal knockdown in the case of CATL, we were able to obtain substantial protein depletion compatible with continued growth  (Figure 5B). Consistent with the results obtained above using chemical inhibition of cathepsin activity, both knockdowns failed to synergise with NHS in killing T. b. brucei (Figure 5C, D). These data suggest that if a protease can resist lysis by human serum, its activity is suppressed.
(A) CATB depletion has no effect on cell growth, while (B) induction of CATL RNAi in 1 µg/ml tetracycline (Tet) results in a significant growth defect; bars represent standard deviation from four and two independent strains, respectively. CATB12MYC and anti-CATL western blots confirm depletion of the individual cathepsins; coomassie-stained gels show loading. Representative NHS EC50 analysis following (C) CATB and (D) CATL depletion in wild-type T. b. brucei; RNAi induced in 1 µg/ml and 2 ng/ml tetracycline, respectively; bars represent standard error.
CATL resists human serum trypanolytic activity in the absence of ICP
The results above show either that repression of cathepsin activity by ICP renders T. b. brucei sensitive to human serum and that cathepsin knockdown is compensated for by down-regulation of ICP activity, or that CATB and CATL play no role in resistance to lysis by human serum. To distinguish between these possibilities, we generated icp null T. b. brucei  (Figure 6A, B). As previously shown for a distinct cathepsin inhibitor , the icp null strains displayed a minor but significant increase in FMK024 EC50 (Figure 6C, D), confirming up-regulation of a cathepsin activity required for robust growth, most-likely that due to the essential CATL (see above). The icp null strains were, on average, 7.2-fold less sensitive to NHS (Figure 6E, F), validating this RNAi screening output and also consistent with a recent report .
(A) Schematic showing segment deleted from the ICP locus (highlighted with an open triangle), and positions of restriction sites (arrow heads), and ICP and downstream (DS) flanking probes. (B) Southern blots confirming ICP deletion from the 2T1 cell line; BSD, blasticidin-S-deaminase; NPT, neomycin phosphotransferase. (C, D) ICP loss has a minor but significant effect on FMK024 sensitivity (mean fold EC50 change, 1.31; five independent strains). (E) NHS EC50 analysis of a representative icp null strain; bars represent standard error. (F) Reduced NHS sensitivity requires deletion of both ICP alleles (five independent strains); loss of one ICP allele has no significant effect on sensitivity to NHS (two independent strains). Bars represent standard error (C, E) and standard deviation (D, F); p values derived from extra sum-of-squares F-test (C, E) or paired t-test (D, F).
In striking contrast to the situation in wild-type T. b. brucei, isobologram and EC50 analyses revealed strong synergy between FMK024 and NHS in killing icp null T. b. brucei (Figure 7A). Indeed, in the presence of 5 to 160 nM FMK024, the NHS sensitivity was almost completely reversed to that of wild-type cells (Figure 7B). These results suggest that one or both of the cathepsins can indeed confer resistance to lysis by human serum, but only effectively in the absence of ICP. We next set out to determine which of the cathepsins is responsible for this phenotype. In order to assess the contribution of the individual cathepsins to resisting NHS, we generated strains for the inducible RNAi-mediated knockdown of either CATB or CATL in an icp null background. Once again, we had to use a sub-lethal knockdown in the case of CATL (see Figure 5B).
(A) FMK024 and NHS act synergistically against icp null T. b. brucei; isobologram analysis showing 50% fractional inhibitory concentrations (FIC) where the dashed line indicates the expected output for no interaction. (B) Treatment with 5–160 nM FMK024 reverses the icp null T. b. brucei NHS resistance phenotype. (C) Representative NHS EC50 analysis following CATB depletion in icp null compared with wild-type and icp null T. b. brucei parental cell lines. (D) NHS EC50 analysis following CATB depletion in four independent strains. (E) Representative EC50 analysis following CATL depletion in icp null cells compared with wild-type and icp null T. b. brucei parental cells. (F) NHS EC50 analysis of two independent CATL RNAi icp null strains. Bars represent standard error (C, E) and standard deviation (D, F); p value derived from paired t-test; CATB and CATL knockdowns were induced in 1 µg or 2 ng/ml tetracycline, respectively.
CATB knockdown in these strains had no impact on NHS-sensitivity (Figure 7C, D). Hence, although CATB may have a role in the degradation of other host-derived proteins, including transferrin , our data suggests that it does not target human serum lytic factors. In contrast, CATL knockdown was associated with a highly significant increase in sensitivity to NHS (Figure 7E, F). Failure to completely reverse the NHS-resistance phenotype following CATL RNAi, may be explained by a second contributing factor or, more likely in our view, is because these experiments had to be carried out under partial knockdown conditions. We conclude that ICP increases sensitivity to NHS primarily by inhibiting CATL activity.
T. b. gambiense and T. b. rhodesiense can resist the APOL1-based trypanolytic factors found in normal human serum, while T. b. brucei fails to do so. We report here an RNAi library screen in bloodstream-form T. b. brucei for resistance to human serum and identify all three known genes, as well as a novel gene, that increase T. b. brucei susceptibility to this innate immune defence mechanism. We go on to show that one of these genes, encoding inhibitor of cysteine peptidase, acts by modulating the essential activity of CATL, a lysosomal cysteine peptidase. These findings illuminate the interactions between ICP, CATL and human serum, and have important implications for human infectivity, as well as for therapies based on cathepsin inhibitors  or serum lytic factors . Finally, we have revealed a novel role for a putative trans-membrane domain protein, Tb927.8.5240, in determining sensitivity to NHS.
A loss-of-function phenotype, associated with HpHbR –, contributes to human serum resistance in T. b. gambiense, the most prevalent cause of sleeping sickness. As expected, our RNAi library screen for human serum resistance identified the gene encoding this protein and also the gene encoding the lysosomal membrane protein, p67, also previously linked to this phenotype through experimental loss-of-function analysis . This confirmed the power and utility of the RNAi-screening approach. Our screen also identified the gene encoding ICP, which was recently linked to human serum sensitivity by others , and a fourth, novel gene (Tb927.8.5240), encoding a predicted multi-pass trans-membrane protein, with an almost identical homolog in T. b. gambiense. These outputs indicate a remarkably low rate of false positives, and suggest a similarly low rate of false negatives when using a multi-genome coverage RNAi library to identify high-confidence hits in T. b. brucei (see Materials and Methods).
To improve our understanding of sensitivity to human serum in African trypanosomes, we focussed on the role of the cysteine peptidase inhibitor, ICP. Our chemical and genetic evidence are entirely consistent, and reveal CATL as the cathepsin primarily responsible for the decreased sensitivity to human serum seen following ICP deletion. Specifically, chemical inhibition or knockdown of the individual cathepsins in an icp null background revealed that only CATL can resist human serum; CATB depletion had no detectable effect on this phenotype. CATL has been shown to accumulate in the lysosome , and is responsible for proteolysis of the transferrin receptor  and of anti-parasite IgG . The lysosome is also the major site of action of APOL1 , the lytic component of both TLF1 and TLF2. Thus, CATL may target TLF, and possibly APOL1, for destruction in the lysosome (Figure 8). ICP, therefore, naturally maintains sensitivity to human serum, possibly by restricting the proteolytic degradation of TLF (or APOL1) in the lysosome. This is consistent with previous pulse chase experiments that found little proteolytic degradation of the lytic factor and its components in T. b. brucei , thereby allowing APOL1 to form membrane-spanning pores, leading to lysosomal swelling and cell lysis. Unmasking of the CATL activity only in the absence of ICP confirms natural control of this cathepsin, as suspected, by ICP.
The four proteins identified in our loss-of-function RNAi library screen are highlighted in green. TLF, including APOL1 (red square), is taken up via HpHbR in the parasite's flagellar pocket, the primary site of endocytosis in T. b. brucei. The lytic factor transits the endosomal system, eventually reaching the lysosome. APOL1 is thought to form pores in the lysosomal membrane, ultimately leading to cellular lysis. CATL activity is normally tightly regulated by ICP; however, we speculate that loss of ICP increases resistance to human serum due to increased proteolysis of TLF, and possibly APOL1, by CATL. The sub-cellular localisation of the putative channel protein, Tb927.8.5240, and its role in determining sensitivity to human serum are unknown. The lysosomal membrane protein p67 is also shown.
Using a similar RNAi-screening approach, uptake of the anti-trypanosomal drug, suramin, was shown to be via receptor-mediated endocytosis in T. b. brucei . The identification of only p67 by both screens suggests distinct uptake and trafficking factors and mechanisms involved in suramin uptake following association with the type-I trans-membrane glycoprotein, ISG75 , and TLF-uptake following association with the GPI-anchored HpHbR . Interestingly, CATL has now been linked to both suramin efficacy  and human serum toxicity (this study) but, while CATL can protect T. b. brucei from killing by human serum, it sensitises T. b. brucei to killing by suramin. This suggests that suramin, a napthylamine, is liberated in active form by lysosomal proteolysis, while we suggest that TLF (APOL1) is degraded by lysosomal proteolysis.
Trypanosomal cathepsins are targets of ongoing drug development , , . Although it was previously suggested that CATB was an appropriate drug target , more recent genetic and chemical evidence indicates that CATL is the essential cysteine peptidase of T. b. brucei and the most appropriate target , . Our current findings also support this view. In this context, it is worth considering the potential impact of therapy targeting the essential cysteine peptidase activity. Our results indicate little impact of exposure to such inhibitors on human serum sensitivity in T. b. brucei. However, cysteine peptidases may be more active in T. b. gambiense  and/or T. b rhodesiense, possibly due to selective pressure through TLF exposure, and CATL inhibition could act synergistically with TLF in this case, increasing sensitivity to lytic activity and presenting a novel rational approach to therapy. On the other hand, T. b. gambiense and T. b. rhodesiense rely upon lysosomal/endosomal VSG variants to resist the toxic effects of APOL1. Reduced proteolysis of these factors  could increase parasite resistance to human serum, meaning that targeting CATL could represent a risky therapeutic strategy. Indeed, FMK024 exposure leads to an accumulation of SRA in the lysosome of T. b. brucei engineered to express this VSG-variant . It will clearly be important to develop an improved understanding of the interplay among these factors in human-infective trypanosomes.
We link four factors to human serum sensitivity using a genome-scale loss-of-function screen in T. b. brucei. These include all three expected factors, based on previous reports, and a novel putative trans-membrane channel. It is interesting to note that, in the case of ICP, we uncovered a gain-of-function phenotype using a loss-of-function screen; this is possible when one protein antagonises the action of another. Our findings indicate that CATL can resist lysis by human serum, and this has important implications, since CATL is a promising potential drug target. In addition, the novel link to a putative trans-membrane channel presents an excellent candidate that may facilitate TLF transit. Notably, the gene encoding TgsGP is not present in T. b. gambiense group 2 , indicating a distinct human serum resistance mechanism in these parasites, and the main route of entry for TLF2 in T. b. brucei is thought to be independent of HpHbR , . The outputs from our screen and our studies on ICP and CATL shed light on mechanisms of toxin delivery and stability in African trypanosomes and should facilitate studies aimed at understanding the multiple mechanisms employed by T. b. gambiense and T. b. rhodesiense to resist lytic factors in humans and other primates.
Materials and Methods
T. b. brucei strains
MITat 1.2 clone 221a 2T1 bloodstream-form T. b. brucei were maintained and manipulated as previously described . Transformants were selected in blasticidin (10 µg/ml), hygromycin (2.5 µg/ml) or G418 (2 µg/ml), as appropriate. For growth assays, cells were seeded at ∼105/ml, counted using a haemocytometer, and diluted back every 24 hours, as necessary, for up to seven days in the absence of antibiotics.
NHS sensitivity analysis
To determine NHS EC50, cells were seeded at 2×103 ml−1 in 96-well plates in a 2-fold dilution series of NHS (pooled mixed gender; Sera Laboratories International), starting from 0.01%; assays were carried out in the absence of antibiotics. After ∼3 days growth, 20 µl of 125 µg/ml resazurin (Sigma) in PBS was added to each well and the plates incubated for a further 6 hours at 37°C. Fluorescence was determined using a fluorescence plate reader (Molecular Devices) at an excitation wavelength of 530 nm, an emission wavelength of 585 nm and a filter cut-off of 570 nm . To analyse the combined effect of NHS and FMK024 treatment, isobologram analysis was carried out using a checkerboard approach, as previously described . Data were processed in Excel, and non-linear regression analysis carried out in GraphPad Prism.
Selective screening of a genome scale T. b. brucei RNAi library
The bloodstream-form T. b. brucei RNAi library  was thawed into 100 ml HMI-11 media (Life Technologies) containing 10% foetal bovine serum (FBS; Sigma) at a density of approximately 1×105/ml. RNAi was induced in 1 µg/ml tetracycline for 24 hours prior to the addition of 0.0005% NHS; RNAi induction and NHS selection were maintained throughout. Daily counts were carried out using a haemocytometer, and the total population was maintained at no lower than 20 million cells for the duration of selection. Once robust growth had been achieved, the inducibility of the selected phenotype was tested  (Figure 1D) and genomic DNA prepared for RNAi target identification.
Sequencing and mapping
The RNAi cassettes remaining in the NHS-selected library were specifically amplified from genomic DNA using the LIB2f/LIB2r primers  producing a ladder of bands ranging in size from 0.25–1.5-kbp following agarose gel-electrophoresis (data not shown). High-throughput sequencing of the amplified DNA was carried out on an Illumina platform (Beijing Genome Institute). Using paired 150-bp sequencing reads; presence or absence of a 14-bp RNAi-construct signature was recorded in the FASTQ header line. Sequence reads were then trimmed to remove lower-quality sequences and mapped to the T. b. brucei reference genome (release 4.2) using bowtie . BAM files were processed using the SAMtools bioinformatics suite . The maps were explored visually in Artemis, and plots were derived using the Artemis graph tool and processed in Adobe Photoshop Elements 8.0. Stacks of reads that included the 14-bp signature on the positive strand were used to define RNAi target fragment junctions and to assign high-confidence hits as those identified by >1 RNAi target fragment.
Plasmid and strain construction
ICP deletion was carried out as described , except that targeting fragments were cloned in pBSD, and the blasticidin-S-deaminase cassette was then replaced with a neomycin phosphotransferase cassette to generate pBSDΔICP and pNPTΔICP constructs. We C-terminally cMyc-tagged an endogenous copy of CATB at a native allele . A 990 bp CATB C-terminal fragment minus the stop codon was cloned into pNATx12MYC ; the construct was linearised with PstI prior to transfection. Stem-loop RNAi constructs targeting CATB (Tb927.6.560), CATL (Tb927.6.960-1060) or Tb927.8.5240 were generated in pRPaiSL ; 206 bp (CATB), 578 bp (CATL) and 417 bp (Tb927.8.5240) target fragments were designed using the RNAit primer design algorithm to minimise off-target effects . pRPaiSL constructs were linearised with AscI to enable targeted integration at the rDNA spacer ‘landing pad’ locus in 2T1 bloodstream form T. b. brucei . Details of all oligonucleotides are available on request. Linearised constructs were transferred to icp null or wild-type 2T1 T. b. brucei using a nucleofector apparatus (Lonza) in conjunction with cytomix or T-cell nucleofection solutions. Protein expression following RNAi depletion was analysed by SDS-PAGE and western blotting with anti-CATL and anti-cMyc, using standard protocols .
cDNA synthesis and qPCR analysis following Tb927.8.5240 RNAi
For each cell line and treatment, 2 µg RNA was DNase-treated and reverse-transcribed using the Superscript VILO cDNA synthesis kit (Invitrogen). 100 ng (RNA-equivalent) cDNA was subjected to qPCR using the Quantitect SYBR Green PCR kit (Qiagen) and primer pairs specific for telomerase reverse transcriptase (TERT; Tb927.11.10190) and Tb927.8.5240 (details of primer sequences are available on request). TERT was used as a reference for normalisation of gene expression, as previously described . qPCR reactions were carried out in a Rotor-gene 3000 (Corbett Research), using the following cycling conditions: 95°C (15 minutes), followed by 40 cycles of 94°C (15 seconds), 58°C (30 seconds), and 72°C (30 seconds). Standard curves, derived from a series of 10-fold dilutions of the target PCR products, were used to determine reaction efficiency. Fold-change in gene expression was calculated by the ΔΔCt method .
Analysis of Tb927.8.5240 knockdown by reverse-transcriptase quantitative PCR. Mean Ct data were used to calculate the ΔΔCt values and fold-change in gene expression of Tb927.8.5240 following RNAi induction, relative to the normalisation gene (TERT).
Thanks to Jay Bangs (SUNY, Buffalo) for providing the anti-CATL antibody, and Martin C. Taylor (LSHTM) for helpful advice regarding reverse transcriptase qPCR.
Conceived and designed the experiments: SA RBC DH. Performed the experiments: SA RBC. Analyzed the data: SA RBC JAG TGC DH. Wrote the paper: SA RBC DH. Sequence mapping and analysis: JAG TGC.
- 1. Vanhollebeke B, Pays E (2010) The trypanolytic factor of human serum: many ways to enter the parasite, a single way to kill. Mol Microbiol 76: 806–814. doi: 10.1111/j.1365-2958.2010.07156.x
- 2. Stephens NA, Hajduk SL (2011) Endosomal localization of the serum resistance-associated protein in African trypanosomes confers human infectivity. Eukaryot Cell 10: 1023–1033. doi: 10.1128/ec.05112-11
- 3. Jackson AP, Sanders M, Berry A, McQuillan J, Aslett MA, et al. (2010) The genome sequence of Trypanosoma brucei gambiense, causative agent of chronic human african trypanosomiasis. PLoS Negl Trop Dis 4: e658. doi: 10.1371/journal.pntd.0000658
- 4. Simarro PP, Diarra A, Ruiz Postigo JA, Franco JR, Jannin JG (2011) The human African trypanosomiasis control and surveillance programme of the World Health Organization 2000–2009: the way forward. PLoS Negl Trop Dis 5: e1007. doi: 10.1371/journal.pntd.0001007
- 5. Hajduk SL, Moore DR, Vasudevacharya J, Siqueira H, Torri AF, et al. (1989) Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein. J Biol Chem 264: 5210–5217.
- 6. Rifkin MR (1978) Identification of the trypanocidal factor in normal human serum: high density lipoprotein. Proc Natl Acad Sci U S A 75: 3450–3454. doi: 10.1073/pnas.75.7.3450
- 7. Raper J, Fung R, Ghiso J, Nussenzweig V, Tomlinson S (1999) Characterization of a novel trypanosome lytic factor from human serum. Infect Immun 67: 1910–1916.
- 8. Tomlinson S, Jansen AM, Koudinov A, Ghiso JA, Choi-Miura NH, et al. (1995) High-density-lipoprotein-independent killing of Trypanosoma brucei by human serum. Mol Biochem Parasitol 70: 131–138. doi: 10.1016/0166-6851(95)00019-w
- 9. Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, Nolan DP, Lins L, et al. (2003) Apolipoprotein L-I is the trypanosome lytic factor of human serum. Nature 422: 83–87. doi: 10.1038/nature01461
- 10. Bullard W, Kieft R, Capewell P, Veitch NJ, Macleod A, et al. (2012) Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors. Virulence 3: 72–76. doi: 10.4161/viru.3.1.18295
- 11. Vanhollebeke B, De Muylder G, Nielsen MJ, Pays A, Tebabi P, et al. (2008) A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosoma brucei in humans. Science 320: 677–681. doi: 10.1126/science.1156296
- 12. Molina-Portela MP, Lugli EB, Recio-Pinto E, Raper J (2005) Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes. Mol Biochem Parasitol 144: 218–226. doi: 10.1016/j.molbiopara.2005.08.018
- 13. Perez-Morga D, Vanhollebeke B, Paturiaux-Hanocq F, Nolan DP, Lins L, et al. (2005) Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes. Science 309: 469–472. doi: 10.1126/science.1114566
- 14. Kieft R, Capewell P, Turner CM, Veitch NJ, MacLeod A, et al. (2010) Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor. Proc Natl Acad Sci U S A 107: 16137–16141. doi: 10.1073/pnas.1007074107
- 15. DeJesus E, Kieft R, Albright B, Stephens NA, Hajduk SL (2013) A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding. PLoS Pathog 9: e1003317. doi: 10.1371/journal.ppat.1003317
- 16. Symula RE, Beadell JS, Sistrom M, Agbebakun K, Balmer O, et al. (2012) Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance. PLoS Negl Trop Dis 6: e1728. doi: 10.1371/journal.pntd.0001728
- 17. Xong HV, Vanhamme L, Chamekh M, Chimfwembe CE, Van Den Abbeele J, et al. (1998) A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense. Cell 95: 839–846. doi: 10.1016/s0092-8674(00)81706-7
- 18. Capewell P, Clucas C, Dejesus E, Kieft R, Hajduk S, et al. (2013) The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense. PLoS Pathog 9: e1003686. doi: 10.1371/journal.ppat.1003686
- 19. Uzureau P, Uzureau S, Lecordier L, Fontaine F, Tebabi P, et al. (2013) Mechanism of Trypanosoma brucei gambiense resistance to human serum. Nature 501: 430–4. doi: 10.1038/nature12516
- 20. Peck RF, Shiflett AM, Schwartz KJ, McCann A, Hajduk SL, et al. (2008) The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes. Mol Microbiol 68: 933–946. doi: 10.1111/j.1365-2958.2008.06195.x
- 21. Hager KM, Pierce MA, Moore DR, Tytler EM, Esko JD, et al. (1994) Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes. J Cell Biol 126: 155–167. doi: 10.1083/jcb.126.1.155
- 22. Capewell P, Veitch NJ, Turner CM, Raper J, Berriman M, et al. (2011) Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1. PLoS Negl Trop Dis 5: e1287. doi: 10.1371/journal.pntd.0001287
- 23. Berberof M, Perez-Morga D, Pays E (2001) A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense. Mol Biochem Parasitol 113: 127–138. doi: 10.1016/s0166-6851(01)00208-0
- 24. Alsford S, Turner DJ, Obado SO, Sanchez-Flores A, Glover L, et al. (2011) High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 21: 915–924. doi: 10.1101/gr.115089.110
- 25. Alsford S, Eckert S, Baker N, Glover L, Sanchez-Flores A, et al. (2012) High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature 482: 232–236. doi: 10.1038/nature10771
- 26. Sanderson SJ, Westrop GD, Scharfstein J, Mottram JC, Coombs GH (2003) Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens. FEBS Lett 542: 12–16. doi: 10.1016/s0014-5793(03)00327-2
- 27. Alphey MS, Hunter WN (2006) High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei. Acta Crystallogr Sect F Struct Biol Cryst Commun 62: 504–508. doi: 10.1107/s1744309106014849
- 28. Santos CC, Coombs GH, Lima AP, Mottram JC (2007) Role of the Trypanosoma brucei natural cysteine peptidase inhibitor ICP in differentiation and virulence. Mol Microbiol 66: 991–1002. doi: 10.1111/j.1365-2958.2007.05970.x
- 29. Steverding D, Sexton DW, Wang X, Gehrke SS, Wagner GK, et al. (2012) Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target. Int J Parasitol 42: 481–488. doi: 10.1016/j.ijpara.2012.03.009
- 30. Caffrey CR, Hansell E, Lucas KD, Brinen LS, Alvarez Hernandez A, et al. (2001) Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense. Mol Biochem Parasitol 118: 61–73. doi: 10.1016/s0166-6851(01)00368-1
- 31. Mackey ZB, O'Brien TC, Greenbaum DC, Blank RB, McKerrow JH (2004) A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei. J Biol Chem 279: 48426–48433. doi: 10.1074/jbc.m402470200
- 32. O'Brien TC, Mackey ZB, Fetter RD, Choe Y, O'Donoghue AJ, et al. (2008) A parasite cysteine protease is key to host protein degradation and iron acquisition. J Biol Chem 283: 28934–28943. doi: 10.1074/jbc.m805824200
- 33. Alsford S, Kawahara T, Glover L, Horn D (2005) Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects. Mol Biochem Parasitol 144: 142–148. doi: 10.1016/j.molbiopara.2005.08.009
- 34. Ettari R, Tamborini L, Angelo IC, Micale N, Pinto A, et al. (2013) Inhibition of Rhodesain as a Novel Therapeutic Modality for Human African Trypanosomiasis. J Med Chem 56: 5637–58. doi: 10.1021/jm301424d
- 35. Thomson R, Molina-Portela P, Mott H, Carrington M, Raper J (2009) Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes. Proc Natl Acad Sci U S A 106: 19509–19514. doi: 10.1073/pnas.0905669106
- 36. Nkemgu NJ, Grande R, Hansell E, McKerrow JH, Caffrey CR, et al. (2003) Improved trypanocidal activities of cathepsin L inhibitors. Int J Antimicrob Agents 22: 155–159. doi: 10.1016/s0924-8579(03)00096-7
- 37. Shimamura M, Hager KM, Hajduk SL (2001) The lysosomal targeting and intracellular metabolism of trypanosome lytic factor by Trypanosoma brucei brucei. Mol Biochem Parasitol 115: 227–237. doi: 10.1016/s0166-6851(01)00292-4
- 38. Redecke L, Nass K, DePonte DP, White TA, Rehders D, et al. (2013) Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339: 227–230. doi: 10.1126/science.1229663
- 39. Jacobs RT, Nare B, Phillips MA (2011) State of the art in African trypanosome drug discovery. Curr Top Med Chem 11: 1255–1274. doi: 10.2174/156802611795429167
- 40. Gibson W, Nemetschke L, Ndung'u J (2010) Conserved sequence of the TgsGP gene in Group 1 Trypanosoma brucei gambiense. Infect Genet Evol 10: 453–458. doi: 10.1016/j.meegid.2010.03.005
- 41. Raz B, Iten M, Grether-Buhler Y, Kaminsky R, Brun R (1997) The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro. Acta Trop 68: 139–147. doi: 10.1016/s0001-706x(97)00079-x
- 42. Singh PK, Tack BF, McCray PB Jr, Welsh MJ (2000) Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am J Physiol Lung Cell Mol Physiol 279: L799–805.
- 43. Baker N, Alsford S, Horn D (2011) Genome-wide RNAi screens in African trypanosomes identify the nifurtimox activator NTR and the eflornithine transporter AAT6. Mol Biochem Parasitol 176: 55–57. doi: 10.1016/j.molbiopara.2010.11.010
- 44. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25. doi: 10.1186/gb-2009-10-3-r25
- 45. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079. doi: 10.1093/bioinformatics/btp352
- 46. Alsford S, Horn D (2008) Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei. Mol Biochem Parasitol 161: 76–79. doi: 10.1016/j.molbiopara.2008.05.006
- 47. Redmond S, Vadivelu J, Field MC (2003) RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol Biochem Parasitol 128: 115–118. doi: 10.1016/s0166-6851(03)00045-8
- 48. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al.. (1998) Current Protocols in Molecular Biology. New York: John Wiley and Sons, Inc.
- 49. Brenndorfer M, Boshart M (2010) Selection of reference genes for mRNA quantification in Trypanosoma brucei. Mol Biochem Parasitol 172: 52–55. doi: 10.1016/j.molbiopara.2010.03.007
- 50. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262