© 2014. Published by The Company of Biologists Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
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that the original work is properly attributed.
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A small Heat Shock Protein is Essential for Thermotolerance and Intracellular Survival of
Leishmania donovani
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Antje Hombach, Gabi Ommen1, Andrea MacDonald and Joachim Clos*
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Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
Journal of Cell Science
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*) corresponding author: [email protected]
present address: EUROIMMUN AG • Seekamp 31 • D-23560 Lübeck
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Abstract
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Leishmania parasites must survive and proliferate in two vastly different environs – the guts of
poikilothermic sandflies and the antigen-presenting cells of homeothermic mammals. The change of
temperature during transmission from sandflies to mammals is both a key trigger for the
progression of their life cycle and for elevated synthesis of heat shock proteins which have been
implicated in survival at higher temperatures. While the main heat shock protein families have been
studied for their function in the Leishmania life cycle, nothing is known about the roles played by
small heat shock proteins. Here, we present first evidence for the pivotal role played by the
Leishmania donovani 23-kD heat shock protein which is expressed preferentially in the mammalian
stage where it assumes a perinuclear localisation. Loss of HSP23 causes increased sensitivity to
chemical stressors, but renders L. donovani incapable of surviving at 37°C. Consequently, HSP23
null mutants are non-infectious to primary macrophages in vitro. All phenotypic effect can be
abrogated by the introduction of a functional HSP23 transgene into the null mutant, confirming the
specificity of the mutant phenotype. Thus, HSP23 expression is a prerequisite for L. donovani
survival at mammalian host temperatures and a crucial virulence factor.
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Key words: Leishmania, small heat shock protein, alpha-crystallin, stress tolerance, infectivity
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Introduction
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The obligately intracellular protozoan parasites of the genus Leishmania are causative for the
various forms of Leishmaniasis. The clinical manifestations range from self-healing skin lesions
(cutaneous leishmaniasis) to lethal, generalised infections of the reticulo-endothelial system
(visceral leishmaniasis). The infections are found on four continents with an estimated annual
incidence between 900,000 and 1.8 million (Alvar et al., 2012). Leishmania spp. undergo two life
cycle stages - the extracellular promastigote stage in the insect vector and the intracellular
amastigote form, located in the phagosomes of infected macrophages. Thus, the parasites colonise
two very diverse environments in the course of their life cycle. The transmission of Leishmania spp
from a poikilothermic insect vector to a homeothermic mammalian host is linked to an increase in
ambient temperature. The increase in temperature induces a heat stress response that is directly
associated with the stage differentiation of the parasite (Krobitsch et al., 1998). The combination of
an increased temperature and the acidic pH in the phagosomes of infected macrophages causes
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conversion from the promastigote to the amastigote stage (Bates et al., 1992), which is pivotal for
parasite survival.
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The heat shock response is a universal reaction which protects cells and organisms against elevated
temperatures. This reaction correlates with an increased synthesis of so-called heat shock proteins
(HSPs), whose synthesis is also induced by other stressors such as metal ions, low pH and oxidative
cell stress (Morimoto, 1990; Vonlaufen et al., 2008). The expression of HSPs represents a fast and
efficient cellular response to various stresses.
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HSPs are highly conserved proteins and present in all cell types and organisms. Based on their
molecular masses and primary structures, HSPs are divided into different families – HSP110,
HSP100, HSP90, HSP70, HSP60, HSP40, HSP10 and the small HSPs (Feder and Hofmann, 1999;
Smith et al., 1998). Under cell stress conditions HSPs interact with denatured proteins and inhibit
the formation of cytotoxic protein aggregates, thereby maintaining the protein homeostasis of a cell
(Bukau et al., 2006; Feder and Hofmann, 1999; Mayer and Bukau, 1999). Under physiological
conditions, most HSPs possess chaperone activity. They interact with many highly diverse client
proteins to support and control maturation, activation, translocation or degradation of those client
proteins.
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The genomes of Leishmania spp encode the full set of HSP families (Ommen and Clos, 2009)
which are synthesised either constitutively during both life cycle stages (Rosenzweig et al., 2008) or
whose abundance increases during amastigote differentiation, correlating with the stress response of
the parasite (Hübel et al., 1995; Krobitsch, 1998; Schlüter et al., 2000; Zamora-Veyl et al., 2005).
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The best characterised HSP in Leishmania so far is HSP100. Chaperones of the HSP100 family are
known to confer inducible thermotolerance in organisms like bacteria, yeast and higher plants
(Lindquist, 1992; Parsell et al., 1994; Parsell and Lindquist, 1994; Sanchez et al., 1992; Schirmer et
al., 1996). The phenotypic analyses of L. donovani and L. major HSP100 null mutants indicated
that the Leishmania homologues are not involved in thermotolerance. Nevertheless, the HSP100
null mutants proved unable to proliferate and survive inside mouse macrophages after in vitro
infection and, in the case of the L. major HSP100 null mutant, to establish an infection in BALB/c
mice (Hübel et al., 1997; Krobitsch et al., 1998; Krobitsch and Clos, 1999). This is probably due to
Leishmania HSP100 being critical for the proper sorting of proteins into exosomes, and thus for the
immune modulatory function of these export vesicles (Silverman et al., 2010).
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Another HSP whose function is crucial for the maintenance of both parasitic life cycle stages is
HSP90 (Hombach et al., 2013; Wiesgigl and Clos, 2001). This chaperone is an essential, conserved
and constitutively expressed HSP that is associated with the maturation and activation of a variety
of signalling client proteins, such as kinases, transcription factors and hormone receptors (Buchner,
1999; Nathan and Lindquist, 1995; Ochel et al., 2001; Picard, 2002; Pratt and Toft, 2003). HSP90 is
the central member of the so-called foldosome complex. Further members of this complex are other
chaperones, such as HSP70 and HSP40, and co-chaperones such as Sti-1 (HOP) and P23 (Sba1).
The correct interplay of the foldosome members facilitates the maturation and activation of
essential client proteins (Johnson, 2012; Johnson and Brown, 2009; Prodromou, 2012).
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The HSP70 and HSP40 families are numerous and diverse in Leishmania spp. In spite of this, very
little is known about the control and roles of these genes (Hombach and Clos, 2014).
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Small HSPs (sHSPs) represent the most widespread, extremely heterogeneous family of molecular
chaperones. Nevertheless, the sHSPs share some characteristic features that include i) a conserved
-crystallin domain (ACD) of 90 residues, ii) a small molecular mass ranging from 12-43 kDa, iii)
the ability to form large oligomers, iv) a dynamic quaternary structure, v) induction at stress
conditions and vi) chaperone activity (Haslbeck et al., 2005; Horwitz, 2003). The ACD is named
after the human lenticular protein -crystallin that prevents protein aggregation in the human eye.
ACDs are flanked by variable N- and C-terminal domains in sHSP family members.
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The sHSPs have a multitude of crucial functions under normal and pathological conditions. They
represent molecular chaperones with strong anti-aggregation properties, interact with the major
cytoskeletal components to regulate their assembly and protect cells against various stresses by their
ability to prevent damage induced by these stressors and/or their ability to interfere with key proapoptotic proteins (Acunzo et al., 2012; Garrido et al., 2012; Wettstein et al., 2012).
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It is well-known that there is a connection between the large HSPs of various parasitic organisms
and their virulence potential (Banumathy et al., 2003; Hombach et al., 2013; Hübel et al., 1997;
Pallavi et al., 2010). However the functions of sHSPs and their potential roles in parasite
pathogenicity are so far mostly unknown. One study of HSP21 from the pathogenic fungus Candida
albicans indicates that sHSPs are directly involved in the adaption to specific stresses and are
crucial for the pathogenicity of this fungus (Mayer et al., 2012). Five potential sHSPs were already
identified in the intracellular parasite Toxoplasma gondii. Those are located in different cellular
compartments and are differentially expressed during the life cycle of the parasite. All of them
exhibit the characteristic properties of sHSPs such as oligomeric structure and chaperone activity
(de Miguel et al., 2009). For Plasmodium it was suggested that a sHSP, HSP20, is involved in
sporozoite cellular adhesion and migration (Montagna et al., 2012).
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So far nothing is known about the functions of sHSPs in Leishmania spp. For L. mexicana
panamensis it was shown that after heat stress the synthesis of small proteins (22-27 kDa) was
increased (Hunter et al., 1984). However to date none of these proteins were identified. In this study
we report the identification of three ACD-containing proteins and the characterisation of an atypical
sHSP in L. donovani. Due to its molecular weight we named the corresponding gene HSP23
(LinJ.34.0230). By molecular analysis we demonstrated that the expression of this novel sHSP is
induced during stage conversion, is involved in L. donovani adaption to specific stresses and is
essential for the infectivity in a murine macrophage model. This work represents the first detailed
description of a sHSP in kinetoplastid protozoa.
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Materials and Methods
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L. donovani culture
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Promastigote stages of L. donovani 1SR (Rosenzweig et al., 2008) were grown at 25°C and cultured
as described before (Krobitsch et al., 1998). After electroporation the recombinant parasites were
cultivated in supplemented M199+ medium with the appropriate selection antibiotics. For routine
cultivation, cells were grown to late log phase in order to maintain exponential growth. Cell density
was measured using a CASY cell counter (Roche, Penzberg, Germany).
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The specific IC50 concentrations were determined for absolute ethanol (Roth, Karlsruhe, Germany),
antimonyl tartrate (Sigma-Aldrich, München, Germany) and geldanamycin (CAYLA-Invivogen,
Toulouse, France).
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Electrotransfection and selection
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Electrotransfection of L. donovani promastigotes was carried out as described before (Ommen et
al., 2009). Parasites were taken from a late log phase culture, washed twice in ice-cold PBS, once in
electroporation buffer and suspended at a density of 1 x 108 ml-1 in electroporation buffer (Kapler et
al., 1990; Laban and Wirth, 1989). 2 µg of linear DNA or 50 µg of circular DNA were used for the
transfection. The DNA was mixed on ice in a 4 mm electroporation cuvette with 0.4 ml of the cell
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suspension. The mixture was immediately subjected to electroporation using a Gene Pulser
apparatus (BioRad, München, Germany). Electrotransfection was carried out by three pulses at
2.750 V/cm and 25 µF. Following electroporation, cells were kept on ice for 10 min before they
were transferred to 10 ml drug-free Medium. The antibiotics, G418 (50 µg ml-1, Roth, Karlsruhe,
Germany), puromycin (25 µg ml-1, Sigma-Aldrich, München, Germany) and ClonNat (150 µg ml-1,
Werner Bioreagents, Jena, Germany), were added after 24 h. Mock transfection was performed in
identical fashion, without DNA, to obtain negative control cultures for antibiotic selection.
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For cloning, promastigotes were seeded in supplemented M199+ at 0.5 cells per well in microtitre
plates. After 10-14 days, wells positive for promastigote growth were identified and the clonal cells
were transferred to culture flasks.
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In vitro stage differentiation
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Axenic amastigotes of L. donovani 1SR were generated as described before (Krobitsch et al., 1998).
For this purpose the differentiation of promastigotes towards axenic amastigotes was induced by a
combination of 37°C cultivation temperature and a time-coordinated acidification of the culture
medium to pH 5.5. Axenic amastigotes were cultivated at 37°C with 5% CO2. For amastigote
quantification the CASY Cell Counter was run in cumulative cell volume mode to include cell
clusters. Equal cell volume aliquots were applied to sodium dodecyl sulfate polyacrylamide gel
electrophoreses (SDS-PAGE) and Western blot, quantified via real time RT-qPCR or were used for
indirect immunfluorescence microscopy.
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In vitro infection experiments
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Murine bone marrow macrophages (BMMs) were isolated from the femurs and tibias of C57BL/6
mice and matured in Iscove‘s modified Dulbecco‘s medium (IMDM) supplemented with 10% heatinactivated FCS, 5% horse serum and 30% L929 supernatant (Reiling et al., 2010). For infection
experiments BMMs were harvested, washed and seeded into the wells of eight-well chamber slide
(NUNC, Roskilde, Denmark) at a density of 2 x 105 cells per well and incubated for 48-72 h a 37°C
and 5% CO2. Adherent BMMs were infected in a ratio of 1:10 (2 x 106) with stationary-phase
promastigotes. After 4 h of incubation at 37°C in supplemented M199+, free parasites were
removed by multiple washing with PBS and incubation was continued for another 44 h in IMDM at
37°C and 5% CO2. The medium was removed and the cells were washed twice with PBS. Finally
the infected BMMs were fixed with ice-cold methanol. Intracellular parasites were quantified by
DAPI (1.25 µm ml-1, Sigma-Aldrich, München, Germany) staining of the nuclei (L. donovani and
BMM) and kinetoplasts (L. donovani). Infection rates and parasite loads were quantified. The
stained cells were analysed by fluorescent microscopy.
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Construction and preparation of recombinant DNA
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Approximately 1,000 bp of 5' non-coding DNA (5'NC) and 3' non-coding DNA (3'NC) of the
HSP23 gene (LinJ.34.0230) were amplified enzymatically from genomic L. donovani DNA with
primers that added EcoRI and KpnI sites (5'NC) or BamHI and HindIII sites (3'NC) to upstream and
downstream ends. SwaI sites were introduced to flank the constructs. The 5'NC amplificates were
digested with EcoRI and KpnI and ligated into pUC19 plasmid, digested with the same enzymes.
The resulting plasmids were digested with BamHI and HindIII, and the 3'NC amplificates, cut with
the same enzymes, were ligated between those sites. Subsequently, pUC19-HSP23-5’-3’NC was cut
with KpnI and BamHI. Neomycin resistance and puromycin resistance genes were amplified with
primers that added KpnI and BamHI sites to their 5' and 3' ends. Cut with those enzymes, the
resistance markers were ligated into pUC-HSP23-5’-3‘NC to yield pUC-HSP23-5'neo3' and pUCHSP23-5'puro3' (Fig. S2 A). After construction, the plasmids were amplified in E. coli, purified by
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caesium chloride density gradient ultracentrifugation (Sambrook and Russell, 2001), and linearised
with SwaI, to yield the constructs depicted in Fig. S2 B. The fragments containing the
recombination cassette were separated by agarose gel electrophoresis and purified using the
Machery&Nagel NucleoSpin Extract II Kit.
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The pCLN plasmid was described previously (Hombach et al., 2013). To create pCLS, the
neomycin resistance gene was replaced with a nourseothricine resistance marker, amplified from
pIRmcs(+) (Hoyer et al., 2005) to create pCLS. HSP23 (LinJ.34.0230) or P23 (LinJ.35.4540)
coding sequences were amplified introducing KpnI and BamHI sites respectively to the 5'- and 3'
ends and then fusing these into pCLS between the matching restriction sites to create pCLS-HSP23
and pCLS-P23 (Fig. S2 C).
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Genomic DNA preparation and PCR
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Mutant genotypes were verified by PCR analysis of genomic DNA. For the preparation of genomic
DNA the Gentra Systems Puregene Tissue Core Kit A (Qiagen, Hilden, Germany) was used,
following the manufacturer‘s instructions.
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The quality of isolated gDNA, the gene-replacement and the overexpression of HSP23 were
verified by PCR using the primers listed below. PCR-products were separated by agarose gel
electrophoresis and ethidium bromide staining. Primer used are as follows: HSP23-specific-fwd
(ATGTCCACCAGCGGCCCA), HSP23-specific-rev (CTCGAGGAGGACACGTGA), P23specific-fwd (ATGTCTCACCTTCCGATC), P23-specific-rev (TTACGCGTTGAGATCGCTG).
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Expression profiling
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Semi-quantitative real time RT-qPCR was performed essentially as described (Choudhury et al.,
2008). HSP23-gene-specific primers were HSP23-F42 (CCATTCCTGGATTTGGCTCGG) and
HSP23-B36 (GCATATCGCCGTCGTCATCTCC). HSP23 mRNA abundance was calculated
relative to the Leishmania actin signal.
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Recombinant protein expression in E. coli and protein purification
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For the expression of antigen, the coding regions for HSP23 and P23 were amplified using specific
primers that added NdeI sites and EcoRI sites to the 5’- and 3’ ends, respectively. The amplification
products were cleaved with NdeI and EcoRI and ligated into the expression vector pJC45 (Schlüter
et al., 2000). The expression plasmids were transformed into the bacterial strain BL21 (DE3)
[pAPlacIQ] and the His-tagged proteins isolated from bacterial lysates were purified using HisBind
Resin (Novagen, Madison, USA) as described previously (Clos and Brandau, 1994).
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The purified proteins were then used to immunise laying hens. The IgY were purified from the egg
yolk as described previously (Hübel et al., 1995; Polson, 1985; Polson, 1980). The purified
antibodies were tested against recombinant HSP23 or P23 and the lysates of different Leishmania
species.
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The laying hens were treated and kept in accordance with §10a, German Animal Protection Law.
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Mouse antisera against HSP70, HSP90 and HSP100 were described previously (Ommen et al.,
2010).
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Western blot analysis
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Production of SDS cell lysates, discontinuous SDS-PAGE and Western blot were performed
according to standard protocols. Membranes were treated with blocking solution (5% milk powder
and 0.1% Tween 20 in Tris-buffered saline), before they were probed with anti-HSP23 (1:500 in
blocking solution), anti-P23 (1:200 in blocking solution) and mouse anti-alpha-tubulin (1:4000 in
blocking solution, Sigma-Aldrich, München, Deutschland), followed by incubation with antichicken IgG-alkaline phosphatase (1:2000 in blocking solution, Dianova, Hamburg, Germany) or
anti-mouse IgG-alkaline phosphatase (1:2000 in blocking solution, Dianova, Hamburg, Germany)
as secondary antibody. Blots were developed using nitro blue tetrazolium chloride (Roth, Karlsruhe,
Germany) and 5-bromo-4-chloro-3-indolyl phosphate (Roth, Karlsruhe, Germany). Protein bands
were quantified using the software ImageJ 1.42q (Wayne Rasband, National Institute for Health,
USA).
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Native PAGE
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Extraction of non-denatured Leishmania proteins and native gradient gel electrophoresis was
performed as described before (Westwood et al., 1991). 2 x 107 log phase promastigotes and 24h
heat-shocked promastigotes were harvested by centrifugation, washed twice with PBS, and
resuspended in 40 µl extraction buffer (15% glycerol, 0.5 mM 1,10-phenanthroline, 10 mM TrisHCL pH 8.0, 70 mM KCL). After two freeze and thaw cycles, cell lysates were cleared by
centrifugation. The supernatants, containing the soluble protein fractions, were mixed 1:5 with
loading buffer (50% glycerol, 0.1% bromphenol blue).
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The samples were run alongside a high-molecular-weight protein marker for native gels (GE
Healthcare Bio-Sciences, Pittsburgh, USA) on a 6-20% polyacrylamide gradient gel in 0.5 x TBE
buffer. Electrophoresis was allowed to proceed for 24 h at 4°C, at 20 V/cm. This long duration of
electrophoresis was necessary for all proteins to migrate to their exclusion limit regardless of their
net charge (Andersson, 1972; Clos, 1990). After that, the high-molecular-weight protein marker
was separated from the gel and stained with Coomassie Blue R-250. The gel was incubated at 60°C
for 1 h in transfer buffer (48 mM Tris, 39 mM glycine, 0.5% SDS, 10 mM DTT), followed by
Western blot analysis with a specific anti-HSP23 antibody and an anti-HslV antibody (Chrobak et
al. 2012; 1:1000 in blocking solution).
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Immunofluorescence and confocal microscopy
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Log Phase promastigotes, heat-shocked promastigotes and axenic amastigotes (1 x 107 cells) were
sedimented, washed twice with PBS and suspended in 1 ml of PBS. Aliquots of the suspension (2 x
105 cells) were applied on microscopic slides. After fixing the cells for 2 min in ice-cold methanol,
the slides were air-dried for 20 min. Non-adherent cells were removed by a gentle wash (0.1%
Triton-X 100 in PBS) followed by incubation in blocking solution (2% BSA, 0.1% Triton-X 100 in
PBS). Slides were then incubated for 1 h with chicken anti-HSP23 (1:100 in blocking solution) and
with either mouse anti-HSP70 (1:125 in blocking solution), mouse anti-HSP90 (1:250 in blocking
solution) or mouse anti-HSP100 (1:100 in blocking solution). Cell were washed thrice and than
incubated for 1 h with anti-chicken-Alexa594 (Dianova, Hamburg, Germany, 1: 500), with antimouse-FITC (Dianova, Hamburg, Germany, 1:250) and DAPI (Sigma-Aldrich, München,
Germany, 1:25). After washing the slides thrice, Mowiol and coverslips were applied and the slides
were left to dry for 24 h at 4°C.
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Fluorescence microscopy was carried out on an Olympus FluView1000 confocal microscope (SIMscanner and spectral detection).
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Scanning electron microscopy
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L. donovani cells were washed twice in PBS, fixed in 2% Glutaraldehyd in sodium cacaodylate
buffer and post-fixed with 1% osmium. Samples were dehydrated at increasing ethanol
concentrations (30-100%). After critical point drying, samples were treated with gold and analysed
on a Philips SEM 500 electron microscope. Images were taken using a conventional 35 mm camera,
and the developed black-and-white films were digitalised using a HAMA 35 mm film scanner
(Monheim, Germany).
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In silico procedures
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DNA and protein sequence analysis was performed using the MacVector® software version 12.x.
Amino acid sequence alignments and tree building were performed using the MUSCLE algorithm
(Edgar, 2004a; Edgar, 2004b) as part of the MacVector package.
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Numerical data were analysed using the Prism® software (version 5). Greyscale and colour images
were optimised for contrast using Photoshop® CS3 (Adobe) or ImageJ (NIH). Composite figures
were assembled using the Intaglio® software (Purgatory). Significance was determined using the Utest (Mann and Whitney, 1947).
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Results
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Kinetoplastida possess atypical small heat shock proteins
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Since virtually nothing is known about the roles and functions of α-crystallin domain proteins
(ACDPs) in kinetoplastid protozoa, we first performed a search of the Tritryp database
(http://tritrypdb.org/tritrypdb/), using the human P23 co-chaperone sequence and the BLAST
algorithm. We identified 3 distinct putative coding sequences with ACD signature, namely
LinJ.29.2560 (dubbed HSP20), LinJ.35.4540 (dubbed P23) and LinJ.34.0230 (dubbed HSP23), in
accordance with their position within a phylogenetic tree of ACDPs (Fig. 1A). Leishmania HSP20
proteins group with bacterial and plant small HSPs, whilst the putative P23 co-chaperone places to
mammalian and fungal P23 co-chaperones.
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The kinetoplastid HSP23 proteins appear to originate from P23 proteins, but represent a separate
lineage (Fig. 1 A). They and the putative kinetoplastidic P23 orthologs also show an atypical
domain arrangement, in that their ACDs are located very close to the N-termini (Fig. 1 B), much in
contrast with the canonical structure of small HSPs and P23 proteins which carry long N-terminal
domains in front of their ACDs. We also compared the sequences of the ACDs only and found them
considerably diverged, underscoring the generally low degree of sequence conservation in ACDPs
(Fig. 1 C).
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We therefore decided to investigate both L. donovani P23 (A.H. and G.O., unpublished data) and
the atypical HSP23 (this paper).
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HSP23 is a stress-inducible protein in Leishmania
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To detect HSP23 in Leishmania, we first raised specific antibodies against recombinantly expressed
protein. The HSP23 coding sequence was inserted into pJC45 expression vector (Schlüter, 2000)
and expressed as (His)10 fusion protein in E. coli. Total extracts (Fig. S1 A) were purified using a
Ni2+ metal affinity resin (Clos and Brandau, 1994), resulting in a pure, (His)10::HSP23 (Fig. S1 A).
The purified antigen was then mixed with Titermax Gold adjuvant and used to immunise laying
hens. Antibodies (IgY) were purified from egg yolk and tested in Western blot analysis. The
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antibodies detect 0.1 ng of recombinant HSP23 at a 500-fold dilution (Fig. S1 B) and natural
HSP23 in lysates from 3 Leishmania species (Fig. S1 B).
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Small HSPs are known to associate into oligomeric complexes. Therefore, we investigated the
native size of Leishmania HSP23 using Native Gradient PAGE (Andersson, 1972, Ommen 2010).
The results show the majority of HSP23 in a presumably monomeric form. However, several larger
species can be seen, ranging from ~60 kD to ~160 kD, likely representing HSP23 oligomeric
complexes that show increased abundance at higher cultivation temperatures (Fig. 2 A, lanes 3-4).
The formation of such complexes is characteristic of small HSPs. As loading control we used the
HslV peptidase of L. donovani. HslV is expressed constitutively in L. donovani and exists primarily
in a multimeric complex (Chrobak et al., 2012). Accordingly, the HslV signal is not increased in the
samples from heat-shocked promastigotes confirming the observed induction of HSP23 as specific
(Fig. 2 A, lanes 1-2).
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Suspecting HSP23 to play a role in the Leishmania stress response, we also studied the expression
kinetics of HSP23 during an in vitro life cycle (Fig. 2 B). Promastigotes of L. donovani grown at
25°C and pH 7.0 (day 1) were exposed to 37°C (day 2) and shifted to a pH 5.5 (day 3) to induce in
vitro amastigote differentiation (day 4 to 7). At day 7, the axenic amastigotes were seeded into pH
7.0 medium and shifted back to 25°C to facilitate amastigote-to-promastigote conversion (day 9).
Samples were taken at relevant time points and tested for HSP23 RNA levels (Fig. 2 C) and HSP23
protein abundance (Fig. 2 D and Fig. S1 C). Both RNA levels and protein abundance increased
synchronously during the axenic amastigote stage, but returned to normal upon reaching the
promastigote stage again. We conclude that HSP23 is a stress-inducible protein with 3-fold higher
abundance in early amastigotes.
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HSP23 changes abundance and subcellular localisation during its life cycle
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Using the HSP23-specific antibodies in confocal laser microscopy, we compared its localisation
under three different cultivation conditions, i) promastigotes at 25°C (Fig. 3 A), ii) heat stressed
(37°C) promastigotes (Fig. 3 B) and iii) axenic amastigotes (Fig. 3 C). At 25°C, HSP23 shows a
cytoplasmic localisation (A, panels 3 and 4). At 37°C, the staining intensity increases in keeping
with the heat-inducibility we observed. Also, we see a minor shift to the vicinity of the nucleus (B,
panels 3 and 4). In axenic amastigotes (C, panels 3 and 4) HSP23 is found almost exclusively in a
perinuclear localisation, indicting a role in nuclear processes at mammalian temperatures. These
results further indicate a participation of HSP23 in the cellular stress response.
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HSP23 and HSP70 co-localise under heat stress
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To investigate possible interactions of HSP23 with the major chaperones of Leishmania, we also
performed co-localisation studies for HSP23 with HSP70, HSP90 and HSP100 respectively using
the same culture forms as above. Neither HSP90 nor HSP100 showed discernible co-localisation
with HSP23 (Fig. S2), suggesting that neither chaperone interacts selectively with this small HSP.
The co-localisation studies with HSP70, however revealed a convergence of both proteins around
the nucleus under heat stress (Fig. 4 B, panel 5). Promastigotes kept at 25°C showed no such colocalisation patterns (Fig. 4 A, panel 5). Partial co-localisation in a perinuclear position was also
observed for the axenic amastigotes (Fig. 4 C, panel 5). Together the results suggest a convergence
of HSP23 with cytoplasmic HSP70 at mammalian tissue temperature.
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Generation of HSP23 null mutants in L. donovani
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Given that HSP23 is expressed predominantly at higher temperatures, we expected that HSP23 null
mutants might be viable at low cultivation temperatures. Therefore, we targeted the HSP23 alleles
with specific replacement constructs (Fig. S3 A), using a sequential homologous recombination
strategy (Krobitsch and Clos, 2000) (Fig. S3 B). Using the null mutants as background, we
introduced HSP23 and P23 transgenes as episomes (Fig. S3 C). The genotypes were then verified
threefold, i) by HSP23-specific PCR from genomic DNA (Fig. 5 A), ii) by RT-qPCR of 4
individual null mutant clones (Fig. 5 B), and iii) by Western blot analysis using HSP23-specific IgY
(Fig. 5 C).
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The ~600 bp HSP23 coding sequence could be amplified both from wild type and from HSP23-/[HSP23+] gDNA (Fig. 5 A, upper panel, lanes 1, 3). The HSP23-/- gDNA (lane 2) and the control
strains carrying a P23 transgene (lane 4) or the empty pCLS vector (lane 5) did not carry HSP23
coding sequences. We also amplified P23 coding sequences from the same gDNA samples to verify
their quality. All gDNA gave rise to P23 amplicons (Fig. 5 A, lower panel).
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The lack of HSP23 coding sequences was also evident from the results of a semi-quantitative,
HSP23-specific real-time PCR using cDNAs from wild type and null mutant clones (Fig. 5 B). All
4 putative null mutant clones were devoid of HSP23-specific RNA, corroborating the qualitative
PCR in Fig. 5 A.
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Lastly, we verified wild type, null mutant and null mutant-derived transgenic strains by Western
blot using HSP23-specific IgY. Wild type and null mutant with HSP23 transgene show HSP23
protein (Fig. 5 C, upper panel, lanes 1, 3). Neither the null mutant nor its derivatives carrying the
P23 transgene or the empty pCLS vector showed any signal for HSP23 protein (lanes 2, 4, 5). The
quality of the protein samples was verified by Western blot using P23-specific IgY (Fig. 5 C, lower
panel). Thus, using three independent methods, we can verify the HSP23 null mutants and their
derivatives.
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HSP23 is essential for Leishmania stress tolerance
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In order to investigate the phenotype of the null mutant and its derivatives compared with wild type
L. donovani, we performed growth experiments with and without cellular stress. Even at ideal in
vitro growth conditions (25°C, pH 7.0), the null mutant shows no significant growth defect (Fig. 6
A, 6 G), in keeping with its limited expression in promastigotes.
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At 37°C, corresponding to mammalian tissue temperature, the HSP23 null mutant is not viable and
does not proliferate (Fig. 6 B, 6 G) This temperature-sensitive phenotype is complemented by the
HSP23 transgene, but not by P23 overexpression or by the empty expression plasmid pCLS (Fig. 6
B). The temperature sensitivity is highly significant and was corroborated by scanning electron
microscopy which showed the HSP23-/- parasites badly damaged at this temperature (data not
shown).
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The second stimulus for promastigote-to-amastigote conversion, acidic milieu, also affects the
HSP23 null mutant stronger than wild type or HSP23-reconstituted null mutants (Fig. 6 C). Again,
only HSP23 transgenes can abrogate this pH sensitive phenotype.
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We also tested the ethanol tolerance of the null mutant since it causes an unfolded protein stress and
can replace heat shock in the in vitro amastigote differentiation (Barak et al, 2005). At the IC50 (2%
v/v), ethanol causes a growth arrest for the HSP23 null mutant and for the P23 and vector control
strains. Only wild type and the HSP23-reconstituted null mutant show a sustained 50% growth
compared with untreated cells (Fig. 6 D). While Barak et al (2005) observed a morphological
change at 5% ethanol, the wild type did not change its cell length at 2% ethanol. However, the
HSP23 null mutant reacted to this ethanol concentration with a shortening of the cell body and a
reduction of the flagellum (Fig. S4 B), however without visible cell damage.
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HSP23 also protects against another stressor, trivalent antimony (SbIII), the active principle of
pentavalent antimony anti-leishmanials. The HSP23 null mutant shows a markedly reduced growth
compared to the wild type at 112 µM SbIII, the IC50 for this compound (Fig. 6 E). HSP23 expression
in the null mutant abrogates this defect partially. Here too, exposure of the null mutants causes a
shortening of the cell body and the flagellum (Fig. S3 C).
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The HSP90 inhibitor geldanamycin (GA) affects the null mutant marginally stronger than the wild
type, however without restorative effect through the HSP23 addback (Fig. 6 F). Interestingly,
overexpression of P23 which is implied in resistance to GA (Forafonov, 2008) restores growth of
the HSP23 null mutant under GA.
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These data imply that HSP23 protects L. donovani against cell stress, namely elevated temperature,
unfolded protein stress and redox stress. Loss of HSP23 sensitises L. donovani to all these stresses
and in particular results in a temperature-sensitive (TS) phenotype. The structurally related P23
cannot alleviate this phenotype and is clearly functionally diverse from HSP23.
Accepted manuscript
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HSP23 null mutants fail to infect macrophages
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The same set of five L. donovani strains were also tested for their ability to infect bone marrowderived macrophages in vitro. Parasites were added to primary macrophages at a 10:1 ratio. After 4
h, free parasites were washed off, after which the incubation was continued for another 44 h. Cells
were then fixed and stained with DAPI for fluorescence microscopy evaluation of infection rate (%
infected macrophages) and parasite load (parasites per infected macrophage). The loss of HSP23
affects overall infection rate only insignificantly (Fig. 7 A) indicating equal rates of uptake by the
macrophages. The loss of HSP23, however, impairs the ability of L. donovani for intracellular
survival and/or proliferation (Fig. 7 B). While wild type parasites average at 10 amastigotes per host
cell, the HSP23 null mutant shows only marginal parasite load at 3 amastigotes per host cell.
Adding a HSP23 transgene brings the parasite load back up to >10 amastigotes per macrophage,
confirming that the loss of HSP23 is causative for the loss of infectivity. Neither the P23 transgene
nor the vector alone have any positive effect on the parasite counts. This result is in perfect
agreement with the observed loss of temperature tolerance in the null mutant.
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We also tested the ability of wild type, HSP23-/- mutant and the HSP23-/-[HSP23+] add back strain
to survive an in vitro stage differentiation regimen initiated by a 24 h heat shock at 37°C followed
by incubation at 37°C and pH 5.5. The Fig. 7 C-E shows scanning electron micrographs of the
strains under those conditions. All strains show normal morphology at 25°C (C). After 24 h at
37°C, wild type, add-back strain and HSP23 null mutant show a shortening of the flagella and the
cell bodies (D). After four days at 37°C and three days at acidic pH, wild type and add-back strain
display the typical shape of axenic amastigotes, including clustering and small, ovoid cell bodies.
By contrast, nothing remained of the HSP23 null mutant but cell debris (E).
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In addition, attempts to regrow promastigotes from the axenic amastigote cultures failed completely
for the HSP23-/- strain, while wild type and add-back strain underwent successful amastigote-topromastigote conversion (Fig. 7 F)
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We conclude that HSP23 is essential for survival of L. donovani at the temperatures of human and
other mammalian hosts and that the HSP23-/- strain is a true TS mutant.
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Discussion
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Many organisms survive and thrive in highly challenging environments. Even more so, many
organisms must cope with rapidly changing living conditions. Amongst the latter are plants, sessile
marine organisms, but also parasitic organisms such as Leishmania spp. Establishing and
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maintaining adequate stress tolerance is therefore a key for evolutionary success and the population
of ecological niches.
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Traditionally, stress tolerance is mostly seen in the context of stress proteins and their functions
(Sanchez and Lindquist, 1990; Sanchez et al., 1992). The primary stress tolerance effectors were
identified by means of reverse genetics, genetic complementation studies, and systems biology
approaches and belong to the HSP70, HSP100 and sHSP families of chaperones.
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The HSP100 or ClpB chaperones are absent from vertebrates but ubiquitous in eubacteriae, plants,
fungi and protozoa – non-motile organisms in brief (Schirmer et al., 1996). HSP100 interacts with
HSP70 and HSP40 to restore stress sensitive protein complexes. Its expression was found to
correlate with inducible thermotolerance in yeast, fungi, bacteria and plants. It was also discussed as
the effector of thermotolerance in Leishmania (Hübel et al., 1995; Hübel et al., 1997). This role
however is minor at best (Krobitsch and Clos, 1999) while the real impact of HSP100 lies in the
proper sorting of exosomal payload proteins (Silverman et al., 2010).
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Overexpression of HSP70 has also been seen correlated with or resulting in increased stress
resistance of insects, vertebrates and plants (Feder and Hofmann, 1999). At this point, we cannot
exclude a similar role in Leishmania due to experimental constraints. However, activation of
HSP70 synthesis is transient only and does not result in significant abundance (Brandau et al.,
1995). However, HSP70, like other heat shock proteins, is subject to stress-inducible
phosphorylation which may impact on its activity during different life cycle stages (Morales et al.,
2010).
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Our data clearly show that at least one small HSP, HSP23, plays a decisive role in the adaption to
the temperatures found in mammalian tissue. Loss of HSP23 renders L. donovani incapable of
surviving at 37°C and consequently non-infectious to ex vivo macrophages kept at this temperature.
The data showed that high temperatures not only abrogate growth of HSP23 null mutants but
outright destroy the cells, making the HSP23-/- bona fide TS mutants. Consequently, in vitro, axenic
amastigote conversion is also abrogated for the HSP23-/- parasites. Exposing the null mutants to the
differentiation conditions results in cell destruction.
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This TS phenotype of L. donovani HSP23-/- is highly reminiscent of Candida albicans HSP21 null
mutants. Lack of this small HSP renders the pathogenic fungus non-infectious and incapable of
proliferation at mammalian tissue temperatures, likely by interfering with stress-inducible signal
transduction pathways (Mayer et al., 2012).
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On the other hand, deletion of the small HSP BAG1/HSP30 in Toxoplasma gondii did not result in
any phenotypical changes in either stage of that parasite (Bohne et al., 1998). A different but
essential role is played by the Plasmodium berghei HSP20. Loss of the protein results in impaired
migration inside the host tissue by the infecting sporozoite stage, likely impeding liver stage
formation which is a prerequisite for a successful infection (Montagna et al., 2012).
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Temperature tolerance is not the only trait affected by the loss of HSP23. Sensitivity to chemical
stressors such as ethanol, but also to semi-metal ions such as antimony, is increased in the null
mutants. While ethanol is known to cause protein misfolding, the exact effects of antimony on
Leishmania cells is not known. The results hint at a possible effect of antimony on proper protein
folding, a view that is enforced by findings that HSP90 expression rates correlate with antimony
resistance (Alexandratos et al., 2013; Vergnes et al., 2007). It remains to be seen how replacement
of other ACD protein-coding genes in L. donovani, i.e. HSP20 and P23, will affect viability, stress
tolerance and infectivity to macrophages.
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In keeping with its role in protecting Leishmania against temperature stress, HSP23 expression is
significantly up-regulated in axenically grown amastigotes, both at the RNA level and by protein
abundance. This is mirrored by the absolute requirement for HSP23 in the mammalian stage. Only a
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few other HSPs in L. donovani show a similar stage-dependent upregulation – HSP100 (Hübel et
al., 1995; Hübel et al., 1997; Krobitsch et al., 1998), CPN60.2 (Schlüter et al., 2000), and CPN10
(Zamora-Veyl et al., 2005). At least for HSP100, the expression kinetic correlates with a stagespecific role (Hübel et al., 1997; Krobitsch and Clos, 1999; Silverman et al., 2010). Other major
chaperones show only transient induction of synthesis (Brandau et al., 1995; Rosenzweig et al.,
2008) during promastigote to amastigote differentiation resulting in minor changes of their
concentration, in keeping with their ubiquitous role.
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The changed requirement for HSP23 at elevated temperatures also shows by its altered sub-cellular
localisation observed by indirect immunofluorescence. Under heat stress and in axenic amastigotes,
HSP23 relocates to the perinuclear region, showing increased co-localisation with the chaperone
HSP70, another suspected mediator of stress tolerance. A relocation to a nuclear or perinuclear
locale is not uncommon for small HSPs. Human HSP28 (Arrigo et al., 1988), Drosophila
melanogaster HSP27 (Beaulieu et al., 1989), and shrimp HSP26 (Willsie and Clegg, 2002) were
shown to associate with nuclear structures during heat stress, the latter co-localising with HSP70,
much like L. donovani HSP23.
Accepted manuscript
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Future experiments should be directed to the identification of target structures for HSP23. The
native gradient gel electrophoresis already indicates a variety of apparent oligomeric complexes that
contain HSP23, a variety that cannot be explained alone by the defining ability of small HSPs to
form homo-oligomers (Haslbeck et al., 2005), but may reflect target protein interactions.
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While HSP23 appears to fulfil most of the criteria for small HSPs, the protein and its suspected
homologs in other Trypanosomatidae display a less than typical structure, grouping rather with P23like members of the family, unlike the putative HSP20. This clade of atypical small HSPs is so far
restricted to kinetoplastid protozoa, an ancient branch of the eukaryota, and may have emerged
from the P23 line of co-chaperones for functions specific to these protists. Given the structural
differences from mammalian small HSPs and the essential role during the intracellular stage of
Leishmania, steps should be initiated to screen for specific inhibitors. Also, the role of HSP23
orthologs in other kinetoplastid protozoa is of interest, e.g. T. brucei and T. cruzi, where heat stress
is not per se a differentiation signal.
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Acknowledgements
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We acknowledge technical assistance by Dorothea Zander, Nicole Rath and Stefanie Pflichtbeil.
Laura J. Lee provided help with the preparation of the manuscript. We thank former and current
members of the lab for helpful discussions and suggestions.
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Figure 1. Phylogenetic analysis of Leishmania α-crystallin proteins. (A) Sequence alignment
and tree building were done using the Neighbour Joining algorithm and Bootstrap analysis (1000
repeats). Leishmania proteins are highlighted by bold print. The clades of P23-like proteins, αcrystallins, small HSPs and atypical small HSPs are marked on the right. (B) Schematic alignment
of three L. donovani α-crystallin proteins with typical representatives of this protein family. Note
the relative positions of the α-crystallin domains (ACD). (C) Amino acid sequence alignment of the
ACDs from human α-crystallin, mouse small HSP, and 3 L. donovani ACD proteins. Sequence
identities are shown as inverse print; similar amino acids are underlaid in grey; conserved residues
are shown.
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Figure 2. Expression kinetics of HSP23. Native Page analysis of L. donovani log phase
promastigotes grown at 25°C (A, lane 1 and 3) and heat-shocked promastigotes incubated for 24 h
at 37°C (A, lane 2 and 3) showed that the abundance of HSP23 oligomers, but not of the HslV
monomer, increase after heat-stress. To determine the expression profile of HSP23 during the life
cycle of L. donovani a temperature and pH-dependent axenic stage differentiation was performed.
Temperature and pH modulation is shown in (B). The kinetic of HSP23 RNA levels (arbitrary
units) was measured by RT-qPCR (C). HSP23 protein abundance (arbitrary units) was quantified by
Western blot and image analysis (D).
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Figure 3. Subcellular localisation of HSP23 in L. donovani life cycle stages. Log phase
promastigotes at 25°C (A) or 37°C (B), and axenic amastigotes (C) were stained with chicken antiHSP23 (1:100) and DAPI (1:25). Images were taken by confocal laser microscopy and in
differential interference contrast (DIC). Representative cells from each culture were visualised as
DIC (panels A1, B1, C1), DAPI (panels A2, B2, C2), anti-HSP23 (panels A3, B3, C3) and
DAPI/anti-HSP23 overlays (panels A4, B4, C4). The size bar represents 5 µm. k, kinetoplast; n,
nucleus.
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Figure 4. Comparative subcellular localisation analysis. Log phase promastigotes at 25°C (A) or
37°C (B), and axenic amastigotes (C) were stained with mouse anti-HSP70 (1:125), chicken antiHSP23 (1:100) and DAPI (1:25). Images were taken by confocal laser microscopy and in
differential interference contrast (DIC). Representative cells from each culture were visualised as
DIC (panels A1, B1, C1), DAPI (panels A2, B2, C2), anti-HSP70 (panels A3, B3, C3), anti-HSP23
(panels A4, B4, C4) and DAPI/HSP70/HSP23 (panels A5, B5, C5). The size bar represents 5 µm. k,
kinetoplast; n, nucleus
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Figure 5. Verification of L. donovani HSP23 null mutants. The gDNAs from L. donovani strains
HSP23+/+(wild type), HSP23-/-, HSP23-/-HSP23+, HSP23-/-P23+, HSP23-/- pCLS were used as
templates for a P23-gene-specific PCR (A, panel 1) to test the quality of the gDNAs. The same
gDNAs were then used as template for a HSP23-specific PCR (A, panel 2). Position of size markers
[bp] shown on the left. HSP23 mRNA levels (arbitrary units) were quantified by RT-qPCR for
HSP23+/+ and for 4 suspected null mutant clones, HSP23-/- cl1 - cl4 (B). Lysates from L. donovanistrains HSP23+/+, LdHSP23-/-, LdHSP23-/-HSP23+, HSP23-/- P23+, HSP23-/- pCLS were separated
by SDS-PAGE, subjected to Western blot and probed with anti-P23 (1:200) (C, panel 1) or antiHSP23 (1:500) (C, panel 2).
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Figure 6. Proliferation of HSP23 null mutants under stress. 1 x 105 cells ml-1 were seeded into
10 ml of supplemented Medium199+ and grown for 4 days. Cell density on day 4 was then
normalised as [%] of wild type (HSP23+/+) cell density. Parasites were grown at 25°C and pH 7.0
(A), 37°C and pH 7.0 (B), 25°C and pH 5.5 (C). Additional cultures were grown at 25°C and pH
7.0 with the addition of 2% ethanol (D), 112 µM antimonyl tartrate (Sb(III)) (E) or 0.6% (w/v)
geldanamycin (GA). The cell density of wild type cells (HSP23+/+) and HSP23 null mutants
(HSP23-/-) at 25°C and pH 7.0 or 37°C and pH 7.0 was analysed for 4 days (G). Y-axis shows
percentage of wild type growth (A-F) or cell density (x107) (G), * = p<0.05; *** = p<0.001 (Utest).
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Figure 7 Infectivity of HSP23 null mutants in vitro. Bone marrow-derived macrophages (Mθ)
were infected with HSP23+/+, HSP23-/-, HSP23-/-HSP23+, HSP-/-P23+ and HSP23-/- pCLS parasites
at a 1:10 ratio. After 4 h, free parasites were removed and the infected macrophage cultures were
further incubated at 37°C and 5% CO2 for 44 h. Cells were fixed and stained with DAPI. 400
macrophages per infection experiment were checked by fluorescence microscopy for infection rate
(A, % infected Mθ) and parasite load (B, # of parasites per Mθ). Scanning electron microscopy
images of different stages during in vitro axenic amastigote differentiation and promastigote redifferentiation. L. donovani HSP23+/+, HSP23-/- and HSP23-/-HSP23+ log phase promastigotes (C),
heat-shocked promastigotes (D), axenic amastigotes (E) and re-converted promastigotes (F). * =
p<0.05; *** = p<0.001 (U-test).
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