Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Hunter College, CUNY

This review focuses on the epidemiology of human African trypanosomiasis: why it occurs in humans, the current methods of surveillance, and the drugs available to treat it. Emphasis is placed on the identification of human-infective trypanosomes by the blood incubation infectivity test. This test distinguishes between trypanosomes that are non-infective for humans and those that are potentially infective. Currently the test requires incubation of parasites with human serum before injection into mice; any surviving parasites are considered human-infective. The factors in serum that kill all non-human-infective parasites are known as trypanosome lytic factors. The paper details the biochemistry of these factors and recommends standardization of the test based on current knowledge. This test can be used to screen animals with trypanosomiasis, in order to evaluate their role during endemic and epidemic human African trypanosomiasis.

Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test.

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor.

Humans are one of the few species that resist infection by Trypanosoma brucei brucei because the parasites are killed by lytic factors found in human serum. Trypanosome lytic factors (TLFs) are protein/lipid complexes that contain apolipoprotein A-I (apoA-I), and are therefore a class of high density lipoproteins (HDLs). Haptoglobin-related protein (Hpr) is a unique protein component of TLFs, and its expression has only been demonstrated in humans. Trypanolytic activity has only been found in the sera of five primates: humans, gorillas, mandrills, baboons and sooty mangabeys. We describe here previously unidentified components of highly purified human TLF1: apolipoprotein L-I (apoL-I), human cathelicidin antimicrobial peptide 18 (hCAP18) and glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). However, we found that hCAP18 and GPI-PLD, along with apoA-I, are common components of both lytic and non-lytic primate HDLs. In contrast, Hpr, which has been previously implicated as the main lytic component of TLF1, was a unique component of all trypanolytic primate HDLs. Furthermore, a polyclonal antiserum to Hpr neutralized the lytic activity from humans and baboons. ApoL-I, a candidate lytic component of human serum, was not immunologically or genetically detectable in two primate species with lytic activity. Polyclonal antiserum to apoL-I also did not neutralize TLF activity in a total human HDL preparation. These findings suggest that apoL-I is not essential in all primate TLFs, and apoL-I alone is not sufficient for optimal trypanosome lytic activity in human TLF.

Characterization of primate trypanosome lytic factors.

Trypanosome lytic factor 1 (TLF1) is a subclass of human high-density lipoprotein that kills some African trypanosomes thereby protecting humans from infection. We have shown that TLF1 is a 500 kDa HDL complex composed of lipids and at least seven different proteins. Here we present evidence outlining a new paradigm for the mechanism of lysis; TLF1 forms cation-selective pores in membranes. We show that the replacement of external Na+ (23 Da) with the larger tetramethylammonium+, choline+ and tetraethylammonium+ ions (74 Da, 104 Da and 130 Da) ameliorates the osmotically driven swelling and lysis of trypanosomes by TLF1. Confirmation of cation pore-formation was obtained using small unilamellar vesicles incubated with TLF1; these showed the predicted change in membrane potential expected from an influx of sodium ions. Using planar lipid bilayer model membranes made from trypanosome lipids, which allow the detection of single channels, we found that TLF1 forms discrete ion-conducting channels (17 pS) that are selective for potassium ions over chloride ions. We propose that the initial influx of extracellular Na+ down its concentration gradient promotes the passive entry of Cl- through preexisting Cl- channels. The net influx of both Na+ and Cl- create an osmotic imbalance that leads to passive water diffusion. This loss of osmoregulation results in cytoplasmic vacuolization, cell swelling and ultimately trypanosome lysis.

Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes.

Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals.

Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model.

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system.

Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.

The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC(50): 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti-T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC(50) values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis.

Identification of three classes of heteroaromatic compounds with activity against intracellular Trypanosoma cruzi by chemical library screening.

Trypanosome lytic factors (TLFs) are high-density lipoproteins and components of primate innate immunity. TLFs are characterized by their ability to kill extracellular protozoon parasites of the genus Trypanosoma. Two subspecies of Trypanosoma brucei have evolved resistance to TLFs and can consequently infect humans, resulting in the disease African sleeping sickness. The unique protein components of TLFs are a hemoglobin-binding protein, haptoglobin-related protein and a pore-forming protein, apoL-I. The recent advances in our understanding of the roles that these proteins play in the mechanism of TLF-mediated lysis are highlighted in this article. In light of recent data, which demonstrate that TLFs can ameliorate infection by the intracellular pathogen Leishmania, we also discuss the broader function of TLFs as components of innate immunity.

Activity of trypanosome lytic factor: a novel component of innate immunity.

Several species of African trypanosomes cause fatal disease in livestock, but most cannot infect humans due to innate trypanosome lytic factors (TLFs). Human TLFs are pore forming high-density lipoprotein (HDL) particles that contain apolipoprotein L-I (apoL-I) the trypanolytic component, and haptoglobin-related protein (Hpr), which binds free hemoglobin (Hb) in blood and facilitates the uptake of TLF via a trypanosome haptoglobin-hemoglobin receptor. The human-infective Trypanosoma brucei rhodesiense escapes lysis by TLF by expression of serum resistance-associated (SRA) protein, which binds and neutralizes apoL-I. Unlike humans, baboons are not susceptible to infection by T. b. rhodesiense due to previously unidentified serum factors. Here, we show that baboons have a TLF complex that contains orthologs of Hpr and apoL-I and that full-length baboon apoL-I confers trypanolytic activity to mice and when expressed together with baboon Hpr and human apoA-I, provides protection against both animal infective and the human-infective T. brucei rhodesiense in vivo. We further define two critical lysines near the C terminus of baboon apoL-1 that are necessary and sufficient to prevent binding to SRA and thereby confer resistance to human-infective trypanosomes. These findings form the basis for the creation of TLF transgenic livestock that would be resistant to animal and human-infective trypanosomes, which would result in the reduction of disease and the zoonotic transmission of human infective trypanosomes.

Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes.

Prophylactic antiparasitic transgenesis for human parasitic disease?

The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites.

Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1.

Human African trypanosomiasis or sleeping sickness is a vector-borne parasitic disease that has a major impact on human health and welfare in sub-Saharan countries. Based mostly on data from animal models, it is currently thought that trypanosome entry into the brain occurs by initial infection of the choroid plexus and the circumventricular organs followed days to weeks later by entry into the brain parenchyma. However, Trypanosoma brucei bloodstream forms rapidly cross human brain microvascular endothelial cells in vitro and appear to be able to enter the murine brain without inflicting cerebral injury. Using a murine model and intravital brain imaging, we show that bloodstream forms of T. b. brucei and T. b. rhodesiense enter the brain parenchyma within hours, before a significant level of microvascular inflammation is detectable. Extravascular bloodstream forms were viable as indicated by motility and cell division, and remained detectable for at least 3 days post infection suggesting the potential for parasite survival in the brain parenchyma. Vascular inflammation, as reflected by leukocyte recruitment and emigration from cortical microvessels, became apparent only with increasing parasitemia at later stages of the infection, but was not associated with neurological signs. Extravascular trypanosomes were predominantly associated with postcapillary venules suggesting that early brain infection occurs by parasite passage across the neuroimmunological blood brain barrier. Thus, trypanosomes can invade the murine brain parenchyma during the early stages of the disease before meningoencephalitis is fully established. Whether individual trypanosomes can act alone or require the interaction from a quorum of parasites remains to be shown. The significance of these findings for disease development is now testable.

Early invasion of brain parenchyma by African trypanosomes.

African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin-hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three α-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.

Jayne Raper

Department of Biological Sciences

Hunter College, CUNY

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Jayne Raper

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

Hunter College, CUNY

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Department of Biological Sciences