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Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections

Rivera-Correa, Juan; Verdi, Joseph; Sherman, Julian; Sternberg, Jeremy M; Raper, Jayne; Rodriguez, Ana
Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.
PMID: 34587165
ISSN: 1935-2735
CID: 5026972

Interplay of Trypanosome Lytic Factor and innate immune cells in the resolution of cutaneous Leishmania infection

Pant, Jyoti; Samanovic, Marie; Nelson, Maria T; Keceli, Mert K; Verdi, Joseph; Beverley, Stephen M; Raper, Jayne
Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein (HDL) complex that, through the cation channel-forming protein apolipoprotein L-1 (APOL1), provides innate immunity to select kinetoplastid parasites. The immunoprotective effects of TLF have been extensively investigated in the context of its interaction with the extracellular protozoan Trypanosoma brucei brucei, to which it confers sterile immunity. We previously showed that TLF could act against an intracellular pathogen Leishmania, and here we dissected the role of TLF and its synergy with host-immune cells. Leishmania major is transmitted by Phlebotomine sand flies, which deposit the parasite intradermally into mammalian hosts, where neutrophils are the predominant phagocytes recruited to the site of infection. Once in the host, the parasites are phagocytosed and shed their surface glycoconjugates during differentiation to the mammalian-resident amastigote stage. Our data show that mice producing TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major, the infective, fly-transmitted stage. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. In vitro we find that only metacyclic promastigotes co-incubated with TLF in an acidic milieu were lysed. However, amastigotes were not killed by TLF at any pH. These findings correlated with binding experiments, revealing that labeled TLF binds specifically to the surface of metacyclic promastigotes, but not to amastigotes. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates LPG and/or PPG (lpg1- and lpg5A-/lpg5B- respectively) whose absence mimics the amastigote surface, were resistant to TLF-mediated lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon it kills the parasite in the acidic phagosome of phagocytes. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs several hours after phagocytosis by immune cells, creating a relatively short-lived but effective window for TLF to act against Leishmania.
PMID: 34559857
ISSN: 1553-7374
CID: 5061532

Apolipoproteins L1-6 share key cation channel-regulating residues but have different membrane insertion and ion conductance properties

Pant, Jyoti; Giovinazzo, Joseph A; Tuka, Lilit S; Pena, Darwin; Raper, Jayne; Thomson, Russell
The human apolipoprotein L gene family encodes the APOL1-6 proteins, which are effectors of the innate immune response to viruses and protozoan parasites. Due to a high degree of similarity between APOL proteins, it is often assumed that they have similar functions to APOL1, which forms cation channels in planar lipid bilayers and membranes resulting in cytolytic activity. However, the channel properties of the remaining APOL proteins have not been reported. Here, we used transient overexpression and a planar lipid bilayer system to study the function of APOL proteins. By measuring lactate dehydrogenase release, we found that APOL1, APOL3 and APOL6 were cytolytic, whereas APOL2, APOL4 and APOL5 were not. Cells expressing APOL1 or APOL3, but not APOL6, developed a distinctive swollen morphology. In planar lipid bilayers, recombinant APOL1 and APOL2 required an acidic environment for the insertion of each protein into the membrane bilayer to form an ion conductance channel. In contrast, recombinant APOL3, APOL4, and APOL5 readily inserted into bilayers to form ion conductance at neutral pH, but required a positive voltage on the side of insertion. Despite these differences in membrane insertion properties, the ion conductances formed by APOL1-4 were similarly pH-dependent and cation-selective, consistent with conservation of the pore-lining region in each protein. Thus, despite structural conservation, the APOL proteins are functionally different. We propose that these proteins interact with different membranes and under different voltage and pH conditions within a cell to effect innate immunity to different microbial pathogens.
PMID: 34252458
ISSN: 1083-351x
CID: 4938282

Cation channel conductance and pH gating of the innate immunity factor APOL1 is governed by pore lining residues within the C-terminal domain

Schaub, Charles; Verdi, Joseph; Lee, Penny; Terra, Nada; Limon, Gina; Raper, Jayne; Thomson, Russell
The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei Endocytosis and acidification of high-density lipoprotein (HDL)-associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated non-selective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic-recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted Helix-Loop-Helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with inter-species chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that while neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain (CTD), tyrosine-351 and glutamate-355, that influence pH gating properties, as well as a single residue, aspartate-348, which determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C-terminus as the pore-lining segment of this novel channel-forming protein.
PMID: 32727852
ISSN: 1083-351x
CID: 4540352

Inducible Germline IgMs Bridge Trypanosome Lytic Factor Assembly and Parasite Recognition

Verdi, Joseph; Zipkin, Ronnie; Hillman, Elani; Gertsch, Rahel A; Pangburn, Sarah J; Thomson, Russell; Papavasiliou, Nina; Sternberg, Jeremy; Raper, Jayne
Trypanosomiasis is a devastating neglected tropical disease affecting livestock and humans. Humans are susceptible to two Trypanosoma brucei subspecies but protected from other trypanosomes by circulating high-density lipoprotein (HDL) complexes called trypanosome lytic factors (TLFs) 1 and 2. TLFs contain apolipoprotein L-1 contributing to lysis and haptoglobin-related protein (HPR), which can function as a ligand for a parasite receptor. TLF2 also uniquely contains non-covalently associated immunoglobin M (IgM) antibodies, the role and origin of which remain unclear. Here, we show that these TLF2-associated IgMs interact with both HPR and alternate trypanosome surface proteins, including variant surface glycoprotein, likely facilitating complex biogenesis and TLF uptake into parasites. TLF2-IgMs are germline antibodies that, while present at basal concentrations in healthy individuals, are elicited by trypanosome infection in both murine models and human sleeping sickness patients. These data suggest that poly- and self-reactive germline antibodies such as TLF2-associated IgMs play a role in antimicrobial immunity.
PMID: 32416060
ISSN: 1934-6069
CID: 4438392

Apolipoprotein L-1 renal risk variants form active channels at the plasma membrane driving cytotoxicity

Giovinazzo, Joseph A; Thomson, Russell P; Khalizova, Nailya; Zager, Patrick; Malani, Nirav; Rodriguez-Boulan, Enrique Javier; Raper, Jayne; Schreiner, Ryan
Recently evolved alleles of Apolipoprotein L-1 (APOL1) provide increased protection against African trypanosome parasites while also significantly increasing the risk of developing kidney disease in humans. APOL1 protects against trypanosome infections by forming ion channels within the parasite, causing lysis. While the correlation to kidney disease is robust, there is little consensus concerning the underlying disease mechanism. We show in human cells that the APOL1 renal risk variants have a population of active channels at the plasma membrane, which results in an influx of both Na+ and Ca2+. We propose a model wherein APOL1 channel activity is the upstream event causing cell death, and that the activate-state, plasma membrane-localized channel represents the ideal drug target to combat APOL1-mediated kidney disease.
PMID: 32427098
ISSN: 2050-084x
CID: 4444132

All You Ever Wanted to Know About APOL1 and TLFs and Did Not Dare Ask

Verdi, Joseph; Schaub, Charles; Thomson, Russell; Raper, Jayne
Interest in trypanosome lytic factors (TLFs) and apolipoprotein L1, the ion channel-forming protein component of TLFs, has increased tenfold since 2010. This is due to the association of African variants of APOL1 with kidney disease such that interest has reached circles beyond parasitology. We have extensive experience purifying and working with these proteins and protein complexes. Herein we describe our detailed purification protocols to aid the new burgeoning field by providing an opportunity for consistency in reagents used across laboratories. We emphasize that it is imperative to maintain APOL1 protein intact (~42 kDa) to analyze the active ion channel-forming component/protein.
PMID: 32221937
ISSN: 1940-6029
CID: 4369922

African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat

Pinger, Jason; Nešić, Dragana; Ali, Liaqat; Aresta-Branco, Francisco; Lilic, Mirjana; Chowdhury, Shanin; Kim, Hee-Sook; Verdi, Joseph; Raper, Jayne; Ferguson, Michael A J; Papavasiliou, F Nina; Stebbins, C Erec
The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Ã…-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.
PMID: 29988048
ISSN: 2058-5276
CID: 3199802

Persistent alteration in behavioural reactivity to a mild social stressor in rhesus monkeys repeatedly exposed to sevoflurane in infancy

Raper, J; De Biasio, J C; Murphy, K L; Alvarado, M C; Baxter, M G
BACKGROUND:Socio-emotional development is the expression and management of emotions, which in non-human primates can be examined using responses toward increasing levels of threat. Damage to the limbic system alters socio-emotional development in primates. Thus, neuronal and glial cell loss caused by exposure to general anaesthesia early in infancy might also impact socio-emotional development. We recently reported that repeated sevoflurane exposure in the first month of life alters emotional behaviours at 6 months of age and impairs visual recognition memory after the first year of life in rhesus monkeys. The present study evaluated socio-emotional behaviour at 1 and 2 yr of age in those same monkeys to determine the persistence of altered emotional behaviour. METHODS:Rhesus monkeys of both sexes were exposed to sevoflurane anaesthesia three times for 4 h each time in the first 6 weeks of life. At 1 and 2 yr of age, they were tested on the human intruder task, a well-established mild acute social stressor. RESULTS:Monkeys exposed to sevoflurane as infants exhibited normal fear and hostile responses, but exaggerated self-directed (displacement) behaviours, a general indicator of stress and anxiety in non-human primates. CONCLUSIONS:Early repeated sevoflurane exposure in infant non-human primates results in an anxious phenotype that was first detected at 6 months, and persists for at least 2 yr of age. This is the first demonstration of such a prolonged impact of early anaesthesia exposure on emotional reactivity.
PMID: 29576116
ISSN: 1471-6771
CID: 5052932

Apolipoprotein l-1 and the wound healing pathway: A fight for survival [Meeting Abstract]

Giovinazzo, J A; Thomson, R P; Almanzar, A; Singh, S; Kang, J W; Raper, J
Background: APOL1 is an innate immunity protein that forms pores in trypanosomes. Variants of APOL1 have been linked to kidney disease, yet the mechanism responsible remains controversial. We tested the hypothesis that APOL1 toxicity is cell intrinsic and dependent upon secretion via the Golgi. Along the secretory pathway, APOL1 is acidified and then neutralized upon delivery to the plasma membrane, wherein the cation selective pore initiates wound repair via the influx of Ca2+.
Method(s): HEK293 cells were transfected with APOL1 and its variants, including deletion of the signal peptide. 24-48h later cell toxicity and viability were measured. Treatment with ammonium chloride was performed 2h prior to transfection. Recombinant APOL1 was purified from E. coli and reconstituted in planar lipid bilayers to measure ion channel conductivity and selectivity. HEK293 cells that stably express APOL1 were generated using the FlpIn recombinase system. The cells were transfected with the fluorescent calcium reporter gCAMP6f. Expression of APOL1 was induced at several timepoints and then read in a fluorescent plate reader. Activity of beta-hexosaminidase (beta-hex) was assayed in the supernatant of transfected cells at various timepoints after APOL1 expression.
Result(s): Deletion of the signal peptide led to a significant reduction of toxicity across all variants. Pre-treatment of cells with ammonium chloride reduced the toxicity of APOL1 by 50%. In planar lipid bilayers, rAPOL1 of all three major variants allowed for the passage of Ca2+. In stably transfected cells, induction of APOL1 expression lead to an increase in cytoplasmic Ca2+. In a cell, this would cause lysosomes to fuse with the plasma membrane to initiate removal of the wound and repair the membrane. Indeed, release of the lysosomal enzyme beta-hexosaminidase, a marker of wound-healing, was detected prior to cell death.
Conclusion(s): These data support a model of APOL1 mediated cell death that requires acidic activation along the secretory pathway prior to forming pores at the plasma membrane. Increases in Ca2+ flux and the release of lysosomal enzymes prior to cell death indicate activation of the wound healing pathway by APOL1 pore-formation. Maintaining the balance between secretion and excessive pore-formation of APOL1 and the ability to remove and repair the wounds are key to cell survival
ISSN: 1533-3450
CID: 4750342