Verdi, J. | bioRxiv Science

bioRxiv2025

Germline Targeted Baboon Apolipoprotein L-1 Protects Mice Against African Trypanosomes

Certain primates are immune to infection by most African trypanosome parasites due to apolipoprotein L-1 (APOL1), a primate-specific ion channel-forming protein. To broaden our understanding of primate APOL1, we generated a panel of trypanosome-resistant murine models expressing various primate APOL1 proteins. We used these mice to investigate the role of APOL1 in trypanosome immunity in vivo by challenging them with various human and livestock trypanosome isolates. Baboon APOL1 provides partial protection to trypanosome isolates, though its protective capacity was limited by poor expression. A more highly expressed chimeric APOL1 encoding human APOL1 with the baboon APOL1 C-terminus was protective against human-infective trypanosomes, although with a fitness cost likely associated with high expression. We investigated the long-standing assumption that human resistance to Trypanosoma vivax is mediated by APOL1. Surprisingly, APOL1-expressing mice were fully susceptible to T. vivax infection, challenging this hypothesis. These model systems are useful tools for evaluating the possibility of generating genetic engineered livestock for disease control.

microbiology↗

bioRxiv2023

A Structural Classification of the Variant Surface Glycoproteins of the African Trypanosome

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the hosts adaptive immune system. The diversity space of the "VSGnome" at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography with deep-learning structural prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection.

microbiology↗

bioRxiv2020

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

Trypanosome Lytic Factor (TLF) is a primate-specific high-density lipoprotein complex that contains APOL1, the lytic component. Human TLF confers sterile immunity to many animal-infective extracellular Trypanosoma Ssp, which have been extensively investigated. Here, we have dissected the underappreciated role of TLF and neutrophils against intracellular Leishmania in intradermal infection. Our data show that mice producing human or baboon TLF have reduced parasite burdens when infected intradermally with metacyclic promastigotes of L. major. This TLF-mediated reduction in parasite burden was lost in neutrophil-depleted TLF mice, suggesting that early recruitment of neutrophils is required for TLF-mediated killing of L. major. Neutrophils and macrophages are the predominant phagocytes recruited to the site of infection. Our data show that acidification of the macrophage phagosome is essential for TLF-mediated lysis of metacyclic promastigotes. 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. During differentiation to the amastigote stage, the parasites shed their surface glycoconjugates. Metacyclic promastigotes of L. major deficient in the synthesis of surface glycoconjugates (lpg1- and lpg5A-/lpg5B-) were partially resistant to TLF lysis. We propose that TLF binds to the outer surface glycoconjugates of metacyclic promastigotes, whereupon APOL1 forms a pH-gated ion channel in the plasma membrane, resulting in osmotic lysis. We hypothesize that resistance to TLF requires shedding of the surface glycoconjugates, which occurs upon phagocytosis by immune cells. Author SummaryLeishmaniasis is a common term used for disease caused by parasites of the genus Leishmania. Depending on the parasite species and the clinical outcome of the disease, leishmaniasis can be divided into cutaneous, muco-cutaneous and visceral. Of the three, cutaneous leishmaniasis is the most common form, which is usually characterized by a localized lesion due to the infection of immune cells, primarily macrophages of the dermis and local lymph nodes. Sometimes, infected individuals can remain asymptomatic and do not show visible lesions. Moreover, the time between the infection and appearance of lesions are also variable and range from a few weeks to months and a few years in some cases. This subclinical stage of leishmaniasis depends on a variety of factors: parasite virulence, infectious dose, and host immune response. Therefore, it is important to understand the host-parasite interaction and its role in the clinical outcome of the disease. Here, we analyze the interaction between a cutaneous strain of Leishmania and a host innate immune factor called Trypanosome Lytic Factor (TLF). TLF is a type of High-Density Lipoprotein (HDL) complex that circulates in our plasma. TLF kills extracellular African Trypanosomes by lysing the parasites. The lytic ability of TLF is due to the primate specific protein APOL1 that forms pH gated ion channels. APOL1 inserts into biological membranes at acidic pH and forms a closed ion-channel that opens when the membrane associated APOL1 is exposed to neutral pH. Using transgenic mice producing primate TLF, we show both human and baboon TLFs ameliorate cutaneous Leishmania major infection. The reduction in parasite burden correlated with: 1. infectious dose of metacyclic promastigotes and 2. the concentration of circulating TLF in mouse plasma. The early recruitment of neutrophils at the site of infection was required for the reduction of parasite burden by TLF. Macrophages, another major cell that phagocytoses metacyclic promastigotes at the site of infection require an acidified phagosome for TLF mediated killing of L. major. The acidification step is also essential for TLF mediated lysis of axenic metacyclic promastigotes of Leishmania in vitro. The susceptibility of metacyclic promastigotes to TLF mediated lysis is governed by the surface glycoconjugates of Leishmania. We find that surface glycoconjugate deficient Leishmania are resistant to TLF mediated killing. Based on these data, we conclude that the shedding of surface glycoconjugates while transitioning from metacyclic promastigotes to amastigotes results in parasite resistance to TLF mediated lysis. Whether TLF is effective at killing metacyclic promastigotes of other experimentally tractable Leishmania sp. such as L. infantum, and L. donovani, which have slightly different surface glycoconjugate structures is yet to be tested. Our data raise the possibility that TLF can have lytic activity against a broad range of pathogens such as bacteria, viruses, fungi and parasites with surface glycoconjugates that transit through intracellular acidic compartments.

microbiology↗

bioRxiv2020

A Parasite Coat Protein Binds Suramin to Confer Drug Resistance

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly one hundred years. Recent studies revealed that trypanosome strains that express the Variant Surface Glycoprotein VSGsur possess heightened resistance to suramin. We show here that VSGsur binds tightly to suramin, other VSGs do not, and that together with VSG13 it defines a structurally divergent subgroup of these coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

microbiology↗