Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins.

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.

Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3, but also revealed how quickly viral escape can curtail effective options4,5. With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities.

Immunocompromised Cas9 transgenic mice for rapid in vivo assessment of host factors involved in highly pathogenic virus infection.

Targeting host factors for anti-viral development offers several potential advantages over traditional countermeasures that include broad-spectrum activity and prevention of resistance. Characterization of host factors in animal models provides strong evidence of their involvement in disease pathogenesis, but the feasibility of performing high-throughput in vivo analyses on lists of genes is problematic. To begin addressing the challenges of screening candidate host factors in vivo, we combined advances in CRISPR-Cas9 genome editing with an immunocompromised mouse model used to study highly pathogenic viruses. Transgenic mice harboring a constitutively expressed Cas9 allele (Cas9 tg/tg ) with or without knockout of type I interferon receptors served to optimize in vivo delivery of CRISPR single-guide RNA (sgRNA) using Invivofectamine 3.0, a simple and easy-to-use lipid nanoparticle reagent. Invivofectamine 3.0-mediated liver-specific editing to remove activity of the critical Ebola virus host factor Niemann-Pick disease type C1 in an average of 74% of liver cells protected immunocompromised Cas9 tg/tg mice from lethal surrogate Ebola virus infection. We envision that immunocompromised Cas9 tg/tg mice combined with straightforward sgRNA in vivo delivery will enable efficient host factor loss-of-function screening in the liver and other organs to rapidly study their effects on viral pathogenesis and help initiate development of broad-spectrum, host-directed therapies against emerging pathogens.

Development of potent and effective synthetic SARS-CoV-2 neutralizing nanobodies.

The respiratory virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly every aspect of life worldwide, claiming the lives of over 3.9 million people globally, at the time of this publication. Neutralizing humanized nanobody (VHH)-based antibodies (VHH-huFc) represent a promising therapeutic intervention strategy to address the current SARS-CoV-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH-huFc antibodies were identified and evaluated for their capacity to tightly bind to the SARS-CoV-2 receptor-binding domain, to prevent binding of SARS-CoV-2 spike (S) to the cellular receptor angiotensin-converting enzyme 2, and to neutralize viral infection. Preliminary preclinical evaluation of multiple VHH-huFc antibody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-CoV-2. The identified and characterized VHH-huFc antibodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.

Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline.

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC50 values below 10 µM, while candesartan cilexetil had an IC50 value of approximately 67 µM against Mpro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC50) value was the only one greater than the limit of the assay (>100 µM).

Identification of Positive Chemotaxis in the Protozoan Pathogen Trypanosoma brucei.

To complete its infectious cycle, the protozoan parasite Trypanosoma brucei must navigate through diverse tissue environments in both its tsetse fly and mammalian hosts. This is hypothesized to be driven by yet unidentified chemotactic cues. Prior work has shown that parasites engaging in social motility in vitro alter their trajectory to avoid other groups of parasites, an example of negative chemotaxis. However, movement of T. brucei toward a stimulus, positive chemotaxis, has so far not been reported. Here, we show that upon encountering Escherichia coli, socially behaving T. brucei parasites exhibit positive chemotaxis, redirecting group movement toward the neighboring bacterial colony. This response occurs at a distance from the bacteria and involves active changes in parasite motility. By developing a quantitative chemotaxis assay, we show that the attractant is a soluble, diffusible signal dependent on actively growing E. coli Time-lapse and live video microscopy revealed that T. brucei chemotaxis involves changes in both group and single cell motility. Groups of parasites change direction of group movement and accelerate as they approach the source of attractant, and this correlates with increasingly constrained movement of individual cells within the group. Identification of positive chemotaxis in T. brucei opens new opportunities to study mechanisms of chemotaxis in these medically and economically important pathogens. This will lead to deeper insights into how these parasites interact with and navigate through their host environments.IMPORTANCE Almost all living things need to be able to move, whether it is toward desirable environments or away from danger. For vector-borne parasites, successful transmission and infection require that these organisms be able to sense where they are and use signals from their environment to direct where they go next, a process known as chemotaxis. Here, we show that Trypanosoma brucei, the deadly protozoan parasite that causes African sleeping sickness, can sense and move toward an attractive cue. To our knowledge, this is the first report of positive chemotaxis in these organisms. In addition to describing a new behavior in T. brucei, our findings enable future studies of how chemotaxis works in these pathogens, which will lead to deeper understanding of how they move through their hosts and may lead to new therapeutic or transmission-blocking strategies.

Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery.

CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain.

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Timescale Separation of Positive and Negative Signaling Creates History-Dependent Responses to IgE Receptor Stimulation.

The high-affinity receptor for IgE expressed on the surface of mast cells and basophils interacts with antigens, via bound IgE antibody, and triggers secretion of inflammatory mediators that contribute to allergic reactions. To understand how past inputs (memory) influence future inflammatory responses in mast cells, a microfluidic device was used to precisely control exposure of cells to alternating stimulatory and non-stimulatory inputs. We determined that the response to subsequent stimulation depends on the interval of signaling quiescence. For shorter intervals of signaling quiescence, the second response is blunted relative to the first response, whereas longer intervals of quiescence induce an enhanced second response. Through an iterative process of computational modeling and experimental tests, we found that these memory-like phenomena arise from a confluence of rapid, short-lived positive signals driven by the protein tyrosine kinase Syk; slow, long-lived negative signals driven by the lipid phosphatase Ship1; and slower degradation of Ship1 co-factors. This work advances our understanding of mast cell signaling and represents a generalizable approach for investigating the dynamics of signaling systems.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms.

Cell Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite Interface in Trypanosoma brucei.

African trypanosomes are devastating human and animal pathogens transmitted by tsetse flies between mammalian hosts. The trypanosome surface forms a critical host interface that is essential for sensing and adapting to diverse host environments. However, trypanosome surface protein composition and diversity remain largely unknown. Here, we use surface labeling, affinity purification, and proteomic analyses to describe cell surface proteomes from insect-stage and mammalian bloodstream-stage Trypanosoma brucei. The cell surface proteomes contain most previously characterized surface proteins. We additionally identify a substantial number of novel proteins, whose functions are unknown, indicating the parasite surface proteome is larger and more diverse than generally appreciated. We also show stage-specific expression for individual paralogs within several protein families, suggesting that fine-tuned remodeling of the parasite surface allows adaptation to diverse host environments, while still fulfilling universally essential cellular needs. Our surface proteome analyses complement existing transcriptomic, proteomic, and in silico analyses by highlighting proteins that are surface-exposed and thereby provide a major step forward in defining the host-parasite interface.

Cyclic AMP Regulates Social Behavior in African Trypanosomes.

UNLABELLED: The protozoan parasite Trypanosoma brucei engages in surface-induced social behavior, termed social motility, characterized by single cells assembling into multicellular groups that coordinate their movements in response to extracellular signals. Social motility requires sensing and responding to extracellular signals, but the underlying mechanisms are unknown. Here we report that T. brucei social motility depends on cyclic AMP (cAMP) signaling systems in the parasite's flagellum (synonymous with cilium). Pharmacological inhibition of cAMP-specific phosphodiesterase (PDE) completely blocks social motility without impacting the viability or motility of individual cells. Using a fluorescence resonance energy transfer (FRET)-based sensor to monitor cAMP dynamics in live cells, we demonstrate that this block in social motility correlates with an increase in intracellular cAMP levels. RNA interference (RNAi) knockdown of the flagellar PDEB1 phenocopies pharmacological PDE inhibition, demonstrating that PDEB1 is required for social motility. Using parasites expressing distinct fluorescent proteins to monitor individuals in a genetically heterogeneous community, we found that the social motility defect of PDEB1 knockdowns is complemented by wild-type parasites in trans. Therefore, PDEB1 knockdown cells are competent for social motility but appear to lack a necessary factor that can be provided by wild-type cells. The combined data demonstrate that the role of cyclic nucleotides in regulating microbial social behavior extends to African trypanosomes and provide an example of transcomplementation in parasitic protozoa. IMPORTANCE: In bacteria, studies of cell-cell communication and social behavior have profoundly influenced our understanding of microbial physiology, signaling, and pathogenesis. In contrast, mechanisms underlying social behavior in protozoan parasites are mostly unknown. Here we show that social behavior in the protozoan parasite Trypanosoma brucei is governed by cyclic-AMP signaling systems in the flagellum, with intriguing parallels to signaling systems that control bacterial social behavior. We also generated a T. brucei social behavior mutant and found that the mutant phenotype is complemented by wild-type cells grown in the same culture. Our findings open new avenues for dissecting social behavior and signaling in protozoan parasites and illustrate the capacity of these organisms to influence each other's behavior in mixed communities.

Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed "social motility," based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies.

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Insect stage-specific receptor adenylate cyclases are localized to distinct subdomains of the Trypanosoma brucei Flagellar membrane.

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.