Glucose-6-phosphate dehydrogenase and its 3D structures from crystallography and electron cryo-microscopy.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway. It has been extensively studied by biochemical and structural techniques. 13 X-ray crystal structures and five electron cryo-microscopy structures in the PDB are focused on in this topical review. Two F420-dependent glucose-6-phosphate dehydrogenase (FGD) structures are also reported. The significant differences between human and parasite G6PDs can be exploited to find selective drugs against infections such as malaria and leishmaniasis. Furthermore, G6PD is a prognostic marker in several cancer types and is also considered to be a tumour target. On the other hand, FGD is considered to be a target against Mycobacterium tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation.

Animal models of neglected parasitic diseases: In vivo multimodal imaging of experimental trypanosomatid infections.

African trypanosomiases and leishmaniases are significant neglected tropical diseases (NTDs) that affect millions globally, with severe health and socio-economic consequences, especially in endemic regions. Understanding the pathogenesis and dissemination of Trypanosoma brucei and Leishmania spp. parasites within their hosts is pivotal for the development of effective interventions. Whole-body bioluminescence and fluorescence imaging systems (BLI and FLI, respectively), are powerful tools to visualize and quantify the progression and distribution of these parasites in real-time within live animal models. By combining this technology with the engineering of stable T. brucei and Leishmania spp. strains expressing luciferase and/or fluorescent proteins, crucial aspects of the infection process including the parasites' homing, the infection dynamics, the tissue tropism, or the efficacy of experimental treatments and vaccines can be deeply investigated. This methodology allows for enhanced sensitivity and resolution, elucidating previously unrecognized infection niches and dynamics. Importantly, whole-body in vivo imaging is non-invasive, enabling for longitudinal studies during the course of an infection in the same animal, thereby aligning with the "3Rs" principle of animal research. Here, we detail a protocol for the generation of dual-reporter T. brucei and L. major, and their use to infect mice and follow the spatiotemporal dynamics of infection by in vivo imaging systems. Additionally, 3D micro-computed tomography (µCT) coupled to BLI in T. brucei-infected animals is applied to gain insights into the anatomical parasite distribution. This Chapter underscores the potential of these bioimaging modalities as indispensable tools in parasitology, paving the way for novel therapeutic strategies and deeper insights into host-parasite interactions.

Genetic modification of the bee parasite Crithidia bombi for improved visualization and protein localization.

Crithidia bombi is a trypanosomatid parasite that infects several species of bumble bees (Bombus spp.), by adhering to their intestinal tract. Crithidia bombi infection impairs learning and reduces survival of workers and the fitness of overwintering queens. Although there is extensive research on the ecology of this host-pathogen system, we understand far less about the mechanisms that mediate internal infection dynamics. Crithidia bombi infects hosts by attaching to the hindgut via the flagellum, and one previous study found that a nectar secondary compound removed the flagellum, preventing attachment. However, approaches that allow more detailed observation of parasite attachment and growth would allow us to better understand factors mediating this host-pathogen relationship. We established techniques for genetic manipulation and visualization of cultured C. bombi. Using constructs established for Crithidia fasciculata, we successfully generated C. bombi cells expressing ectopic fluorescent transgenes using two different selectable markers. To our knowledge, this is the first genetic modification of this species. We also introduced constructs that label the mitochondrion and nucleus of the parasite, showing that subcellular targeting signals can function across parasite species to highlight specific organelles. Finally, we visualized fluorescently tagged parasites in vitro in both their swimming and attached forms, and in vivo in bumble bee (Bombus impatiens) hosts. Expanding our cell and molecular toolkit for C. bombi will help us better understand how factors such as host diet, immune system, and physiology mediate outcomes of infection by these common parasites.

Compounds with potentialities as novel chemotherapeutic agents in leishmaniasis at preclinical level.

Leishmaniasis are neglected infectious diseases caused by kinetoplastid protozoan parasites from the genus Leishmania. These sicknesses are present mainly in tropical regions and almost 1 million new cases are reported each year. The absence of vaccines, as well as the high cost, toxicity or resistance to the current drugs determines the necessity of new treatments against these pathologies. In this review, several compounds with potentialities as new antileishmanial drugs are presented. The discussion is restricted to the preclinical level and molecules are organized according to their chemical nature, source and molecular targets. In this manner, we present antimicrobial peptides, flavonoids, withanolides, 8-aminoquinolines, compounds from Leish-Box, pyrazolopyrimidines, and inhibitors of tubulin polymerization/depolymerization, topoisomerase IB, proteases, pteridine reductase, N-myristoyltransferase, as well as enzymes involved in polyamine metabolism, response against oxidative stress, signaling pathways, and sterol biosynthesis. This work is a contribution to the general knowledge of these compounds as antileishmanial agents.

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code.

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.

Trypanosomatid diversity in a bat community of an urban area in Campo Grande, Mato Grosso do Sul, Brazil.

Bats have a long evolutionary history with trypanosomatids, but the role of these flying mammals on parasite transmission cycles in urban areas, especially for Trypanosoma and Leishmania species, remains poorly known. The objective of this study was to evaluate the species richness of trypanosomatids parasitizing a bat community in Campo Grande (CG), a state capital within the Cerrado of the Brazilian Midwest. We evaluated 237 bats of 13 species by means of hemoculture and molecular detection in spleen samples. The bat community of CG appears to participate in the transmission cycles of various species of trypanosomatids. We report an overall trypanosomatid detection rate of 34.2% (n = 81), involving 11 out of 13 sampled bat species. We identified six species of trypanosomatids from 61 bats by analyzing SSU rRNA and/or kDNA: Trypanosoma cruzi DTU TcI, T. c. marinkellei, T. dionisii, Leishmania infantum, L. amazonensis, and T. janseni, with this latter being detected by hemoculture for the first time in a bat species. We also detected a Molecular Operational Taxonomic Unit, Trypanosoma sp. DID, in the phyllostomids Glossophaga soricina and Platyrrhinus lineatus. The highest trypanosomatid richness was observed for Sturnira lilium, which hosted three species: L. infantum, T. dionisii and T. janseni. Given that visceral leishmaniasis is endemic in CG, special focus should be placed on L. infantum. Moreover, L. amazonensis and T. cruzi warrant attention, since these are zoonotic parasites responsible for human cases of tegumentary leishmaniasis and Chagas disease, respectively. In this respect, we discuss how bat communities may influence the Leishmania spp. transmission in endemic areas.

Aspartyl protease in the secretome of honey bee trypanosomatid parasite contributes to infection of bees.

BACKGROUND: The exoproteome, which consists of both secreted proteins and those originating from cell surfaces and lysed cells, is a critical component of trypanosomatid parasites, facilitating interactions with host cells and gut microbiota. However, its specific roles in the insect hosts of these parasites remain poorly understood. METHODS: We conducted a comprehensive characterization of the exoproteome in Lotmaria passim, a trypanosomatid parasite infecting honey bees, under culture conditions. We further investigated the functions of two conventionally secreted proteins, aspartyl protease (LpAsp) and chitinase (LpCht), as representative models to elucidate the role of the secretome in L. passim infection of honey bees. RESULTS: Approximately 48% of L. passim exoproteome proteins were found to share homologs with those found in seven Leishmania spp., suggesting the existence of a core exoproteome with conserved functions in the Leishmaniinae lineage. Bioinformatics analyses suggested that the L. passim exoproteome may play a pivotal role in interactions with both the host and its microbiota. Notably, the deletion of genes encoding two secretome proteins revealed the important role of LpAsp, but not LpCht, in L. passim development under culture conditions and its efficiency in infecting the honey bee gut. CONCLUSIONS: Our results highlight the exoproteome as a valuable resource for unraveling the mechanisms employed by trypanosomatid parasites to infect insect hosts by interacting with the gut environment.

A yeast-based high-throughput screen identifies inhibitors of trypanosomatid HRG heme transporters with potent leishmanicidal and trypanocidal activity.

OBJECTIVES: New drugs are required to treat neglected diseases caused by trypanosomatid parasites such as Leishmania, Trypanosoma brucei and Trypanosoma cruzi. An Achilles' heel of these parasites is their heme auxotrophy; they have an absolute dependence on scavenging this molecule from the host, and trypanosomatid HRG heme transporters (TrypHRG) play an important role in this process. As these proteins are essential for the parasites and have low similarity with their human orthologue, they have been proposed as attractive therapeutic targets. Here, we have developed two yeast-based assays that allow an inexpensive high-throughput screening of TrypHRG inhibitors within a cellular context. METHODS: We first assessed that Leishmania major, Leishmania donovani and T. brucei HRG proteins were heterologously expressed in the digestive vacuole membrane of a mutant heme auxotrophic yeast strain. Here, TrypHRG imports hemoglobinderived heme into the cytosol, allowing mutant yeast to grow in the presence of low hemoglobin concentrations and promoting the activity of hemeproteins such as catalase, which was used as a reporter of cytosolic heme levels. RESULTS: In the presence of a TrypHRG inhibitor, both catalase activity (test 1) and yeast growth (test 2) were diminished, being easily monitored. The assays were then tested on a pilot scale for HTS purposes using a collection of repurposing drugs and food antioxidants. Some of the TrypHRG inhibitors identified in yeast presented strong trypanocidal and leishmanicidal activity in the submicromolar range, proving the potential of this approach. CONCLUSIONS: Cumulatively, it was shown that the inhibition bioassays developed were robust and applicable to large-scale HTS.

Genome-wide screens connect HD82 loss-of-function to purine analog resistance in African trypanosomes.

Nucleoside analogs have been used extensively as anti-infective agents, particularly against viral infections, and have long been considered promising anti-parasitic agents. These pro-drugs are metabolized by host-cell, viral, or parasite enzymes prior to incorporation into DNA, thereby inhibiting DNA replication. Here, we report genes that sensitize African trypanosomes to nucleoside analogs, including the guanosine analog, ganciclovir. We applied ganciclovir selective pressure to a trypanosome genome-wide knockdown library, which yielded nucleoside mono- and diphosphate kinases as hits, validating the approach. The two most dominant hits to emerge, however, were Tb927.6.2800 and Tb927.6.2900, which both encode nuclear proteins; the latter of which is HD82, a SAMHD1-related protein and a putative dNTP triphosphohydrolase. We independently confirmed that HD82, which is conserved among the trypanosomatids, can sensitize Trypanosoma brucei to ganciclovir. Since ganciclovir activity depends upon phosphorylation by ectopically expressed viral thymidine kinase, we also tested the adenosine analog, ara-A, that may be fully phosphorylated by native T. brucei kinase(s). Both Tb927.6.2800 and HD82 knockdowns were resistant to this analog. Tb927.6.2800 knockdown increased sensitivity to hydroxyurea, while dNTP analysis indicated that HD82 is indeed a triphosphohydrolase with dATP as the preferred substrate. Our results provide insights into nucleoside/nucleotide metabolism and nucleoside analog metabolism and resistance in trypanosomatids. We suggest that the product of 6.2800 sensitizes cells to purine analogs through DNA repair, while HD82 does so by reducing the native purine pool.IMPORTANCEThere is substantial interest in developing nucleoside analogs as anti-parasitic agents. We used genome-scale genetic screening and discovered two proteins linked to purine analog resistance in African trypanosomes. Our screens also identified two nucleoside kinases required for pro-drug activation, further validating the approach. The top novel hit, HD82, is related to SAMHD1, a mammalian nuclear viral restriction factor. We validated HD82 and localized the protein to the trypanosome nucleus. HD82 appears to sensitize trypanosomes to nucleoside analogs by reducing native pools of nucleotides, providing insights into both nucleoside/nucleotide metabolism and nucleoside analog resistance in trypanosomatids.

Unraveling the diversity of Trypanosoma species from Central Mexico: Molecular confirmation on the presence of Trypanosoma dionisii and novel Neobat linages.

Bats are one of the groups of mammals with the highest number of associated Trypanosoma taxa. There are 50 Trypanosoma species and genotypes infecting more than 75 species of bats across five continents. However, in Mexico, the inventory of species of the genus Trypanosoma associated with bats is limited to only two species (Trypanosoma vespertilionis and Trypanosoma cruzi) even though 140 species of bats inhabit this country. Specifically, 91 bat species have been recorded in the state of Veracruz, but records of trypanosomatids associated with this mammalian group are absent. Due to the complex Trypanosoma-bat relationship, the high diversity of bat species in Veracruz, as well as the lack of records of trypanosomatids associated with bats for this state, the aim of this work was to analyze the diversity of species of the genus Trypanosoma and their presence from a bat community in the central area of the state of Veracruz, Mexico. During the period of January to August 2022 in the Tequecholapa Environmental Management Unit where bats were collected using mist nets and blood samples were obtained from their thumbs. We extracted genetic material and amplified a fragment of 800 bp of the 18S ribosomal gene of the genus Trypanosoma by conventional PCR. The positive amplicons were sequenced, and phylogenetic reconstruction was performed to identify the parasite species. A total of 285 bats (149♀, 136♂) belonging to 13 species from 10 genera and a single family (Phyllostomidae) were collected. Twenty-three specimens from six species tested positive for the presence of Trypanosoma dionisii, Trypanosoma sp. Neobat 4, and a potential novelty species provisionally named as Trypanosoma sp. Neobat 6. The results of the present work increase the number of species of the genus Trypanosoma infecting bats in Mexico and in the Neotropical region.

Probing ligand selectivity in pathogens.

Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms?

Trypanosoma cruzi Fibrillarins: Two paralogous proteins with non-identical signals for nuclear transport.

Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.

Detection of Lotmaria passim in honeybees from Emilia Romagna (Italy) based on a culture method.

Lotmaria passim is considered an emerging field of study in honeybee pathology, since it can threaten the health of the colony leading to a higher mortality rate. However, there is a lack of knowledge regarding the diffusion of this trypanosomatid in Italy. In this study, we highlight the presence of L. passim in the province of Bologna through its culture isolation from honeybee guts and microscopic observation.

Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia.

Visceral leishmaniasis (VL) is a neglected disease considered a serious public health problem, especially in endemic countries. Several studies have discovered monoxenous trypanosomatids (Leptomonas and Crithidia) in patients with VL. In different situations of leishmaniasis, investigations have examined cases of co-infection between Leishmania spp. and Crithidia spp. These coinfections have been observed in a wide range of vertebrate hosts, indicating that they are not rare. Diagnostic techniques require improvements and more robust tools to accurately detect the causative agent of VL. This study aimed to develop a real-time quantitative dye-based PCR (qPCR) assay capable of distinguishing Leishmania infantum from Crithidia-related species and to estimate the parasite load in samples of VL from humans and animals. The primer LinJ31_2420 targets an exclusive phosphatase of L. infantum; the primer Catalase_LVH60-12060_1F targets the catalase gene of Crithidia. Therefore, primers were designed to detect L. infantum and Crithidia sp. LVH60A (a novel trypanosomatid isolated from VL patients in Brazil), in samples related to VL. These primers were considered species-specific, based on sequence analysis using genome data retrieved from the TriTryp database and the genome assembling of Crithidia sp. LVH60A strain, in addition to experimental and clinical data presented herein. This novel qPCR assay was highly accurate in identifying and quantifying L. infantum and Crithidia sp. LVH60A in samples obtained experimentally (in vitro and in vivo) or collected from hosts (humans, dogs, cats, and vectors). Importantly, the screening of 62 cultured isolates from VL patients using these primers surprisingly revealed that 51 parasite cultures were PCR+ for Crithidia sp. In addition, qPCR assays identified the co-infection of L. infantum with Crithidia sp. LVH60A in two new VL cases in Brazil, confirming the suspicion of co-infection in a previously reported case of fatal VL. We believe that the species-specific genes targeted in this study can be helpful for the molecular diagnosis of VL, as well as for elucidating suspected co-infections with monoxenous-like trypanosomatids, which is a neglected fact of a neglected disease.

Proteasome as a Drug Target in Trypanosomatid Diseases.

Some diseases caused by trypanosomatid parasites, like Leishmaniasis, Chagas Disease, and Human African Trypanosomiasis (HTA), are challenging to manage, mainly concerning pharmacological therapy because they are associated with vulnerable populations. Unfortunately, there is a lack of significant investments in the search for new drugs. Therefore, one of the strategies to aid the discovery of new drugs is to identify and inhibit molecular targets essential to the parasite's survival, such as the proteasome, which degrades most proteins in the parasite cells. Our study has presented several proteasome inhibitors with various pharmacophoric cores, and two of them, 5, and 13, have stood out in the clinical phase of treatment for leishmaniasis.

Exploring N-myristoyltransferase as a promising drug target against parasitic neglected tropical diseases.

Neglected tropical diseases (NTDs) constitute a group of approximately 20 infectious diseases that mainly affect the impoverished population without basic sanitation in tropical countries. These diseases are responsible for many deaths worldwide, costing billions of dollars in public health investment to treat and control these infections. Among them are the diseases caused by protozoa of the Trypanosomatid family, which constitute Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (sleeping sickness), and Leishmaniasis. In addition, there is a classification of other diseases, called the big three, AIDS, tuberculosis, and malaria, which are endemic in countries with tropical conditions. Despite the high mortality rates, there is still a gap in the treatment. The drugs have a high incidence of side effects and protozoan resistance, justifying the investment in developing new alternatives. In fact, the Target-Based Drug Design (TBDD) approach is responsible for identifying several promising compounds, and among the targets explored through this approach, N-myristoyltransferase (NMT) stands out. It is an enzyme related to the co-translational myristoylation of N-terminal glycine in various peptides. The myristoylation process is a co-translation that occurs after removing the initiator methionine. This process regulates the assembly of protein complexes and stability, which justifies its potential as a drug target. In order to propose NMT as a potential target for parasitic diseases, this review will address the entire structure and function of this enzyme and the primary studies demonstrating its promising potential against Leishmaniasis, T. cruzi, T. brucei, and malaria. We hope our information can help researchers worldwide search for potential drugs against these diseases that have been threatening the health of the world's population.

Co-infection of Leishmania infantum and a Crithidia-related species in a case of refractory relapsed visceral leishmaniasis with non-ulcerated cutaneous manifestation in Brazil.

We report a refractory and relapsed visceral leishmaniasis case in a male child patient followed from 2016 to 2020, whose clinical isolates from multiple relapses were analyzed at the genome level. To the best of our knowledge, it is the first report that both visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis have concomitantly manifested in the same patient. Importantly, sequence analysis revealed that the patient was co-infected with Leishmania infantum and a Crithidia-related parasite, which was previously found in a fatal case of visceral leishmaniasis from the same endemic region.

Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Molecular detection of Lotmaria passim in honeybees in Japan.

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%-66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.

Manipulation of multiple floral traits demonstrates role in pollinator disease transmission.

Plants modulate multitrophic ecological interactions, and variation in plant traits can affect these interactions. Pollinators are exposed to pathogens at flowers and acquire or transmit pathogens at different rates on different plant species, but the traits mediating those interactions are almost entirely unknown. We experimentally manipulated five plant traits that span scales including flower, inflorescence, and plant, to determine their effects on pathogen transmission between foraging bees. Specifically, we manipulated two morphological traits (corolla lip length and flower orientation within an inflorescence) and three resource distribution traits (inflorescence nectar, plant patch nectar, and plant aggregation) in tents to test how plant traits affect bee pathogen transmission. We also quantified foraging behavior and fecal deposition patterns as potential mechanisms driving differences in transmission, and assessed trait manipulation consequences for bee reproduction. We found that pathogen transmission was reduced when we trimmed the corolla lip, evenly dispersed nectar distribution within an inflorescence, or aggregated plants in space. Some traits also affected bee reproduction; tents with trimmed corollas had more larval production than control tents, and tents with evenly distributed nectar across plant patches had more larval production than tents with clumped resources. Thus, some trait manipulations both reduced transmission and increased bee microcolony reproduction, although our design does not allow us to discern whether these are related or separate effects. Taken together, our results demonstrate causal effects of several floral traits on pathogen transmission and pollinator reproduction, indicating the importance of intraspecific plant trait variation for pollinator health and population dynamics.