Anna K. Snyder

Insight into the Transmission Biology and Species-Specific Functional Capabilities of Tsetse (Diptera: Glossinidae) Obligate Symbiont Wigglesworthia

ABSTRACT Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species. IMPORTANCE Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species. Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species.

Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts

Host-associated microbial interactions may involve genome complementation, driving-enhanced communal efficiency and stability. The tsetse fly (Diptera: Glossinidae), the obligate vector of African trypanosomes (Trypanosoma brucei subspp.), harbours two enteric Gammaproteobacteria symbionts: Wigglesworthia glossinidia and Sodalis glossinidius. Host coevolution has streamlined the Wigglesworthia genome to complement the exclusively sanguivorous tsetse lifestyle. Comparative genomics reveal that the Sodalis genome contains the majority of Wigglesworthia genes. This significant genomic overlap calls into question why tsetse maintains the coresidence of both symbionts and, furthermore, how symbiont homeostasis is maintained. One of the few distinctions between the Wigglesworthia and Sodalis genomes lies in thiamine biosynthesis. While Wigglesworthia can synthesize thiamine, Sodalis lacks this capability but retains a thiamine ABC transporter (tbpAthiPQ) believed to salvage thiamine. This genetic complementation may represent the early convergence of metabolic pathways that may act to retain Wigglesworthia and evade species antagonism. We show that thiamine monophosphate, the specific thiamine derivative putatively synthesized by Wigglesworthia, impacts Sodalis thiamine transporter expression, proliferation and intracellular localization. A greater understanding of tsetse symbiont interactions may generate alternative control strategies for this significant medical and agricultural pest, while also providing insight into the evolution of microbial associations within hosts.

The phylogeny of Sodalis-like symbionts as reconstructed using surface-encoding loci.

Phylogenetic analyses of 16S rRNA support close relationships between the Gammaproteobacteria Sodalis glossinidius, a tsetse (Diptera: Glossinidae) symbiont, and bacteria infecting diverse insect orders. To further examine the evolutionary relationships of these Sodalis-like symbionts, phylogenetic trees were constructed for a subset of putative surface-encoding genes (i.e. ompA, spr, slyB, rcsF, ycfM, and ompC). The ompA and ompC loci were used toward examining the intra- and interspecific diversity of Sodalis within tsetse, respectively. Intraspecific analyses of ompA support elevated nonsynonymous (dN) polymorphism with an excess of singletons, indicating diversifying selection, specifically within the tsetse Glossina morsitans. Additionally, interspecific ompC comparisons between Sodalis and Escherichia coli demonstrate deviation from neutrality, with higher fixed dN observed at sites associated with extracellular loops. Surface-encoding genes varied in their phylogenetic resolution of Sodalis and related bacteria, suggesting conserved vs. host-specific roles. Moreover, Sodalis and its close relatives exhibit genetic divergence at the rcsF, ompA, and ompC loci, indicative of initial molecular divergence. The application of outer membrane genes as markers for further delineating the systematics of recently diverged bacteria is discussed. These results increase our understanding of insect symbiont evolution, while also identifying early genome alterations occurring upon integration of microorganisms with eukaryotic hosts.

Interwoven Biology of the Tsetse Holobiont

Microbial symbionts can be instrumental to the evolutionary success of their hosts. Here, we discuss medically significant tsetse flies (Diptera: Glossinidae), a group comprised of over 30 species, and their use as a valuable model system to study the evolution of the holobiont (i.e., the host and associated microbes). We first describe the tsetse microbiota, which, despite its simplicity, harbors a diverse range of associations. The maternally transmitted microbes consistently include two Gammaproteobacteria, the obligate mutualists Wigglesworthia spp. and the commensal Sodalis glossinidius, along with the parasitic Alphaproteobacteria Wolbachia. These associations differ in their establishment times, making them unique and distinct from previously characterized symbioses, where multiple microbial partners have associated with their host for a significant portion of its evolution. We then expand into discussing the functional roles and intracommunity dynamics within this holobiont, which enhances our understanding of tsetse biology to encompass the vital functions and interactions of the microbial community. Potential disturbances influencing the tsetse microbiome, including salivary gland hypertrophy virus and trypanosome infections, are highlighted. While previous studies have described evolutionary consequences of host association for symbionts, the initial steps facilitating their incorporation into a holobiont and integration of partner biology have only begun to be explored. Research on the tsetse holobiont will contribute to the understanding of how microbial metabolic integration and interdependency initially may develop within hosts, elucidating mechanisms driving adaptations leading to cooperation and coresidence within the microbial community. Lastly, increased knowledge of the tsetse holobiont may also contribute to generating novel African trypanosomiasis disease control strategies.

The Tsetse Fly Obligate Mutualist Wigglesworthia morsitans Alters Gene Expression and Population Density via Exogenous Nutrient Provisioning

ABSTRACT The obligate mutualist Wigglesworthia morsitans provisions nutrients to tsetse flies. The symbionts response to thiamine (B1) supplementation of blood meals, specifically towards the regulation of thiamine biosynthesis and population density, is described. Despite an ancient symbiosis and associated genome tailoring, Wigglesworthia responds to nutrient availability, potentially accommodating a decreased need.

“Wigglesworthia morsitans” Folate (Vitamin B9) Biosynthesis Contributes to Tsetse Host Fitness

ABSTRACT Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between “Wigglesworthia morsitans” (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B9) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control.

Aspergillus Collagen-Like Genes (acl): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

ABSTRACT The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects.

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates.

Use of the Internal Transcribed Spacer (ITS) Regions to Examine Symbiont Divergence and as a Diagnostic Tool for Sodalis-Related Bacteria

Bacteria excel in most ecological niches, including insect symbioses. A cluster of bacterial symbionts, established within a broad range of insects, share high 16S rRNA similarities with the secondary symbiont of the tsetse fly (Diptera: Glossinidae), Sodalis glossinidius. Although 16S rRNA has proven informative towards characterization of this clade, the gene is insufficient for examining recent divergence due to selective constraints. Here, we assess the application of the internal transcribed spacer (ITS) regions, specifically the ITSglu and ITSala,ile, used in conjunction with 16S rRNA to enhance the phylogenetic resolution of Sodalis-allied bacteria. The 16S rRNA + ITS regions of Sodalis and allied bacteria demonstrated significant divergence and were robust towards phylogenetic resolution. A monophyletic clade of Sodalis isolates from tsetse species, distinct from other Enterobacteriaceae, was consistently observed suggesting diversification due to host adaptation. In contrast, the phylogenetic distribution of symbionts isolated from hippoboscid flies and various Hemiptera and Coleoptera were intertwined suggesting either horizontal transfer or a recent establishment from an environmental source. Lineage splitting of Sodalis-allied bacteria into symbiotic and free-living sister groups was also observed. Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis-allied symbionts in the field. These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis. These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.