The endosymbionts of tsetse flies: manipulating host-parasite interactions

Through understanding the mechanisms by which tsetse endosymbionts potentiate trypanosome susceptibility in tsetse, it may be possible to engineer modified endosymbionts which, when introduced into tsetse, render these insects incapable of transmitting parasites. In this study we have assayed the effect of three different antibiotics on the endosymbiotic microflora of tsetse (Glossina morsitans morsitans). We showed that the broad-spectrum antibiotics, ampicillin and tetracycline, have a dramatic impact on tsetse fecundity and pupal emergence, effectively rendering these insects sterile. This results from the loss of the tsetse primary endosymbiont, Wigglesworthia glossinidia, which is eradicated by ampicillin and tetracycline treatment. Using the sugar analogue and antibiotic, streptozotocin, we demonstrated specific elimination of the tsetse secondary endosymbiont, Sodalis glossinidius, with no observed detrimental effect upon W. glossinidia. The specific eradication of S. glossinidius had a negligible effect upon the reproductive capability of tsetse but did effect a significant reduction in fly longevity. Furthermore, elimination of S. glossinidius resulted in increased refractoriness to trypanosome infection in tsetse, providing further evidence that S. glossinidius plays an important role in potentiating trypanosome susceptibility in this important disease vector. In the light of these findings, we highlight progress made towards developing recombinant Sodalis strains engineered to avoid potentiating trypanosome susceptibility in tsetse. In particular, we focus on the chitinase/N-acetyl-D-glucosamine catabolic machinery of Sodalis which has previously been implicated in causing immune inhibition in tsetse.     

Attenuation of the sensing capabilities of PhoQ in transition to obligate insect-bacterial association

Sodalis glossinidius, a maternally inherited endosymbiont of the tsetse fly, maintains genes encoding homologues of the PhoP-PhoQ two-component regulatory system. This two-component system has been extensively studied in facultative bacterial pathogens and is known to serve as an environmental magnesium sensor and a regulator of key virulence determinants. In the current study, we show that the inactivation of the response regulator, phoP, renders S. glossinidius sensitive to insect derived cationic antimicrobial peptides (AMPs). The resulting mutant strain displays reduced expression of genes involved in the structural modification of lipid A that facilitates resistance to AMPs. In addition, the inactivation of phoP alters the expression of type-III secretion system (TTSS) genes encoded within three distinct chromosomal regions, indicating that PhoP-PhoQ also serves as a master regulator of TTSS gene expression. In the absence of phoP, S. glossinidius is unable to superinfect either its natural tsetse fly host or a closely related hippoboscid louse fly. Furthermore, we show that the S. glossinidius PhoQ sensor kinase has undergone functional adaptations that result in a substantially diminished ability to sense ancestral signals. The loss of PhoQ's sensory capability is predicted to represent a novel adaptation to the static symbiotic lifestyle, allowing S. glossinidius to constitutively express genes that facilitate resistance to host derived AMPs.     

The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia

Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin. Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia. To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. Peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W. glossinidia 60 kDa chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W. glossinidia. Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.     

Candidatus Sodalis melophagi sp. nov.: phylogenetically independent comparative model to the tsetse fly symbiont Sodalis glossinidius

Bacteria of the genus Sodalis live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies Sodalis glossinidius, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new Sodalis species which could provide a useful comparative model to the tsetse symbiont. It lives in association with Melophagus ovinus, an insect related to tsetse flies, and resembles S. glossinidius in several important traits. Similar to S. glossinidius, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus Arsenophonus. As a typical secondary symbiont, Candidatus Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, groEL and spaPQR region) we show that the bacterium branched within the genus Sodalis, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name Candidatus Sodalis melophagi for this new bacterium.     

A new Sodalis lineage from bloodsucking fly Craterina melbae (Diptera, Hippoboscoidea) originated independently of the tsetse flies symbiont Sodalis glossinidius

Symbiotic bacterium closely related to the secondary symbiont of tsetse flies, Sodalis glossinidius, has been described from the bloodsucking fly Craterina melbae. Phylogenetic analysis of two genes, 16S rRNA gene and component of type three secretion system, placed the bacterium closer to the Sitophilus-derived branch of Sodalis than to the tsetse symbionts. This indicates that the Craterina-derived lineage of Sodalis originated independent of the tsetse flies symbionts and documents the capability of Sodalis bacteria either to switch between different host groups or to establish the symbiosis by several independent events.     

Functional analysis of the twin-arginine translocation pathway in Sodalis glossinidius, a bacterial symbiont of the tsetse fly

This study demonstrates a functional twin-arginine (Tat) translocation pathway present in the tsetse fly symbiont Sodalis glossinidius and its potential to export active heterologous proteins to the periplasm. Functionality was demonstrated using green fluorescent protein (GFP) fused to the Tat signal peptide of Escherichia coli trimethylamine N-oxide reductase (TorA).     

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly (Glossina) after bacterial challenge

Proteins containing a glutamic acid-proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% (G. m. morsitans) and 46% (G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

Interactions among multiple genomes: tsetse, its symbionts and trypanosomes

Insect-borne diseases exact a high public health burden and have a devastating impact on livestock and agriculture. To date, control has proved to be exceedingly difficult. One such disease that has plagued sub-Saharan Africa is caused by the protozoan African trypanosomes (Trypanosoma species) and transmitted by tsetse flies (Diptera: Glossinidae). This presentation describes the biology of the tsetse fly and its interactions with trypanosomes as well as its symbionts. Tsetse can harbor up to three distinct microbial symbionts, including two enterics (Wigglesworthia glossinidia and Sodalis glossinidius) as well as facultative Wolbachia infections, which influence host physiology. Recent investigations into the genome of the obligate symbiont Wigglesworthia have revealed characteristics indicative of its long co-evolutionary history with the tsetse host species. Comparative analysis of the commensal-like Sodalis with free-living enterics provides examples of adaptations to the host environment (physiology and ecology), reflecting genomic tailoring events during the process of transitioning into a symbiotic lifestyle. From an applied perspective, the extensive knowledge accumulated on the genomic and developmental biology of the symbionts coupled with our ability to both express foreign genes in these microbes in vitro and repopulate tsetse midguts with these engineered microbes now provides a means to interfere with the host physiological traits which contribute to vector competence promising a novel tool for disease management.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

The stability of plasmid pSG 1912 inheritance by the cells of Streptomyces globisporus 1912 and of heterologous streptomycete strains]

Streptomyces globisporus 1912 strain contains plasmid pSG1912 (11, 2kb) determining some phenotypic properties. The plasmid is able to exist and to provide for exhibition of these properties both in autonomous and integrated state. It is established that plasmid pSG1912 and constructed derivatives are stably inherited by cells of S. globisporus 1912 and heterologous hosts, and besides, molecular size and determined properties being retained.     

Epidemiological characterization of Streptococcus pyogenes isolated from patients with multiple onsets of pharyngitis

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.     

An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans

Tsetse flies (Diptera:Glossinidae) are vectors of African trypanosomes, the protozoan agents of devastating diseases in humans and animals. Prior studies in trypanosome infected Glossina morsitans morsitans have shown induced expression and synthesis of several antimicrobial peptides in fat body tissue. Here, we have expressed one of these peptides, Attacin (GmAttA1) in Drosophila (S2) cells in vitro. We show that the purified recombinant protein (recGmAttA1) has strong antimicrobial activity against Escherichia coli-K12, but not against the enteric gram-negative symbiont of tsetse, Sodalis glossinidius. The recGmAttA1 also demonstrated inhibitory effects against both the mammalian bloodstream form and the insect stage Trypanosoma brucei in vitro (minimal inhibitory concentration MIC50 0.075 microM). When blood meals were supplemented with purified recGmAttA1 during the course of parasite infection, the prevalence of trypanosome infections in tsetse midgut was significantly reduced. Feeding fertile females GmAttA1 did not affect the fecundity or the longevity of mothers, nor did it affect the hatchability of their offspring. We discuss a paratransgenic strategy, which involves the expression of trypanocidal molecules such as recGmAttA1 in the midgut symbiont Sodalis in vivo to reduce trypanosome transmission.     

A novel phospholipase from Trypanosoma brucei

Phospholipase A(1) activities have been detected in most cells where they have been sought and yet their characterization lags far behind that of the phospholipases A(2), C and D. The study presented here details the first cloning and characterization of a cytosolic PLA(1) that exhibits preference for phosphatidylcholine (GPCho) substrates. Trypanosoma brucei phospholipase A(1) (TbPLA(1)) is unique from previously identified eukaryotic PLA(1) because it is evolutionarily related to bacterial secreted PLA(1). A T. brucei ancestor most likely acquired the PLA(1) from a horizontal gene transfer of a PLA(1) from Sodalis glossinidius, a bacterial endosymbiont of tsetse flies. Nano-electrospray ionization tandem mass spectrometry analysis of TbPLA(1) mutants established that the enzyme functions in vivo to synthesize lysoGPCho metabolites containing long-chain mostly polyunsaturated and highly unsaturated fatty acids. Analysis of purified mutated recombinant forms of TbPLA(1) revealed that this enzyme is a serine hydrolase whose catalytic mechanism involves a triad consisting of the amino acid residues Ser-131, His-234 and Asp-183. The TbPLA(1) homozygous null mutants generated here constitute the only PLA(1) double knockouts from any organism.     

Bacterial endosymbiont of the slender pigeon louse, Columbicola columbae, allied to endosymbionts of grain weevils and tsetse flies

The current study focuses on a symbiotic bacterium found in the slender pigeon louse, Columbicola columbae (Insecta: Phthiraptera). Molecular phylogenetic analyses indicated that the symbiont belongs to the gamma subdivision of the class Proteobacteria and is allied to Sodalis glossinidius, the secondary symbiont of tsetse flies (Glossina spp.) and also to the primary symbiont of grain weevils (Sitophilus spp.). Relative-rate tests revealed that the symbiont of C. columbae exhibits accelerated molecular evolution in comparison with the tsetse fly symbiont and the weevil symbiont. Whole-mount in situ hybridization was used to localize the symbiont and determine infection dynamics during host development. In first- and second-instar nymphs, the symbionts were localized in the cytoplasm of oval bacteriocytes that formed small aggregates on both sides of the body cavity. In third-instar nymphs, the bacteriocytes migrated to the central body and were finally located in the anterior region of the lateral oviducts, forming conspicuous tissue formations called ovarial ampullae. In adult females, the symbionts were transmitted from the ovarial ampullae to developing oocytes in the ovarioles. In adult males, the bacteriocytes often disappeared without migration. A diagnostic PCR survey of insects collected from Japan, the United States, Australia, and Argentina detected 96.5% (109/113) infection, with a few uninfected male insects. This study provides the first microbial characterization of a bacteriocyte-associated symbiont from a chewing louse. Possible biological roles of the symbiont are discussed in relation to the host nutritional physiology associated with the feather-feeding lifestyle.     

Vector competence of Glossina palpalis gambiensis for Trypanosoma brucei s.l. and genetic diversity of the symbiont Sodalis glossinidius

Tsetse flies transmit African trypanosomes, responsible for sleeping sickness in humans and nagana in animals. This disease affects many people with considerable impact on public health and economy in sub-Saharan Africa, whereas trypanosomes' resistance to drugs is rising. The symbiont Sodalis glossinidius is considered to play a role in the ability of the fly to acquire trypanosomes. Different species of Glossina were shown to harbor genetically distinct populations of S. glossinidius. We therefore investigated whether vector competence for a given trypanosome species could be linked to the presence of specific genotypes of S. glossinidius. Glossina palpalis gambiensis individuals were fed on blood infected either with Trypanosoma brucei gambiense or Trypanosoma brucei brucei. The genetic diversity of S. glossinidius strains isolated from infected and noninfected dissected flies was investigated using amplified fragment length polymorphism markers. Correspondence between occurrence of these markers and parasite establishment was analyzed using multivariate analysis. Sodalis glossinidius strains isolated from T. brucei gambiense-infected flies clustered differently than that isolated from T. brucei brucei-infected individuals. The ability of T. brucei gambiense and T. brucei brucei to establish in G. palpalis gambiensis insect midgut is statistically linked to the presence of specific genotypes of S. glossinidius. This could explain variations in Glossina vector competence in the wild. Then, assessment of the prevalence of specific S. glossinidius genotypes could lead to novel risk management strategies.     

Comparative genomics of insect-symbiotic bacteria: influence of host environment on microbial genome composition

Commensal symbionts, thought to be intermediary amid obligate mutualists and facultative parasites, offer insight into forces driving the evolutionary transition into mutualism. Using macroarrays developed for a close relative, Escherichia coli, we utilized a heterologous array hybridization approach to infer the genomic compositions of a clade of bacteria that have recently established symbiotic associations: Sodalis glossinidius with the tsetse fly (Diptera, Glossina spp.) and Sitophilus oryzae primary endosymbiont (SOPE) with the rice weevil (Coleoptera, Sitophilus oryzae). Functional biologies within their hosts currently reflect different forms of symbiotic associations. Their hosts, members of distant insect taxa, occupy distinct ecological niches and have evolved to survive on restricted diets of blood for tsetse and cereal for the rice weevil. Comparison of genome contents between the two microbes indicates statistically significant differences in the retention of genes involved in carbon compound catabolism, energy metabolism, fatty acid metabolism, and transport. The greatest reductions have occurred in carbon catabolism, membrane proteins, and cell structure-related genes for Sodalis and in genes involved in cellular processes (i.e., adaptations towards cellular conditions) for SOPE. Modifications in metabolic pathways, in the form of functional losses complementing particularities in host physiology and ecology, may have occurred upon initial entry from a free-living to a symbiotic state. It is possible that these adaptations, streamlining genomes, act to make a free-living state no longer feasible for the harnessed microbe.     

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.