Colin Dale

The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism-mutualism continuum.

Type III secretion systems and the evolution of mutualistic endosymbiosis

The view that parasites can develop cooperative symbiotic relationships with their hosts is both appealing and widely held; however, there is no molecular genetic evidence of such a transition. Here we demonstrate that a mutualistic bacterial endosymbiont of grain weevils maintains and expresses inv/spa genes encoding a type III secretion system homologous to that used for invasion by bacterial pathogens. Phylogenetic analyses indicate that inv/spa genes were present in presymbiotic ancestors of the weevil endosymbionts, occurring at least 50 million years ago. The function of inv/spa genes in maintaining symbiosis is demonstrated by the up-regulation of their expression under both in vivo and in vitro conditions that coincide with host cell invasion.

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.

Low and homogeneous copy number of plasmid‐borne symbiont genes affecting host nutrition in Buchnera aphidicola of the aphid Uroleucon ambrosiae

The bacterial endosymbiont of aphids, Buchnera aphidicola, often provides amino acids to its hosts. Plasmid amplification of leucine (leuABCD) and tryptophan (trpEG) biosynthesis genes may be a mechanism by which some Buchnera over‐produce these nutrients. We used quantitative polymerase chain reaction to assess the leuABCD/trpEG copy variability within Uroleucon ambrosiae, an aphid with a wide diet breadth and range. Both leuABCD and trpEG abundances are: (i) similar for aphids across 15 populations, and (ii) low compared to Buchnera from other aphid species (particularly trpEG). Consequently, the plasmid location of trpEG combined with Buchneras chromosomal polyploidy may functionally limit, rather than increase, tryptophan production within Uroleucon ambrosiae.

Isolation and culture of tsetse secondary endosymbionts

Insect symbionts have attracted attention recently for their potential use as a vehicle for the expression of anti-parasitic gene products in arthropod disease vectors (Beard et al., 1993; Aldhous, 1994). Much research has focused on a bacterial endosymbiont found in the haemolymph and various tissues of the tsetse fly (Glossina spp.). This symbiont, referred to as ‘rickettsia-like-organisms’ (RLO) or ‘secondary symbionts’ are present in some tsetse with two other bacterial endosymbionts: the mycetome bacteroids and Wolbachia spp. The giant bacteroids of tsetse are found exclusively within the midgut mycetome and are believed to provide essential vitamins required to supplement the fly’s haematophagous diet (Nogge, 1978). Wolbachia spp., found only in ovarian tissues, have been implicated as agents of ‘cytoplasmic incompatibility’ in many insects including tsetse and it has been suggested that this phenomenon could be utilized to drive engineered symbionts through wild fly populations (Aldhous, 1994).

Physical Analysis of Chromosome Size Variation

Early characterization of genetic material from a wide range of organisms involved the determination of base composition and genome size. Aside from the intrinsic value of such information, these properties were studied because they could be obtained for the large number of samples where cytogenetic and transmission genetic analysis was onerous or obscure. As it turned out, these general features divulged some of the most fundamental aspects of gene and genome organization and evolution. The base compositional differences among bacteria led to theories about mutational processes that foreshadowed the neutral theory of molecular evolution [1–3] and, among eukaryotes, to the discovery of the isochore structuring within chromosomes [4]. With respect to genome-size variation, the results were equally consequential. Across life forms, there seemed to be little relationship between the amount of genetic material and the degree of organismal complexity (the so-called “C-value paradox”), which has led to inquiries about the amounts, the accumulation, and the function of non-coding DNA in genomes [5–9]. Within bacteria, genomesize would appear to have direct consequences on the biology of an organism: because of the high coding content of bacterial DNA, variation in genome-size implies differences in the absolute number of genes.