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

Abstract

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.

Figure 1

Time course of infection. A. albopictus C6/36 cells were infected with wild-type Sodalis glossinidius strain M1 and examined at intervals by Gimenez staining and light microscopy. (A–E) 8, 24, 48, 72, and 96 h after infection, respectively.

Figure 2

Organization of the inv/spa genes of Sodalis glossinidius and selected enteric pathogens. Homologous genes are decorated with the same color. Arrows indicate the direction of transcription of the inv/spa genes. The Yersinia pestis genes without color have no defined homologues in the other bacterial species. Solid lines above the gene organization represent the PCR products amplified in this study with the primers listed in Table 1. Gene designations are those published by Hueck (31).

Figure 3

Southern hybridization of S. glossinidius strain M1 genomic DNA with a Sodalis invC probe generated by PCR. (A) Genomic DNA digested with BamHI/XhoI (lane 1) and EcoRI/ClaI (lane 2) separated on a 0.7% agarose gel. (B) Southern blot of A probed with the 500-bp Sodalis invC PCR product. Molecular size markers are indicated (Left) .

Figure 4

Phylogenetic relationships between Inv/Spa proteins from selected enteric pathogens as revealed by phylogenetic reconstruction from protein distance matrices calculated with protdist in the phylip package. Only clades supported by over 70% of bootstrap resamples are shown as resolved. Sodalis Inv/Spa proteins consistently group with Salmonella and Shigella according to all four gene trees.