From the Cover: The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

Although parasitism and mutualism may have radically different implications for host fitness, endosymbiotic bacteria participating in these relationships are known to share many similarities, including an intracellular habitat (1). The exploitation of an intracellular habitat is thought to have been one of the most important events in bacterial evolution, permitting significant environmental niche expansion and defining the arrival of intracellular pathogens and mutualistic endosymbionts (2, 3). Although there is a good understanding of the mechanisms contributing to bacterial pathogenesis, very little is known about interactions between bacterial endosymbionts and their host cells. Theoretical studies assume that there may be a tradeoff between the effectiveness of horizontal and vertical modes of transmission (4, 5). It has been predicted that mutualists evolve from parasites through an evolutionary continuum in which parasite virulence is attenuated and transmission strategy switches from horizontal to vertical (6). According to this theory, we might expect to find that pathogens and mutualistic endosymbionts harbor similar virulence determinants and utilize the same machinery to facilitate invasion and survival in host cells. In the present study, we explore these issues by investigating genes that coordinate insect cell invasion in Sodalis glossinidius, an intracellular secondary endosymbiont of the tsetse fly (Glossina spp.). Three distinct endosymbiotic bacteria have been identified previously in the tissues of tsetse (7). Whereas one of these bacteria is known to be a parasitic Wolbachia, the remaining two are thought to be mutualists and have been classified as the primary and secondary endosymbionts of tsetse (named Wigglesworthia glossinidia and S. glossinidius, respectively) (8, 9). Sodalis is a bacterium found exclusively in tsetse flies residing both inter- and intracellularly in a number of different host tissues, including midgut, fat body, and hemolymph (9, 10). The symbiotic role of Sodalis remains unclear, because it has proved difficult to selectively eliminate either Sodalis or Wigglesworthia from tsetse without inducing sterility in the host. Phylogenetic reconstructions based on the 16S rDNA locus reveal that Sodalis is a member of the family Enterobacteriaceae, which is closely related to other intracellular secondary bacterial endosymbionts found in other insects such as the flour weevil Sitophilus zeamais and the aphid Acrythosiphon pisum (11–13). We are particularly interested in Sodalis as a study model because it is known that the association between this bacterium and tsetse has only recently been established. This association is evident from symbiont–host coevolution studies demonstrating the absence of phylogenetic congruence in the evolution of Sodalis and tsetse (11). Sodalis provides an excellent model for the study of host–symbiont interactions because of the availability of an in vitro Sodalis–insect cell coculture system (14). In addition, Sodalis is the only maternally transmitted insect endosymbiont to have been isolated and maintained in pure culture (9). In this study, we demonstrate the use of Tn5 transposon mutagenesis as a tool for generating random Sodalis mutants. With the use of an in vitro negative selection procedure, we have identified Sodalis mutants deficient in their ability to attach to and invade insect cells both in vitro and in vivo. Characterization of a noninvasive Tn5 Sodalis mutant has revealed that Sodalis relies on components of a type III secretion system to facilitate entry into insect cells.