OpnS,an Outer Membrane Porin of Xenorhabdus nematophila,Confers a Competitive Advantage for Growth in the Insect Host

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.The bacterium Xenorhabdus nematophila forms a mutualistic association with the entomopathogenic nematode Steinernema carpocapsae (2). The nonfeeding infective juvenile form of the nematode (IJ) exists in the soil and carries the bacteria in a specialized receptacle region in the anterior intestine (4, 39). The IJ invades susceptible insect species and enters the hemocoel, where exposure to insect hemolymph stimulates the movement of bacteria down the intestine and out of the anus (36, 39). Together, the nematode and bacteria kill the insect host. X. nematophila not only helps to kill the insect but also promotes bioconversion of host macromolecules and tissues to provide nutrients for nematode reproduction and secretes diverse antimicrobial products to suppress competition for the nutrient resources of the insect cadaver (11, 13, 18, 19, 38). In turn, the nematode vectors X. nematophila to new insect hosts and protects it from the competitive environment of the soil. Colonization of the nematode receptacle is predominantly a monoculture process that is initiated by a single cell followed by bacterial proliferation (24, 39). The level of colonization varies from a few cells to several hundreds per nematode and is higher in nematodes reproducing in insects than on bacterial lawns, suggesting that the insect environment provides additional nutrients for bacterial growth (16, 39).Hydrophilic nutrients and antibiotics passively diffuse across the outer membrane of gram-negative bacteria through general porins and substrate-specific channels (17, 29). The most extensively studied general porins, OmpF and OmpC of Escherichia coli (30), are 16-stranded β-barrel proteins that are reciprocally regulated by changes in external osmolarity (12, 21, 41). Although the flow rate through OmpF is greater than OmpC (28), comparison of the resolved crystal structures does not reveal significant physiochemical differences between the two porins (3). The biological significance of the differential regulation of porins with distinct functional properties remains unclear. The major outer membrane protein of X. nematophila, OpnP, was shown to be produced at high levels in exponentially growing cells and is a homologue of OmpF and OmpC (14). OpnP production was not affected by changes in medium osmolarity, and the flow rate measured for the OpnP porin was more similar to the restrictive porin OmpC than to the more permissive OmpF porin (3). As cells transitioned to stationary phase, de novo synthesis of OpnP decreased, while the synthesis of the outer membrane protein, designated OpnS, increased (15, 22).Porin function and regulation have been studied in both pathogenic and symbiotic bacteria. In Vibrio cholerae two well-studied porins, OmpU and OmpT, that possess distinct functional properties have been shown to be differentially regulated (37). OmpU confers resistance to sodium deoxycholate (DC), a major component of bile, as well as polymixin B, detergents, and antimicrobial peptides, while the expression of OmpT alone sensitizes the cell to DC (26, 33). OmpU was thought to be expressed when V. cholerae colonizes the intestine, suggesting that it was required for host colonization (33); however, subsequent findings indicated that neither OmpU nor OmpT were essential for intestinal colonization (34). Recent findings indicated that OmpU may sense membrane perturbations and activate DegS which in turn modulates σ^E activity (25, 26). In the symbiotic bacterium Vibrio fischeri the deletion of ompU was shown to reduce the efficiency of colonization of the light organ of the Euprymna scolopes squid and increase sensitivity to bile, antimicrobial peptides, and detergent (1). Interestingly, the ompU strain did not display a competitive defect for colonization in the presence of the wild-type strain.In the present study the growth-phase-regulated outer membrane protein OpnS of X. nematophila was identified as a general porin that conferred a competitive advantage for growth in the insect host. OpnP and OpnS were the only general porins identified in the genome of X. nematophila. The reciprocal expression of OpnP and OpnS suggest that they serve distinct biological roles.