Identification ofAeromonas veroniiGenes Required for Colonization of the Medicinal Leech,Hirudo verbana

Abstract

Most digestive tracts contain a complex consortium of beneficial microorganisms, making it challenging to tease apart the molecular interactions between symbiont and host. The digestive tract of Hirudo verbana, the medicinal leech, is an ideal model system because it harbors a simple microbial community in the crop, comprising the genetically amenable Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) was used to identify genes required for digestive tract colonization. Of 3,850 transposon (Tn) mutants screened, 46 were identified as colonization mutants. Previously we determined that the complement system of the ingested blood remained active inside the crop and prevented serum-sensitive mutants from colonizing. The identification of 26 serum-sensitive mutants indicated a successful screen. The remaining 20 serum-resistant mutants are described in this study and revealed new insights into symbiont-host interactions. An in vivo competition assay compared the colonization levels of the mutants to that of a wild-type competitor. Attenuated colonization mutants were grouped into five classes: surface modification, regulatory, nutritional, host interaction, and unknown function. One STM mutant, JG736, with a Tn insertion in lpp, encoding Braun's lipoprotein, was characterized in detail. This mutant had a >25,000-fold colonization defect relative to colonization by the wild-type strain at 72 h and, in vitro, an increased sensitivity to sodium dodecyl sulfate, suggesting the presence of an additional antimicrobial property in the crop. The classes of genes identified in this study are consistent with findings from previous STM studies involving pathogenic bacteria, suggesting parallel molecular requirements for beneficial and pathogenic host colonization.

High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

“…A total of 5,088 transposon mutants of WT CO92 were created, which included 96 mutants for each of the 53 unique 40-bp signature tags (34). As a source of the tags, 53 E. coli S-17 strains, each harboring the plasmid pUTminiTn5Km2STM with a unique tag, were used as donor strains and conjugated with WT CO92 (35). Initially, 56 tags were chosen, as previously described (35), and were tested for their cross-hybridization.…”

Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis

“…streptomycin versus rifampicin), and the corresponding bacterial loads were then determined. The competitive index (CI) was calculated as follows: CI5(mutant output/WT output)/(mutant input/WT input) (Silver et al, 2007).…”

The GenusAeromonas: Taxonomy, Pathogenicity, and Infection

“…on May 10, 2018 by guest http://cmr.asm.org/ presently unknown (266). Such a model offers exciting opportunities to potentially discover important genes and gene products critical to colonization of digestive tracts in susceptible hosts.…”

“…The microbiota of the leech's crop is normally populated by only two resident symbiotic microbial species, one of which (Aeromonas) can be grown in vitro and is amenable to genetic manipulation. Silver et al (266) have identified several classes of genes that play important roles in colonization of the leech digestive tract, including bacterial cell surface modifications, regulatory factors, nutritional elements (amino acid and phosphate transporters), and genes involved in type three secretion systems (TTSS). Mutants in specific genes, such as that encoding Braun's major outer membrane lipoprotein or a gene encoding a cytoplasmic membrane component of a TTSS, compete 10,000-to 25,000-fold less efficiently against wild-type or competitor strains in colonization of the leech's crop (265,266).…”

“…Silver et al (266) have identified several classes of genes that play important roles in colonization of the leech digestive tract, including bacterial cell surface modifications, regulatory factors, nutritional elements (amino acid and phosphate transporters), and genes involved in type three secretion systems (TTSS). Mutants in specific genes, such as that encoding Braun's major outer membrane lipoprotein or a gene encoding a cytoplasmic membrane component of a TTSS, compete 10,000-to 25,000-fold less efficiently against wild-type or competitor strains in colonization of the leech's crop (265,266). Even more interesting is the fact that Graf has identified seven or more genes in A. veronii that are colonization mutants and whose function is 56 JANDA AND ABBOTT CLIN.…”