Identification of Aeromonas veronii genes 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.

FIG. 1.

(A) lpp locus. The region used for complementation (black line) encompasses lpp as well as 92 bp downstream and 89 bp upstream of lpp. The transposon insertion site is indicated by the lollipop. (B) Amino acid alignment of Lpp proteins from A. veronii, A. salmonicida, A. hydrophila, Shewanella amazonensis, E. coli, and S. enterica.

FIG. 2.

Complementation of JG736. The observed phenotype was linked to the disruption of lpp by complementing the JG736 colonization defect. The parent strain, HM21R, and JG736, each carrying either the broad-host-range vector pMMB207 or pAS11 (pMMB207 containing the complementing region) were tested against the competitor strain, HM21RS, and assessed at 72 h. The CI [calculated as (mutant_output/competitor_output)/(mutant_input/competitor_input)] for each animal is shown. Horizontal lines represent median CI values. Asterisks indicate a statistically significant difference between data sets (P < 0.05 by the Mann-Whitney test); ns, not significantly different.

FIG. 3.

Decreased ability of the lpp mutant, JG736, to colonize the leech. JG736 was coinoculated with the competitor strain in a 1:1 ratio. The CI [(mutant_output/competitor_output)/(mutant_input/competitor_input)] was calculated for each animal and plotted over time. A CI of 1 (dashed line) indicates that the mutant and competitor strain colonize to equal levels. A CI below 1 indicates that the mutant is outcompeted and has a colonization defect. The decrease in the CI is statistically significant from 15 h onward (P < 0.05 by a single-sided, two-tailed t test).