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.
"Within ILF of the crop, the digestive-tract symbionts reside and proliferate. The symbionts require many molecular tools for successful colonization and persistence within this environment (Graf, 2006; Silver et al., 2007a; Nelson et al., 2012). These molecular tools can be critical during the first 24 h of incubation, especially the ability of A. veronii to lyse erythrocytes (Maltz and Graf, 2011). "
[Show abstract][Hide abstract] ABSTRACT: It is known that many pathogens produce high-affinity iron uptake systems like siderophores and/or genes for utilizing iron bound to heme-containing molecules, which facilitate iron-acquisition inside a host. In mutualistic digestive-tract associations, iron uptake systems have not been as well studied. We investigated the importance of two iron utilization systems within the beneficial digestive-tract association Aeromonas veronii and the medicinal leech, Hirudo verbana. Siderophores were detected in A. veronii using chrome azurol S. Using a mTn5, a transposon insertion in viuB generated a mutant unable to acquire iron using siderophores. The A. veronii genome was then searched for genes potentially involved in iron utilization bound to heme-containing molecules. A putative outer membrane heme receptor (hgpB) was identified with a transcriptional activator, termed hgpR, downstream. The hgpB gene was interrupted in both the parent strain and the viuB mutant with an antibiotic resistance cassette, yielding a hgpB mutant and a mutant with both iron uptake systems inactivated. In vitro assays indicated that hgpB is involved in utilizing iron bound to heme and that both iron utilization systems are important for A. veronii to grow in blood. In vivo colonization assays revealed that the ability to acquire iron from heme-containing molecules is critical for A.veronii to colonize the leech gut. Since iron and specifically heme utilization is important in this mutualistic relationship and has a role as a possible virulence factor in other organisms, genomes from different Aeromonas strains (both clinical and environmental) were queried with iron utilization genes of A. veronii. This analysis revealed the heme utilization genes are widely distributed among aeromonads. In addition, aeromonads posses a suite of genes involved in iron acquisition. These data further confirm symbiotic and pathogenic relationships possess similar mechanisms for interacting with animal hosts.
Frontiers in Microbiology 08/2015; 6:763. DOI:10.3389/fmicb.2015.00763 · 3.99 Impact Factor
"Moreover, an A. veronii T3SS mutant analysed by Silver et al. (2007) was found to be targeted by hemocytes (macrophage-like cells of invertebrates) and phagocytosed, whereas the wild type was able to evade this host immune response. The same study also demonstrated that the A. veronii mutant was less virulent in a mouse septicaemia model, signifying that secreted factors of the T3SS have a role in aeromonad colonisation in each instance, even though the relationship with each host is drastically different (Silver et al., 2007). "
"is may not be a universal trend among symbioses . For example , in the leech crop symbiosis , co - competition experiments between the wild - type symbiont and nonsym biont species ( or mutants ) of Aeromonas show that non native ( or mutant ) species are always present in appreciable numbers in an individual crop along with wild - type bacteria ( Silver et al . , 2007a ) . This suggests that colonization of an individual crop is not limited to one or a few symbionts ( Laufer et al . , 2008 ) . However , the number of individual bacterial cells that initiate colonization or form microcolo nies has not been investigated directly ."
[Show abstract][Hide abstract] ABSTRACT: Mutually beneficial interactions between microorganisms and animals are a conserved and ubiquitous feature of biotic systems. In many instances animals, including humans, are dependent on their microbial associates for nutrition, defense, or development. To maintain these vital relationships, animals have evolved processes that ensure faithful transmission of specific microbial symbionts between generations. Elucidating mechanisms of transmission and symbiont specificity has been aided by the study of experimentally tractable invertebrate animals with diverse and highly evolved associations with microorganisms. Here, we review several invertebrate model systems that contribute to our current understanding of symbiont transmission, recognition, and specificity. Although the details of transmission and symbiont selection vary among associations, comparisons of diverse mutualistic associations are revealing a number of common themes, including restriction of symbiont diversity during transmission and glycan-lectin interactions during partner selection and recruitment.
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