GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host

The GacS/GacA two-component system regulates the expression of bacterial traits during host association. Although the importance of GacS/GacA as a regulator of virulence is well established, its role in benign associations is not clear, as mutations in either the gacS or gacA gene have little impact on the success of colonization in nonpathogenic associations studied thus far. Using as a model the symbiotic association of the bioluminescent marine bacterium Vibrio fischeri with its animal host, the Hawaiian bobtail squid, Euprymna scolopes, we investigated the role of GacA in this beneficial animal-microbe interaction. When grown in culture, gacA mutants were defective in several traits important for symbiosis, including luminescence, growth in defined media, growth yield, siderophore activity, and motility. However, gacA mutants were not deficient in production of acylated homoserine lactone signals or catalase activity. The ability of the gacA mutants to initiate squid colonization was impaired but not abolished, and they reached lower-than-wild-type population densities within the host light organ. In contrast to their dark phenotype in culture, gacA mutants that reached population densities above the luminescence detection limit had normal levels of luminescence per bacterial cell in squid light organs, indicating that GacA is not required for light production within the host. The gacA mutants were impaired at competitive colonization and could only successfully cocolonize squid light organs when present in the seawater at higher inoculum densities than wild-type bacteria. Although severely impaired during colonization initiation, gacA mutants were not displaced by the wild-type strain in light organs that were colonized with both strains. This study establishes the role of GacA as a regulator of a beneficial animal-microbe association and indicates that GacA regulates utilization of growth substrates as well as other colonization traits.     

The GacA global regulator is required for the appropriate expression of Erwinia chrysanthemi 3937 pathogenicity genes during plant infection

Pathogenicity of the phytopathogenic enterobacterium Erwinia chrysanthemi , the causal agent of soft rot disease on many plants, is a complex process involving several factors whose production is regulated by a complex, intertwined regulatory network. In this work we characterized the GacA regulator, member of the GacS–GacA two-component system, as a global regulator which is required for disease expression but not for bacterial multiplication in planta during the first stages of the plant infection. GacA was shown to control the expression of plant cell wall-degrading enzymes and hrp genes in vitro . Analysis of virulence gene expression during infection of Arabidopsis thaliana revealed a coordinated expression of these virulence genes at 12 h post infection and showed that GacA is required for the appropriate production of virulence factors in planta . GacA might partly act by negatively controlling the expression of the pecT gene encoding the global repressor PecT, indicating a hierarchy in the pathways involved in the E. chrysanthemi regulatory network.     

Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.     

The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp‐encoded regulator SypA in Vibrio fischeri

Bacteria utilize multiple regulatory systems to modulate gene expression in response to environmental changes, including two‐component signalling systems and partner‐switching networks. We recently identified a novel regulatory protein, SypE, that combines features of both signalling systems. SypE contains a central response regulator receiver domain flanked by putative kinase and phosphatase effector domains with similarity to partner‐switching proteins. SypE was previously shown to exert dual control over biofilm formation through the opposing activities of its terminal effector domains. Here, we demonstrate that SypE controls biofilms in Vibrio fischeri by regulating the activity of SypA, a STAS (sulphate transporter and anti‐sigma antagonist) domain protein. Using biochemical and genetic approaches, we determined that SypE both phosphorylates and dephosphorylates SypA, and that phosphorylation inhibits SypA's activity. Furthermore, we found that biofilm formation and symbiotic colonization required active, unphosphorylated SypA, and thus SypA phosphorylation corresponded with a loss of biofilms and impaired host colonization. Finally, expression of a non‐phosphorylatable mutant of SypA suppressed both the biofilm and symbiosis defects of a constitutively inhibitory SypE mutant strain. This study demonstrates that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilm development and symbiotic colonization.     

Evidence that AphB, essential for the virulence of Vibrio vulnificus, is a global regulator

The Vibrio vulnificus aphB mutant was significantly less virulent than the wild type and was impaired in motility and adherence to host cells. Microarray analysis revealed that AphB of V. vulnificus (AphB(Vv)) influences the expression of over 10% of the V. vulnificus genome. The combined results indicated that AphB(Vv) is a global regulator contributing to the pathogenesis of V. vulnificus.     

Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.     

The N-acetyl-D-glucosamine repressor NagC of Vibrio fischeri facilitates colonization of Euprymna scolopes

To successfully colonize and persist within a host niche, bacteria must properly regulate their gene expression profiles. The marine bacterium Vibrio fischeri establishes a mutualistic symbiosis within the light organ of the Hawaiian squid, Euprymna scolopes. Here, we show that the repressor NagC of V. fischeri directly regulates several chitin- and N-acetyl-D-glucosamine-utilization genes that are co-regulated during productive symbiosis. We also demonstrate that repression by NagC is relieved in the presence of N-acetyl-D-glucosamine-6-phosphate, the intracellular form of N-acetyl-D-glucosamine. We find that gene repression by NagC is critical for efficient colonization of E. scolopes. Further, our study shows that NagC regulates genes that affect the normal dynamics of host colonization.     

The Euprymna scolopes-Vibrio fischeri symbiosis: a biomedical model for the study of bacterial colonization of animal tissue

The diversity of microorganisms found in the marine environment reflects the immense size, range of physical conditions and energy sources, and evolutionary age of the sea. Because associations with living animal tissue are an important and ancient part of the ecology of many microorganisms, it is not surprising that the study of marine symbioses (including both cooperative and pathogenic interactions) has produced numerous discoveries of biotechnological and biomedical significance. The association between the bioluminescent bacterium Vibrio fischeri and the sepiolid squid Euprymna scolopes has emerged as a productive model system for the investigation of the mechanisms by which cooperative bacteria initiate colonization of specific host tissues. The results of the last decade of research on this system have begun to reveal surprising similarities between this association and the pathogenic associations of disease-causing Vibrio species, including those of interest to human health and aquaculture. Studies of the biochemical and molecular events underlying the development of the squid-vibrio symbiosis can be expected to continue to increase our understanding of the factors controlling both benign and pathogenic bacterial associations.     

Vibrio fischeri lux genes play an important role in colonization and development of the host light organ

The bioluminescent bacterium Vibrio fischeri and juveniles of the squid Euprymna scolopes specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (lux) genes of symbiotic V. fischeri have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (luxA, luxI, and luxR), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these lux mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in trans, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of lux mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.     

The Slt2-type MAP kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth, conidiation, plant surface sensing and host tissue colonization

Mitogen-activated protein kinases (MAPKs) play important roles in signal transduction and regulation of various aspects of saprotrophic and pathogenic growth in plant pathogenic fungi. We have generated a Botrytis cinerea knock-out mutant in the bmp3 gene encoding a homologue of the yeast Slt2 cell wall integrity MAPK. The Δ bmp3 mutant showed reduced vegetative growth on various media, strongly impaired conidiation and loss of sclerotia formation. Growth retardation of the mutant was enhanced in media with low osmolarity, whereas nearly wild-type growth rates were observed under high osmolarity conditions. The Δ bmp3 mutant did not show increased susceptibility to cell wall damage induced by glucanase, Calcofluor White or Nikkomycin Z, but was more susceptible to the oxidizing agent paraquat and the phenylpyrrole fungicide fludioxonil. Δ bmp3 conidia showed normal germination and germ tube growth on agar, but excessive germ tube elongation on hard surfaces and reduced penetration efficiency, indicating a defect in surface sensing. After penetration, development of necrotic lesions induced by the Δ bmp3 mutant was retarded. All these defects were restored by genetic complementation of the mutant with the wild-type bmp3 gene.     

Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus-pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.     

The twin arginine translocation system contributes to symbiotic colonization of Euprymna scolopes by Vibrio fischeri

In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.