Migration of <i>Paraburkholderia terrae</i> BS001 along old fungal hyphae in soil at various pH levels

The movement of bacterial cells along with fungal hyphae in soil (the mycosphere) has been reported in several previous studies. However, how local soil conditions affect bacterial migration direction in the mycosphere has not been extensively studied. Here, we investigated the influence of two soil parameters, pH and soil moisture content, on the migration, and survival, of Paraburkholderia terrae BS001 in the mycosphere of Lyophyllum sp. strain Karsten in microcosms containing a loamy sand soil. The data showed that bacterial movement along the hyphal networks took place in both the "forward" and the "backward" directions. Low soil pH strongly restricted bacterial survival, as well as dispersal in both directions, in the mycosphere. The backward movement was weakly correlated with the amount of fungal tissue formed in the old mycelial network. The initial soil moisture content, set at 12 versus 17% (corresponding to 42 and 60% of the soil water holding capacity), also significantly affected the bacterial dispersal along the fungal hyphae. Overall, the presence of fungal hyphae was found to increase the soil pH (under conditions of acidity), which possibly exerted protective effects on the bacterial cells. Finally, we provide a refined model that describes the bacterial migration patterns with fungal hyphae based on the new findings in this study

Chemotaxis and adherence to fungal surfaces are key components of the behavioral response of Burkholderia terrae BS001 to two selected soil fungi

Burkholderia terrae BS001 has previously been proposed to be a 'generalist' associate of soil fungi, but its strategies of interaction have been largely ignored. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-Delta sctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-Delta sctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-Delta sctD to Lyophyllum sp. strain Karsten and to cell envelope-extracted ceramide monohexosides (CMHs) revealed that CMHs in both conidia and hyphae could bind strain BS001 cells. As BS001-Delta sctD adhered significantly less to the CMHs than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such avid adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope, in particular of the former.</p

The role of hydrophobicity and surface receptors at hyphae of <i>Lyophyllum</i> sp. strain Karsten in the interaction with <i>Burkholderia terrae</i> BS001:Implications for interactions in soil

The soil bacterium Burkholderia terrae strain BS001 can interact with varying soil fungi, using mechanisms that range from the utilization of carbon/energy sources such as glycerol to the ability to reach novel territories in soil via co-migration with growing fungal mycelia. Here, we investigate the intrinsic properties of the B. terrae BS001 interaction with the basidiomycetous soil fungus Lyophyllum sp. strain Karsten. In some experiments, the ascomycetous Trichoderma asperellum 302 was also used. The hyphae of Lyophyllum sp. strain Karsten were largely hydrophilic on water-containing media versus hydrophobic when aerial, as evidenced by contact angle analyses (CA). Co-migration of B. terrae strain BS001 cells with the hyphae of the two fungi occurred preferentially along the - presumably hydrophilic - soil-dwelling hyphae, whereas aerial hyphae did not allow efficient migration, due to reduced thickness of their surrounding mucous films. Moreover, the cell numbers over the length of the hyphae in soil showed an uneven distribution, i.e. the CFU numbers increased from minima at the inoculation point to maximal numbers in the middle of the extended hyphae, then decreasing towards the terminal side. Microscopic analyses of the strain BS001 associations with the Lyophyllum sp. strain Karsten hyphae in the microcosms confirmed the presence of B. terrae BS001 cells on the mucous matter that was present at the hyphal surfaces of the fungi used. Cell agglomerates were found to accumulate at defined sites on the hyphal surfaces, which were coined ‘fungal-interactive’ hot spots. Evidence was further obtained for the contention that receptors for a physical bacterium-fungus interaction occur at the Lyophyllum sp. strain Karsten hyphal surface, in which the specific glycosphingolipid ceramide mono hexoside (CMH) plays an important role. Thus, bacterial adherence may be mediated by heterogeneously-distributed fungal-specific receptors, implying the CMH moieties. This study sheds light on the physical aspects of the B. terrae BS001 – L. sp strain Karsten interaction, highlighting heterogeneity along the hyphae with respect to hydrophobicity and the presence of potential anchoring sites

Glucosylceramides From Lomentospora prolificans Induce a Differential Production of Cytokines and Increases the Microbicidal Activity of Macrophages

Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions, such as involvement in fungal growth and morphological transitions in several fungi. The elucidation of the primary structure of the fungal surface glycoconjugates could contribute for the understanding of the mechanisms of pathogenicity. In this work, GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages. These results indicate that GlcCer species from L. prolificans are a potent immune response activator

Glucosylceramides from Cladosporium and Their Roles in Fungi&ndash;Plant Interaction

Cladosporium species are widely distributed filamentous fungi. One of the most important species is C. herbarum, which is related to infections in a variety of plants and of concern in plantations. Fungal cerebrosides, such as glucosylceramide (GlcCer), have been described as playing important roles in fungal growth and pathogenesis, but GlcCer from C. herbarum has not been characterized so far. For this reason, the present study aimed to elucidate the chemical structure of GlcCer from C. herbarum and its role in the interaction with Passiflora edulis. Mass spectrometry characterization of purified GlcCer revealed two major molecular ions, m/z 760 and m/z 774, and it reacts with monoclonal anti-GlcCer antibodies and is exposed on the fungal surface. P. edulis treatment with GlcCer induced increased levels of superoxide as well as the expression of some genes related to plant defense, such as PR3, POD, LOX and PAL. GlcCer also enhanced growth parameters, such as plant height and root weight. All these results suggest that C. herbarum GlcCer can stimulate plant defense mechanisms, which could help plants to face fungal infections

Glucosylceramides from <i>Cladosporium</i> and Their Roles in Fungi–Plant Interaction

Cladosporium species are widely distributed filamentous fungi. One of the most important species is C. herbarum, which is related to infections in a variety of plants and of concern in plantations. Fungal cerebrosides, such as glucosylceramide (GlcCer), have been described as playing important roles in fungal growth and pathogenesis, but GlcCer from C. herbarum has not been characterized so far. For this reason, the present study aimed to elucidate the chemical structure of GlcCer from C. herbarum and its role in the interaction with Passiflora edulis. Mass spectrometry characterization of purified GlcCer revealed two major molecular ions, m/z 760 and m/z 774, and it reacts with monoclonal anti-GlcCer antibodies and is exposed on the fungal surface. P. edulis treatment with GlcCer induced increased levels of superoxide as well as the expression of some genes related to plant defense, such as PR3, POD, LOX and PAL. GlcCer also enhanced growth parameters, such as plant height and root weight. All these results suggest that C. herbarum GlcCer can stimulate plant defense mechanisms, which could help plants to face fungal infections