Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization

9siIn host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.openopenSegond D; Abi Khalil E; Buisson C; Daou N; Kallassy M; Lereclus D; Arosio P; Bou-Abdallah F; Nielsen Le Roux C.Segond, D; Abi Khalil, E; Buisson, C; Daou, N; Kallassy, M; Lereclus, D; Arosio, Paolo; Bou Abdallah, F; Nielsen Le Roux, C

Artisanal and farmer bread making practices differently shape fungal species community composition in French sourdoughs

Preserving microbial diversity in food systems is one of the many challenges to be met to achieve food security and quality. Although industrialization led to the selection and spread of specific fermenting microbial strains, there are still ongoing artisanal processes that may allow the conservation of a wider species diversity and genetic diversity. We examined whether the diversity of artisanal practices could lead to an increased level in fungal species diversity for bread making. We used an interdisciplinary participatory research approach including bakers, psycho-sociologists and microbiologists to analyze French bread making practices and describe fungal communities in naturally fermented sourdough of 27 bakers and 12 farmer bakers. Bread making practices were classified in two groups: the farmer-like practice group and the artisanal-like practice group. The well-known bakery yeast, Saccharomyces cerevisiae, was dominant (i.e. with a relative abundance over 50%) in only 24% of sourdoughs while other yeast species, belonging to the Kazachstania genus, were dominant in 54% of sourdoughs. Bread making practices were found to drive the distribution of fungal species across sourdoughs. The most striking bread making practice effect was the occurrence of Kazachstania humilis in sourdoughs made with artisanal-like practices and the occurrence of Kazachstania bulderi in sourdoughs made with farmer-like practices. Phenotypic divergences between sourdough and non-sourdough strains were found for K. humilis but not for K. bulderi. Overall, our results showed that preserving bread making practice diversity allows the preservation of a higher species and phenotypic diversity in microbial communities

Contrôle de l'homéostasie du fer chez Arabidopsis thaliana en réponse à l'infection par la bactérie phytopathogène Erwinia chrysanthemi

Le fer est un élément essentiel pour quasiment tous les organismes vivants. Dans les interactions animaux-microorganismes, il est au cœur d une rude compétition. Les animaux ont ainsi acquis la capacité de mobiliser leur fer en le rendant moins disponible aux agents pathogènes. Afin d étudier l homéostasie du fer chez les plantes au cours d une infection, nous avons utilisé le pathosystème Erwinia chrysanthemi/Arabidopsis thaliana comme modèle de choix. E. chrysanthemi est une entérobactérie responsable de pourritures molles sur un large spectre d hôtes et qui requiert ses systèmes d acquisition du fer à haute affinité utilisant les sidérophores pour progresser dans la plante. La régulation de plusieurs acteurs de l homéostasie du fer et leur implication dans la résistance à l infection ont été étudiées. Nous avons montré que la ferritine AtFER1, une protéine impliquée dans le stockage du fer, et les transporteurs de métaux AtNRAMP3 et AtNRAMP4, qui participent à l homéostasie intracellulaire du fer, contribuent à la résistance de la plante à l infection par E. chrysanthemi. De plus, l expression des gènes AtFER1 et AtNRAMP3 est induite dans les feuilles infectées. Au niveau racinaire, une induction coordonnée de l expression d AtNRAMP3 et des systèmes d acquisition du fer a lieu au cours de l infection par E. chrysanthemi. Ces inductions impliquent notamment les sidérophores bactériens. Enfin, nous avons établi que la disponibilité du fer dans les tissus végétaux influe sur le développement de la maladie causée par E. chrysanthemi. L ensemble de nos données illustre le lien étroit qui existe chez A. thaliana entre l homéostasie du fer et la résistance à l infection.Iron is an essential nutritional resource for most forms of life. In animal-pathogen interactions, a rough competition for this metal takes place. Animals have acquired the ability to mobilize their iron, rendering it less available to pathogens. We used the Erwinia chrysanthemi/ Arabidopsis thaliana as a model to study iron homeostasis during infection. Indeed, E. chrysanthemi causes systemic rotting symptoms on a wide host range and requires its high affinity iron uptake system based on siderophores to progress in plant tissue. The regulation of several actors of iron homeostasis and their involvement in plant resistance to infection have been studied. We showed that ferritin AtFER1, a protein involved in iron storage, and the metal transporters AtNRAMP3 and AtNRAMP4 which participate to intracellular iron homeostasis, contribute to plant resistance to E. chrysanthemi infection. Moreover, AtFER1 and AtNRAMP3 gene expression is upregulated in infected leaves and the bacterial siderophores contribute to this upregulation. In roots, E. chrysanthemi infection triggers coordinate upregulation of AtNRAMP3 and several iron deficiency genes. This finding suggests the existence of a systemic signal reminiscent of an iron deficiency signal activated by pathogen infection and involving notably the siderophores. Finally, we showed that iron availability in plant tissues affects the development of the disease caused by E. chrysanthemi. Altogether, our data confirm the tight connection between iron homeostasis and resistance to pathogen attack in A. thaliana.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Sourdough yeast-bacteria interactions can change ferulic acid metabolism during fermentation

International audienceThe metabolism of ferulic acid (FA) was studied during fermentation with different species and strains of lactic acid bacteria (LAB) and yeasts, in synthetic sourdough medium. Yeast strains of Kazachstania humilis, Kazachstania bulderi, and Saccharomyces cerevisiae, as well as lactic acid bacteria strains of Fructilactobacillus sanfranciscensis, Lactiplantibacillus plantarum, Lactiplantibacillus xiangfangensis, Levilactobacillus hammesii, Latilactobacillus curvatus and Latilactobacillus sakei were selected from French natural sourdoughs. Fermentation in presence or absence of FA was carried out in LAB and yeasts monocultures, as well as in LAB/yeast co-cultures. Our results indicated that FA was mainly metabolized into 4-vinylguaiacol (4-VG) by S. cerevisiae strains, and into dihydroferulic acid (DHFA) and 4-VG in the case of LAB. Interactions of LAB and yeasts led to the modification of FA metabolism, with a major formation of DHFA, even by the strains that do not produce it in monoculture. Interestingly, FA was almost completely consumed by the F. sanfranciscensis bFs17 and K. humilis yKh17 pair and converted into DHFA in 89.5 ± 19.6% yield, while neither bFs17, nor yKh17 strains assimilated FA in monocultur

Ferulic acid content variation from wheat to bread

International audienceWheat is the most important food grain source for humans with an estimated global production of around 747.8 million tons and a consumption that reached 753.9 million tons for the year 2018/2019 (United States Department of Agriculture). This increasing consumption is partly correlated with the nutritional value of the wheat grain which mainly contains carbohydrates (65%-75%), proteins (7%-12%), and lipids (2%-6%) (Pomeranz, 1988). The wheat grain is composed of different parts, of which the bran accounts for 14%-19% of its weight (Javed et al., 2012). Studies on the effect of wheat, oat, and corn brans on human organism showed that wheat bran contains most of the micronutrients and bioactive compounds of the grain (Hemery et al., 2007), and therefore, it might contribute to reduce certain diseases of the digestive system. Wheat bran comprises the outer tissues of the wheat kernel and includes histologically and chemically distinct layers and tissues, whose aleurone layer accounts for 50% of all bran tissue. Aleurone cell walls contain 29% B-glucans and 65% linear arabinoxylans (Javed et al., 2012; Shewry & Hey, 2015). They are a source of vitamins and bioactive compounds such as phenolic compounds (bound phenolic compounds: 4.73-2020 µg/g) (Onipe et al., 2015). Phenolic compounds of wheat bran mainly belong to phenolic acids group with ferulic acid (4-hydroxy-3-methoxy cinnamic acid) as major component (it represents up to 90% of total phenolic acids) (Adom et al., 2003). Ferulic acid (FA) mainly occurs in trans configuration and is esterified to arabinose (linked in the O-5 position), stanols, sterols, and glucose (Naczk & Shahidi, 2006). It is also able to be associated to lignin vi

Interactions between Kazachstania humilis Yeast Species and Lactic Acid Bacteria in Sourdough

Sourdoughs harbor simple microbial communities usually composed of a few prevailing lactic acid bacteria species (LAB) and yeast species. However, yeast and LAB found in sourdough have been described as highly diverse. Even if LAB and yeast associations have been widely documented, the nature of the interactions between them has been poorly described. These interactions define the composition and structure of sourdough communities, and therefore, the characteristics of the final bread product. In this study, the nature of the interactions between strains of two commonly found sourdough yeast species, Kazachstania humilis and Saccharomyces cerevisiae, and lactic acid bacteria isolated from sourdoughs has been analyzed. Population density analysis showed no evidence of positive interactions, but instead revealed neutral or negative asymmetric interaction outcomes. When in coculture, the yeasts´ population size decreased in the presence of LAB regardless of the strain, while the LAB´s population size was rarely influenced by the presence of yeasts. However, a higher maltose depletion was shown in maltose-negative K. humilis and maltose-positive obligately heterofermentative LAB cocultures compared to monocultures. In addition, tested pairs of obligately heterofermentative LAB and K. humilis strains leavened dough as much as couples of LAB and S. cerevisiae strains, while K. humilis strains never leavened dough as much as S. cerevisiae when in monoculture. Taken together, our results demonstrate that even if higher fermentation levels with increased maltose depletion were detected for K. humilis and obligately heterofermentative LAB pairs, these interactions cannot be ecologically classified as positive, leading us to rethink the established hypothesis of coexistence by facilitation in sourdoughs

Microbial Siderophores Exert a Subtle Role in Arabidopsis during Infection by Manipulating the Immune Response and the Iron Status1[W]

Siderophores (ferric ion chelators) are secreted by organisms in response to iron deficiency. The pathogenic enterobacterium Erwinia chrysanthemi produces two siderophores, achromobactin and chrysobactin (CB), which are required for systemic dissemination in host plants. Previous studies have shown that CB is produced in planta and can trigger the up-regulation of the plant ferritin gene AtFER1. To further investigate the function of CB during pathogenesis, we analyzed its effect in Arabidopsis (Arabidopsis thaliana) plants following leaf infiltration. CB activates the salicylic acid (SA)-mediated signaling pathway, while the CB ferric complex is ineffective, suggesting that the elicitor activity of this siderophore is due to its iron-binding property. We confirmed this hypothesis by testing the effect of siderophores structurally unrelated to CB, including deferrioxamine. There was no activation of SA-dependent defense in plants grown under iron deficiency before CB treatment. Transcriptional analysis of the genes encoding the root ferrous ion transporter and ferric chelate reductase, and determination of the activity of this enzyme in response to CB or deferrioxamine, showed that these compounds induce a leaf-to-root iron deficiency signal. This root response as well as ferritin gene up-regulation in the leaf were not compromised in a SA-deficient mutant line. Using the Arabidopsis-E. chrysanthemi pathosystem, we have shown that CB promotes bacterial growth in planta and can modulate plant defenses through an antagonistic mechanism between SA and jasmonic acid signaling cascades. Collectively, these data reveal a new link between two processes mediated by SA and iron in response to microbial siderophores

NRAMP genes function in Arabidopsis thaliana resistance to Erwinia chrysanthemi infection.

International audienceAtNRAMP3 and AtNRAMP4 are two Arabidopsis metal transporters sharing about 50% sequence identity with mouse NRAMP1. The NRAMP1/Slc11A1 metal ion transporter plays a crucial role in the innate immunity of animal macrophages targeted by intracellular bacterial pathogens. AtNRAMP3 and AtNRAMP4 localize to the vacuolar membrane. We found that AtNRAMP3 is upregulated in leaves challenged with the bacterial pathogens Pseudomonas syringae and Erwinia chrysanthemi, whereas AtNRAMP4 expression is not modified. Using single and double nramp3 and nramp4 mutants, as well as lines ectopically expressing either of these genes, we show that AtNRAMP3 and, to a lesser extent, AtNRAMP4 are involved in Arabidopsis thaliana resistance against the bacterial pathogen E. chrysanthemi. The susceptibility of the double nramp3 nramp4 mutant is associated with the reduced accumulation of reactive oxygen species and ferritin (AtFER1), an iron storage protein known to participate in A. thaliana defense. Interestingly, roots from infected plants accumulated transcripts of AtNRAMP3 as well as the iron-deficiency markers IRT1 and FRO2. This finding suggests the existence of a shoot-to-root signal reminiscent of an iron-deficiency signal activated by pathogen infection. Our data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants