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

Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes

Geographical and temporal distribution of human giardiasis in Ontario, Canada

BACKGROUND: Giardia is the most frequently identified intestinal parasite in North America. Although information on geographical distribution of giardiasis is critical in identifying communities at high risk, little has been done in this area. Therefore, the objective of this study was to investigate the geographical and temporal distribution of human giardiasis in Ontario in order to identify possible high risk areas and seasons. Two spatial scales of analyses and two disease measures were used with a view to identifying the best of each in assessing geographical patterns of giardiasis in Ontario. Global Moran's I and Moran Local Indicators of Spatial Associations were used to test for evidence of global and local spatial clustering, respectively. RESULTS: There were seasonal patterns with summer peaks and a significant (P < 0.001) decreasing temporal trend. Significant (P < 0.05) global spatial clustering of high rates was observed at the Census Sub-division spatial scale but not at the Census Division scale. The Census Sub-division scale was a better scale of analyses but required spatial empirical Bayesian smoothing of the rates. A number of areas with significant local clustering of giardiasis rates were identified. CONCLUSIONS: The study identified spatial and temporal patterns in giardiasis distribution. This information is important in guiding decisions on disease control strategies. The study also showed that there is benefit in performing spatial analyses at more than one spatial scale to assess geographical patterns in disease distribution and that smoothing of disease rates for mapping in small areas enhances visualization of spatial patterns

IlsA, A Unique Surface Protein of Bacillus cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Identification de nouveaux facteurs hôtes-dépendants chez Bacillus cereus Caractérisation moléculaire et fonctionnelle d'IlsA, une protéine de surface essentielle pour l'acquisition du fer au cours de l'infection

Bacillus cereus is frequently associated with food-borne gastroenteritis and can be responsible for severe opportunistic infections. Adaptation factors involved in the pathogenicity of B. cereus are still unknown. The ability to acquire ion during infection, is an important adaptation factor that allow bacteria to overcome the lack of iron imposed by the host. Our work has led to the identification of new factors involved in the adaptation of B. cereus in the host and to the characterization of a novel protein IlsA (Iron regulated leucine-rich surface protein) highly expressed in the host. These factors were identified using the IVET (In Vivo Expression Technology) system, which was adapted to the strain B. cereus ATCC 14579 and screened after infection in the insect larvae Galleria mellonella. This system detects transient activated promoters. The protein structure analysis of IlsA shows four distinct domains: an N-terminal signal peptide for export, a NEAT domain thought to be involved in iron transport, a leucine-rich repeat domain that may interact with host cells, and an SLH domain presumably binding the protein to the bacterial surface. The presence of a fur box in the promoter region of ilsA suggests an iron dependent regulation. Indeed, the transcriptional analysis showed that ilsA is expressed under iron restricted conditions in vitro and in vivo. In addition, we have shown that IlsA is located on the bacterial surface and that is required for iron acquisition from the host proteins: hemoglobin, heme and ferritin, by direct binding to them. Furthermore, the protein sequence of the NEAT domain of IlsA, suggests that it could be responsible for the interaction with the heme. Disruption of ilsA reduced the survival and the virulence of B. cereus in the insect and in a mice macrophage cell line. Our results indicate that IlsA is an adaptation factor that play an essential role in iron acquisition during infection and thus contribute to the pathogenesis of B. cereus in both invertebrates and vertebratesBacillus cereus est fréquemment associé à des toxi-infections alimentaires et peut être responsable de pathologies opportunistes sévères. Les facteurs d'adaptations de B. cereus chez l'hôte, liés à son pouvoir pathogène, sont encore inconnus. La capacité d'acquérir le fer lors d'une infection, est une importante réponse adaptative des bactéries, leur permettant de surmonter le manque de fer imposé par l'hôte. Nos travaux ont permis l'identification de nouveaux facteurs impliqués dans l'adaptation de B. cereus chez l'hôte, ainsi que la caractérisation d'une nouvelle protéine IlsA (Iron regulated leucine-rich surface protein) fortement exprimée in vivo. L'identification de ces facteurs a été réalisée à l'aide du système IVET (In Vivo Expression Technology), adapté à la souche B. cereus ATCC 14579 et analysé après infection chez la larve du lépidoptère Galleria mellonella. Ce système permet la détection des promoteurs activés de façon transitoire. L'analyse de la structure protéique d'IlsA, montre quatre domaines conservés: un peptide signal d'export N-terminal, un domaine NEAT potentiellement impliqué dans le transport du fer, suivi d'une région riche en leucine (LRR) susceptible d'interagir avec les protéines de l'hôte, et un domaine SLH de liaison à la surface bactérienne. La présence d'une boîte fur dans la région promotrice d'ilsA suggère une régulation dépendante du fer. Les analyses transcriptionnelles ont montré qu'ilsA est en effet, exprimé dans les conditions de carence en fer in vitro et in vivo. De plus, nous avons démontré qu'IlsA est localisée à la surface et qu'elle est nécessaire pour l'acquisition de fer à partir des protéines présentes chez l'hôte : l'hémoglobine, l'hème et la ferritine, et ceci en se liant directement avec elles. En outre, l'étude de la séquence protéique du domaine NEAT d'IlsA, suggère qu'il serait responsable de l'interaction avec l'hème. Par ailleurs, nous avons montré que l'inactivation d'ilsA affecte la survie et la virulence de B. cereus chez l'insecte, et chez les macrophages murins. Nos résultats indiquent qu'IlsA est un facteur d'adaptation essentiel pour l'acquisition de fer au cours de l'infection, contribuant à la pathogénie de B. cereus chez les invertébrés et vertébrés

Identification de nouveaux facteurs hôtes-dépendants chez Bacillus cereus (caractérisation moléculaire et fonctionnelle d'IlsA, une protéine de surface essentielle pour l'acquisition du fer au cours de l'infection)

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Heterogeneous Photoredox Catalysis Based on Silica Mesoporous Material and Eosin Y: Impact of Material Support on Selectivity of Radical Cyclization

Heterogenization of the photocatalyst appears to be a valuable solution to reach sustainable processes. Rapid and efficient synthesis of supported photocatalyst is still a remaining challenge and the choice of the support material is crucial. The present study aims at preparing a new generation of hybrid inorganic/organic photocatalysts based on silica mesoporous material and Eosin Y. These results highlight the influence of non-covalent interactions between the material support and the reagent impacting the selectivity of the reaction

Role of the global regulator Rex in control of NAD + ‐regeneration in Clostridioides (Clostridium) difficile

International audienceFor the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the glycine reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile

ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

<div><p>Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.</p> </div