Bacillus anthracis, "la maladie du charbon", Toxins, and Institut Pasteur.

Institut Pasteur and Bacillus anthracis have enjoyed a relationship lasting almost 120 years, starting from its foundation and the pioneering work of Louis Pasteur in the nascent fields of microbiology and vaccination, and blooming after 1986 following the molecular biology/genetic revolution. This contribution will give a historical overview of these two research eras, taking advantage of the archives conserved at Institut Pasteur. The first era mainly focused on the production, characterisation, surveillance and improvement of veterinary anthrax vaccines; the concepts and technologies with which to reach a deep understanding of this research field were not yet available. The second period saw a new era of B. anthracis research at Institut Pasteur, with the anthrax laboratory developing a multi-disciplinary approach, ranging from structural analysis, biochemistry, genetic expression, and regulation to bacterial-host cell interactions, in vivo pathogenicity, and therapy development; this led to the comprehensive unravelling of many facets of this toxi-infection. B. anthracis may exemplify some general points on how science is performed in a given society at a given time and how a scientific research domain evolves. A striking illustration can be seen in the additive layers of regulations that were implemented from the beginning of the 21st century and their impact on B. anthracis research. B. anthracis and anthrax are complex systems that raise many valuable questions regarding basic research. One may hope that B. anthracis research will be re-initiated under favourable circumstances later at Institut Pasteur.

[Unconventional Laveran]. / Laveran décalé.

Charles Louis Alphonse Laveran - 18 June 1845 - 18 May 1922: first French Nobel Prize in Medicine, "in recognition of his work on the role played by protozoa in causing diseases". One hundred years after his death, only written records remain of his work and life. The witnesses to this period are no more. Alphonse Laveran has become an "object" of history.He was deeply involved in a turbulent historical period, marked by crises of regime change (Monarchy/Empire/Republic), military events (French colonial expansion in North Africa from 1830, the wars of 1870 and 1914-1918) and their consequences (the medical impact of infections in the colonial empire and during armed conflicts, the Dreyfus affair, among others), the advent of Pasteurian "microbiology" and the deciphering of the causes and modes of transmission of infectious diseases. A player on the edge of the military and civilian worlds, with their own, sometimes incompatible, visions of the aims and objectives to be pursued, Alphonse Laveran lived through these upheavals in a society in the throes of change, in his family and scientific environment.Paradoxically, the primary sources available to us for learning about this scientist and man are both abundant and "scarce" for us in the 21st century. His scientific publications and many of his speeches at various academies, committees and meetings are for the most part public and accessible, giving us a vision of a professional in scientific and medical research in action, presenting and convincing people of his ideas and theoretical and practical insights. The writings of his contemporaries, both public and private, shed light on - distort? - the man's many facets. On the other hand, there are few surviving sources on the man and his vision of life, his life and that of his family and friends.We will rely on the archives that have been preserved, in particular by the organisations that welcomed him during his military and civilian career, as well as by his wife Marie Laveran and his colleague Marie Phisalix, one of the first doctors of medicine in France and a renowned herpetologist. These two female figures have preserved and contributed to his memory. Let's take a closer look at the man behind the scientist, as we can imagine him through the traces that remain.

Early expression of capsule during Bacillus anthracis germination.

Bacillus anthracis is a spore-forming bacterium that produces two major virulence factors, a tripartite toxin with two enzymatic toxic activities and a pseudo-proteic capsule. One of the main described functions of the poly-gamma-d-glutamate capsule is to enable B. anthracis bacilli to escape phagocytosis. Thus, kinetics of expression of the capsule filaments at the surface of the emerging bacillus during germination is an important step for the protection of the nascent bacilli. In this study, through immunofluorescence and electron microscopic approaches, we show the emergence of the capsule through a significant surface of the exosporium in the vast majority of the germinating spores, with co-detection of BclA and capsular material. This suggests that, due to an early capsule expression, the extracellular life of B. anthracis might occur earlier than previously thought, once germination is triggered. This raises the prospect that an anti-capsular vaccine may play a protective role at the initial stage of infection by opsonisation of the nascent encapsulated bacilli before their emergence from the exosporium.

Management and characteristics of patients suffering from Clostridiodes difficile infection in primary care.

BACKGROUND: Clostridioides difficile infection (CDI) is rising and increases patient healthcare costs due to extended hospitalisation, tests and medications. Management of CDI in French primary care is poorly reported. OBJECTIVES: To characterise patients suffering from CDI, managed in primary care and describe their clinical outcomes. METHODS: Retrospective observational study based on survey data among 500 randomly selected General Practitioners (GPs) surveyed in France from September 2018 to April 2019. GPs were asked to complete a multiple-choice questionnaire for each reported patient presenting a CDI. Responses were analysed according to clinical characteristics. Treatment strategies were compared according to the outcome: recovery or recurrent infection. RESULTS: Participation rate was 8.6% (n = 43/500) with two incomplete questionnaires. Data from 41 patients with an actual diagnosis of CDI were analysed. Recovery was observed in 61% of patients with a confirmed diagnosis of CDI. In the recovery group, this was exclusively a primary episode, most patients (72%) had no comorbidities, were significantly younger (p = 0.02) than the ones who relapsed and 92% were successfully treated with oral metronidazole. Duration of diarrhoea after antimicrobial treatment initiation was significantly shorter in the recovery group (≤ 48 h) (p = 0.03). Cooperation with hospital specialists was reported in 28% of the recovery group versus 87.5% of the recurrent group (p = 0.0003). Overall, GPs managed successfully 82.9% of cases without need of hospital admission. CONCLUSION: GPs provide relevant ambulatory care for mild primary episodes of CDI using oral metronidazole. Persistent diarrhoea despite an appropriate anti-Clostridiodes regimen should be interpreted as an early predictor of relapse.

Analyzing In Silico the Relationship Between the Activation of the Edema Factor and Its Interaction With Calmodulin.

Molecular dynamics (MD) simulations have been recorded on the complex between the edema factor (EF) of Bacilllus anthracis and calmodulin (CaM), starting from a structure with the orthosteric inhibitor adefovir bound in the EF catalytic site. The starting structure has been destabilized by alternately suppressing different co-factors, such as adefovir ligand or ions, revealing several long-distance correlations between the conformation of CaM, the geometry of the CaM/EF interface, the enzymatic site and the overall organization of the complex. An allosteric communication between CaM/EF interface and the EF catalytic site, highlighted by these correlations, was confirmed by several bioinformatics approaches from the literature. A network of hydrogen bonds and stacking interactions extending from the helix V of of CaM, and the residues of the switches A, B and C, and connecting to catalytic site residues, is a plausible candidate for the mediation of allosteric communication. The greatest variability in volume between the different MD conditions was also found for cavities present at the EF/CaM interface and in the EF catalytic site. The similarity between the predictions from literature and the volume variability might introduce the volume variability as new descriptor of allostery.

Muscle Injury Induces Postoperative Cognitive Dysfunction.

Postoperative cognitive dysfunction (POCD) is a major complication affecting patients of any age undergoing surgery. This syndrome impacts everyday life up to months after hospital discharge, and its pathophysiology still remains unclear. Translational research focusing on POCD is based on a wide variety of rodent models, such as the murine tibial fracture, whose severity can limit mouse locomotion and proper behavioral assessment. Besides, influence of skeletal muscle injury, a lesion encountered in a wide range of surgeries, has not been explored in POCD occurrence. We propose a physical model of muscle injury in CX3CR1GFP/+ mice (displaying green fluorescent microglial cells) to study POCD, with morphological, behavioral and molecular approaches. We highlighted: alteration of short- and long-term memory after muscle regeneration, wide microglial reactivity in the brain, including hippocampus area, 24 hours after muscle injury, and an alteration of central brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) balance, 28 days after muscle injury. Our results suggest for the first time that muscle injury can have early as well as late impacts on the brain. Our CX3CR1GFP/+ model can also facilitate microglial investigation, more specifically their pivotal role in neuroinflammation and synaptic plasticity, in the pathophysiology of POCD.

Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection.

BACKGROUND: Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. METHODS: To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. RESULTS: One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. CONCLUSIONS: In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.

Questionable Efficacy of Therapeutic Antibodies in the Treatment of Anthrax.

Inhalational anthrax caused by Bacillus anthracis, a spore-forming Gram-positive bacterium, is a highly lethal infection. Antibodies targeting the protective antigen (PA) binding component of the toxins have recently been authorized as an adjunct to antibiotics, although no conclusive evidence demonstrates that anthrax antitoxin therapy has any significant benefit. We discuss here the rational basis of anti-PA development regarding the pathogenesis of the disease. We argue that inductive reasoning may induce therapeutic bias. We identified anthrax animal model analysis as another bias. Further studies are needed to assess the benefit of anti-PA antibodies in the treatment of inhalational anthrax, while a clearer consensus should be established around what evidence should be proven in an anthrax model.

Corrigendum: Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections.

[This corrects the article on p. 208 in vol. 7, PMID: 28589102.].

Effect of replacing main-chain ureas with thiourea and guanidinium surrogates on the bactericidal activity of membrane active oligourea foldamers.

Membrane-active foldamers have recently emerged as potential mimics of antimicrobial peptides (AMPs). Amphiphilic cationic helical N,N'-linked oligoureas are one such class of AMP mimics with activities in vitro against a broad range of bacteria including Bacillus anthracis, a Gram-positive sporulating bacillus and causing agent of anthrax. Here we have used site-selective chemical modifications of the oligourea backbone to gain additional insight into the relationship between structure and function and modulate anthracidal activity. A series of analogues in which urea linkages at selected positions are replaced by thiourea and guanidium surrogates have been prepared on solid support and tested against different bacterial forms of B. anthracis (germinated spores and encapsulated bacilli). Urea→thiourea and urea→guanidinium replacements close to the negative end of the helix dipole led to analogues with increased potency and selectivity for B. anthracis versus mammalian cells.

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections.

Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

Mechanisms of Invariant NKT Cell Activity in Restraining Bacillus anthracis Systemic Dissemination.

Exogenous activation of invariant NKT (iNKT) cells by the superagonist α-galactosylceramide (α-GalCer) can protect against cancer, autoimmune diseases, and infections. In the current study, we investigated the effect of α-GalCer against Bacillus anthracis infection, the agent of anthrax. Using an experimental model of s.c. B. anthracis infection (an encapsulated nontoxigenic strain), we show that concomitant administration of α-GalCer delayed B. anthracis systemic dissemination and prolonged mouse survival. Depletion of subcapsular sinus CD169-positive macrophages by clodronate-containing liposome was associated with a lack of iNKT cell activation in the draining lymph nodes (dLNs) and prevented the protective effect of α-GalCer on bacterial dissemination out of the dLNs. Production of IFN-γ triggered chemokine (C-C motif) ligand 3 synthesis and recruitment of neutrophils in the dLNs, leading to the restraint of B. anthracis dissemination. Our data highlight a novel immunological pathway leading to the control of B. anthracis infection, a finding that might lead to improved therapeutics based on iNKT cells.

Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection.

The synthesis of bioinspired unnatural backbones leading to foldamers can provide effective peptide mimics with improved properties in a physiological environment. This approach has been applied to the design of structural mimics of membrane active antimicrobial peptides (AMPs) for which activities in vitro have been reported. Yet activities and pharmacokinetic properties in vivo in animal models have remained largely unexplored. Here, we report helical oligourea AMP mimics that are active in vitro against bacterial forms of Bacillus anthracis encountered in vivo, as well as in vivo in inhalational and cutaneous mouse models of B. anthracis infection. The pharmacokinetic profile and the tissue distribution were investigated by ß-radio imager whole-body mapping in mice. Low excretion and recovery of the native oligourea in the kidney following intravenous injection is consistent with high stability in vivo. Overall these results provide useful information that support future biomedical development of urea-based foldamer peptide mimics.

In vivo dynamics of active edema and lethal factors during anthrax.

Lethal and edema toxins are critical virulence factors of Bacillus anthracis. However, little is known about their in vivo dynamics of production during anthrax. In this study, we unraveled for the first time the in vivo kinetics of production of the toxin components EF (edema factor) and LF (lethal factor) during cutaneous infection with a wild-type toxinogenic encapsulated strain in immuno-competent mice. We stratified the asynchronous infection process into defined stages through bioluminescence imaging (BLI), while exploiting sensitive quantitative methods by measuring the enzymatic activity of LF and EF. LF was produced in high amounts, while EF amounts steadily increased during the infectious process. This led to high LF/EF ratios throughout the infection, with variations between 50 to a few thousands. In the bloodstream, the early detection of active LF and EF despite the absence of bacteria suggests that they may exert long distance effects. Infection with a strain deficient in the protective antigen toxin component enabled to address its role in the diffusion of LF and EF within the host. Our data provide a picture of the in vivo complexity of the infectious process.

Physical Sequestration of Bacillus anthracis in the Pulmonary Capillaries in Terminal Infection.

The lung is the terminal target of Bacillus anthracis before death, whatever the route of infection (cutaneous, inhalational, or digestive). During a cutaneous infection in absence of toxins, we observed encapsulated bacteria colonizing the alveolar capillary network, bacteria and hemorrhages in alveolar and bronchiolar spaces, and hypoxic foci in the lung (endothelial cells) and brain (neurons and neuropil). Circulating encapsulated bacteria were as chains of approximately 13 µm in length. Bacteria of such size were immediately trapped within the lung capillary network, but bacteria of shorter length were not. Controlling lung-targeted pathology would be beneficial for anthrax treatment.

Crossing of the epithelial barriers by Bacillus anthracis: the Known and the Unknown.

Anthrax, caused by Bacillus anthracis, a Gram-positive spore-forming bacterium, is initiated by the entry of spores into the host body. There are three types of human infection: cutaneous, inhalational, and gastrointestinal. For each form, B. anthracis spores need to cross the cutaneous, respiratory or digestive epithelial barriers, respectively, as a first obligate step to establish infection. Anthrax is a toxi-infection: an association of toxemia and rapidly spreading infection progressing to septicemia. The pathogenicity of Bacillus anthracis mainly depends on two toxins and a capsule. The capsule protects bacilli from the immune system, thus promoting systemic dissemination. The toxins alter host cell signaling, thereby paralyzing the immune response of the host and perturbing the endocrine and endothelial systems. In this review, we will mainly focus on the events and mechanisms leading to crossing of the respiratory epithelial barrier, as the majority of studies have addressed inhalational infection. We will discuss the critical gaps of knowledge that need to be addressed to gain a comprehensive view of the initial steps of inhalational anthrax. We will then discuss the few data available on B. anthracis crossing the cutaneous and digestive epithelia.

Micropatterned macrophage analysis reveals global cytoskeleton constraints induced by Bacillus anthracis edema toxin.

Bacillus anthracis secretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.