Benoit Marteyn

Life on the inside: the intracellular lifestyle of cytosolic bacteria

Bacterial pathogens exploit a huge range of niches within their hosts. Many pathogens can invade non-phagocytic cells and survive within a membrane-bound compartment. However, only a small number of bacteria, including Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis and Rickettsia spp., can gain access to and proliferate within the host cell cytosol. Here, we discuss the mechanisms by which these cytosolic pathogens escape into the cytosol, obtain nutrients to replicate and subvert host immune responses.

Modulation of Shigella virulence in response to available oxygen in vivo

Bacteria coordinate expression of virulence determinants in response to localized microenvironments in their hosts. Here we show that Shigella flexneri, which causes dysentery, encounters varying oxygen concentrations in the gastrointestinal tract, which govern activity of its type three secretion system (T3SS). The T3SS is essential for cell invasion and virulence. In anaerobic environments (for example, the gastrointestinal tract lumen), Shigella is primed for invasion and expresses extended T3SS needles while reducing Ipa (invasion plasmid antigen) effector secretion. This is mediated by FNR (fumarate and nitrate reduction), a regulator of anaerobic metabolism that represses transcription of spa32 and spa33, virulence genes that regulate secretion through the T3SS. We demonstrate there is a zone of relative oxygenation adjacent to the gastrointestinal tract mucosa, caused by diffusion from the capillary network at the tips of villi. This would reverse the anaerobic block of Ipa secretion, allowing T3SS activation at its precise site of action, enhancing invasion and virulence.

Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione

Background Enteric glial cells (EGCs) are important regulators of intestinal epithelial barrier (IEB) functions. EGC-derived S-nitrosoglutathione (GSNO) has been shown to regulate IEB permeability. Whether EGCs and GSNO protect the IEB during infectious insult by pathogens such as Shigella flexneri is not known. Methods S flexneri effects were characterised using in vitro coculture models of Caco-2 cells and EGCs (or GSNO), ex vivo human colonic mucosa, and in vivo ligated rabbit intestinal loops. The effect of EGCs on S flexneri-induced changes in the invasion area and the inflammatory response were analysed by combining immunohistochemical, ELISA and PCR methods. Expression of small G-proteins was analysed by western blot. Expression of ZO-1 and localisation of bacteria were analysed by fluorescence microscopy. Results EGCs significantly reduced barrier lesions and inflammatory response induced by S flexneri in Caco-2 monolayers. The EGC-mediated effects were reproduced by GSNO, but not by reduced glutathione, and pharmacological inhibition of pathways involved in GSNO synthesis reduced EGC protecting effects. Furthermore, expression of Cdc42 and phospho-PAK in Caco-2 monolayers was significantly reduced in the presence of EGCs or GSNO. In addition, changes in ZO-1 expression and distribution induced by S flexneri were prevented by EGCs and GSNO. Finally, GSNO reduced S flexneri-induced lesions of the IEB in human mucosal colonic explants and in a rabbit model of shigellosis. Conclusion These results highlight a major protective function of EGCs and GSNO in the IEB against S flexneri attack. Consequently, this study lays the scientific basis for using GSNO to reduce barrier susceptibility to infectious or inflammatory challenge.

Shigella: a model of virulence regulation in vivo.

Much is known about the molecular effectors of pathogenicity of gram-negative enteric pathogens, among which Shigella can be considered a model. This is due to its capacity to recapitulate the multiple steps required for a pathogenic microbe to survive close to its mucosal target, colonize and then invade its epithelial surface, cause its inflammatory destruction and simultaneously regulate the extent of the elicited innate response to likely survive the encounter and achieve successful subsequent transmission. These various steps of the infectious process represent an array of successive environmental conditions to which the bacteria need to successfully adapt. These conditions represent the selective pressure that triggered the “arms race” in which Shigella acquired the genetic and molecular effectors of its pathogenic armory, including the regulatory hierarchies that regulate the expression and function of these effectors. They also represent cues through which Shigella achieves the temporo-spatial expression and regulation of its virulence effectors. The role of such environmental cues has recently become obvious in the case of the major virulence effector of Shigella, the type three secretion system (T3SS) and its dedicated secreted virulence effectors. It needs to be better defined for other major virulence components such as the LPS and peptidoglycan which are used as examples here, in addition to the T3SS as models of regulation as it relates to the assembly and functional regulation of complex macromolecular systems of the bacterial surface. This review also stresses the need to better define what the true and relevant environmental conditions can be at the various steps of the progression of infection. The “identity” of the pathogen differs depending whether it is cultivated under in vitro or in vivo conditions. Moreover, this “identity” may quickly change during its progression into the infected tissue. Novel concepts and relevant tools are needed to address this challenge in microbial pathogenesis.

Breathing life into pathogens: the influence of oxygen on bacterial virulence and host responses in the gastrointestinal tract

The gastrointestinal tract provides a variety of environmental challenges to any bacterium seeking to successfully colonize or cause disease in a host. A major obstacle is the varied oxygen concentrations encountered at different sites in the intestine. Here we review the mechanisms bacterial pathogens utilize to sense oxygen within the gastrointestinal tract, and recent insights into how this acts as a signal to trigger virulence and to modulate host responses.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers

Intestinal pathogens use the hosts excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non‐antibiotic‐based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4‐MD‐2‐LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)‐6 by Gram‐negative bacteria. In a rabbit model of shigellosis, PAMAM‐DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL‐6 and IL‐8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)‐DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM‐DG potently inhibited Shigella Lipid A‐induced IL‐6 expression. In rabbits, PETIM‐DG prevented Shigella‐induced epithelial gut wall damage, reduced local IL‐6 and IL‐8 expression, and minimized bacterial invasion. There was no change in β‐defensin, IL‐10, interferon‐β, transforming growth factor‐β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.

Shigella Diversity and Changing Landscape: Insights for the Twenty-First Century.

Shigella is a pathovar of Escherichia coli comprising four groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii, each of them, with the exception of S.sonnei, comprising several serotypes. Shigella accounts for the majority of dysentery causing infections occurring world-wide each year. Recent advancements in the Shigella field have led to a better understanding of the molecular mechanisms underlying host epithelial cell invasion and immune cell function manipulation, mainly using S. flexneri as a model. Host-cell invasion is the final step of the infection process, as Shigellas virulence strategy relies also on its ability to survive hostile conditions during its journey through the gastro-intestinal tract, to compete with the host microbiota and to cross the intestinal mucus layer. Hence, the diversity of the virulence strategies among the different Shigella species has not yet been deeply investigated, which might be an important step to understand the epidemiological spreading of Shigella species worldwide and a key aspect for the validation of novel vaccine candidates. The recent development of high-throughput screening and sequencing methods will facilitate these complex comparison studies. In this review we discuss several of the major avenues that the Shigella research field has taken over the past few years and hopefully gain some insights into the questions that remain surrounding this important human pathogen.

ZapE Is a Novel Cell Division Protein Interacting with FtsZ and Modulating the Z-Ring Dynamics

ABSTRACT Bacterial cell division requires the formation of a mature divisome complex positioned at the midcell. The localization of the divisome complex is determined by the correct positioning, assembly, and constriction of the FtsZ ring (Z-ring). Z-ring constriction control remains poorly understood and (to some extent) controversial, probably due to the fact that this phenomenon is transient and controlled by numerous factors. Here, we characterize ZapE, a novel ATPase found in Gram-negative bacteria, which is required for growth under conditions of low oxygen, while loss of zapE results in temperature-dependent elongation of cell shape. We found that ZapE is recruited to the Z-ring during late stages of the cell division process and correlates with constriction of the Z-ring. Overexpression or inactivation of zapE leads to elongation of Escherichia coli and affects the dynamics of the Z-ring during division. In vitro, ZapE destabilizes FtsZ polymers in an ATP-dependent manner. IMPORTANCE Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future. Bacterial cell division has mainly been characterized in vitro. In this report, we could identify ZapE as a novel cell division protein which is not essential in vitro but is required during an infectious process. The bacterial cell division process relies on the assembly, positioning, and constriction of FtsZ ring (the so-called Z-ring). Among nonessential cell division proteins recently identified, ZapE is the first in which detection at the Z-ring correlates with its constriction. We demonstrate that ZapE abundance has to be tightly regulated to allow cell division to occur; absence or overexpression of ZapE leads to bacterial filamentation. As zapE is not essential, we speculate that additional Z-ring destabilizing proteins transiently recruited during late cell division process might be identified in the future.