Septins and K63 ubiquitin chains are present in separate bacterial microdomains during autophagy of entrapped Shigella.

During host cell invasion, Shigella escapes to the cytosol and polymerizes actin for cell-to-cell spread. To restrict cell-to-cell spread, host cells employ cell-autonomous immune responses including antibacterial autophagy and septin cage entrapment. How septins interact with the autophagy process to target Shigella for destruction is poorly understood. Here, we employed a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study Shigella septin cage entrapment in its near-native state. Quantitative cryo-SXT showed that Shigella fragments mitochondria and enabled visualization of X-ray-dense structures (∼30 nm resolution) surrounding Shigella entrapped in septin cages. Using Airyscan confocal microscopy, we observed lysine 63 (K63)-linked ubiquitin chains decorating septin-cage-entrapped Shigella. Remarkably, septins and K63 chains are present in separate bacterial microdomains, indicating they are recruited separately during antibacterial autophagy. Cryo-SXT and live-cell imaging revealed an interaction between septins and LC3B-positive membranes during autophagy of Shigella. Together, these findings demonstrate how septin-caged Shigella are targeted for autophagy and provide fundamental insights into autophagy-cytoskeleton interactions.

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor.

In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Shigella MreB promotes polar IcsA positioning for actin tail formation.

Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation of the Shigella autotransporter protein IcsA is essential for actin tail formation, which is necessary for the bacterium to travel from cell-to-cell; yet how proteins are targeted to the bacterial cell pole is poorly understood. The bacterial actin homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis and Caulobacter crescentus, but has never been visualised in rod-shaped pathogenic bacteria during infection of host cells. Here, using single-cell analysis of intracellular Shigella, we discover that MreB accumulates at the cell pole of bacteria forming actin tails, where it colocalises with IcsA. Pharmacological inhibition of host cell actin polymerisation and genetic deletion of IcsA is used to show, respectively, that localisation of MreB to the cell poles precedes actin tail formation and polar localisation of IcsA. Finally, by exploiting the MreB inhibitors A22 and MP265, we demonstrate that MreB polymerisation can support actin tail formation. We conclude that Shigella MreB promotes polar IcsA positioning for actin tail formation, and suggest that understanding the bacterial cytoskeleton during host-pathogen interactions can inspire development of new therapeutic regimes for infection control.This article has an associated First Person interview with the first author of the paper.

Intact Cell Lipidomics Reveal Changes to the Ratio of Cardiolipins to Phosphatidylinositols in Response to Kanamycin in HeLa and Primary Cells.

Antimicrobial resistance is a major threat the world is currently facing. Development of new antibiotics and the assessment of their toxicity represent important challenges. Current methods for addressing antibiotic toxicity rely on measuring mitochondrial damage using ATP and/or membrane potential as a readout. In this study, we propose an alternative readout looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionization mass spectrometry. As a proof of principle, we evaluated the impact of known antibiotics (levofloxacin, ethambutol, and kanamycin) on the lipidome of HeLa cells and mouse bone marrow-derived macrophages. Our methodology revealed that clinically relevant concentrations of kanamycin alter the ratio of cardiolipins to phosphatidylinositols. Unexpectedly, only kanamycin had this effect even though all antibiotics used in this study led to a decrease in the maximal mitochondrial respiratory capacity. Altogether, we report that intact cell-targeted lipidomics can be used as a qualitative method to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells. Moreover, these results demonstrate there is no direct correlation between the ratio of cardiolipins to phosphatidylinositols and the maximal mitochondrial respiratory capacity.

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.

Regulation of integrin α5ß1-mediated Staphylococcus aureus cellular invasion by the septin cytoskeleton.

Staphylococcus aureus, a Gram-positive bacterial pathogen, is an urgent health threat causing a wide range of clinical infections. Originally viewed as a strict extracellular pathogen, accumulating evidence has revealed S. aureus to be a facultative intracellular pathogen subverting host cell signalling to support invasion. The majority of clinical isolates produce fibronectin-binding proteins A and B (FnBPA and FnBPB) to interact with host integrin α5ß1, a key component of focal adhesions. S. aureus binding of integrin α5ß1 promotes its clustering on the host cell surface, triggering activation of focal adhesion kinase (FAK) and cytoskeleton rearrangements to promote bacterial invasion into non-phagocytic cells. Here, we discover that septins, a component of the cytoskeleton that assembles on membranes, are recruited as collar-like structures with actin to S. aureus invasion sites engaging integrin α5ß1. To investigate septin recruitment to the plasma membrane in a bacteria-free system, we used FnBPA-coated latex beads and showed that septins are recruited upon activation of integrin α5ß1. SEPT2 depletion reduced S. aureus invasion, but increased surface expression of integrin α5 and adhesion of S. aureus to host cells. Consistent with this, SEPT2 depletion increased cellular protein levels of integrin α5 and ß1 subunits, as well as FAK. Collectively, these results provide insights into regulation of integrin α5ß1 and invasion of S. aureus by the septin cytoskeleton.

Septins promote caspase activity and coordinate mitochondrial apoptosis.

Apoptosis is a form of regulated cell death essential for tissue homeostasis and embryonic development. Apoptosis also plays a key role during bacterial infection, yet some intracellular bacterial pathogens (such as Shigella flexneri, whose lipopolysaccharide can block apoptosis) can manipulate cell death programs as an important survival strategy. Septins are a component of the cytoskeleton essential for mitochondrial dynamics and host defense, however, the role of septins in regulated cell death is mostly unknown. Here, we discover that septins promote mitochondrial (i.e., intrinsic) apoptosis in response to treatment with staurosporine (a pan-kinase inhibitor) or etoposide (a DNA topoisomerase inhibitor). Consistent with a role for septins in mitochondrial dynamics, septins promote the release of mitochondrial protein cytochrome c in apoptotic cells and are required for the proteolytic activation of caspase-3, caspase-7, and caspase-9 (core components of the apoptotic machinery). Apoptosis of HeLa cells induced in response to infection by S. flexneri ΔgalU (a lipopolysaccharide mutant unable to block apoptosis) is also septin-dependent. In vivo, zebrafish larvae are significantly more susceptible to infection with S. flexneri ΔgalU (as compared to infection with wildtype S. flexneri), yet septin deficient larvae are equally susceptible to infection with S. flexneri ΔgalU and wildtype S. flexneri. These data provide a new molecular framework to understand the complexity of mitochondrial apoptosis and its ability to combat bacterial infection.

Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.

Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.

Mechanistic insight into bacterial entrapment by septin cage reconstitution.

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.

Measuring Plasmid Stability in Gram-Negative Bacteria.

In this chapter, a highly sensitive method to measure plasmid stability in Gram-negative bacteria is described. This procedure is based on the counterselection of plasmid-containing cells using an aph-parE cassette. When bacteria carrying the aph-parE module in the plasmid of interest are grown in media containing rhamnose as the only carbon source, the PparE promoter is induced, ParE is synthesized, and plasmid-containing cells are eliminated; bacteria that have lost the plasmid survive. The absence of the kanamycin resistance marker (aph) can be used to confirm the loss of the plasmid in rhamnose grown bacteria.

A requirement for septins and the autophagy receptor p62 in the proliferation of intracellular Shigella.

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. During infection of epithelial cells, Shigella escape from the phagosome to the cytosol, where they reroute host cell glycolysis to obtain nutrients for proliferation. Septins, a poorly understood component of the cytoskeleton, can entrap cytosolic Shigella targeted to autophagy in cage-like structures to restrict bacterial proliferation. Although bacterial entrapment by septin caging has been the subject of intense investigation, the role of septins and the autophagy machinery in the proliferation of noncaged Shigella is mostly unknown. Here, we found that intracellular Shigella fail to efficiently proliferate in SEPT2-, SEPT7-, or p62/SQSTM1-depleted cells. Consistent with a failure to proliferate, single cell analysis of bacteria not entrapped in septin cages showed that the number of metabolically active Shigella in septin- or p62-depleted cells is reduced. Targeted metabolomic analysis revealed that host cell glycolysis is dysregulated in septin-depleted cells, suggesting a key role for septins in modulation of glycolysis. Together, these results suggest that septins and the autophagy machinery may regulate metabolic pathways that promote the proliferation of intracellular Shigella not entrapped in septin cages.

Mitochondria promote septin assembly into cages that entrap Shigella for autophagy.

Septins are cytoskeletal proteins implicated in cytokinesis and host-pathogen interactions. During macroautophagy/autophagy of Shigella flexneri, septins assemble into cage-like structures to entrap actin-polymerizing bacteria and restrict their dissemination. How septins assemble to entrap bacteria is not fully known. We discovered that mitochondria support septin cage assembly to promote autophagy of Shigella. Consistent with roles for the cytoskeleton in mitochondrial dynamics, we showed that DNM1L/DRP1 (dynamin 1 like) can interact with septins to enhance mitochondrial fission. Remarkably, Shigella fragment mitochondria and escape from septin cage entrapment in order to avoid autophagy. These results uncover a close relationship between mitochondria and septin assembly, and identify a new role for mitochondria in bacterial autophagy.

Septins Recognize and Entrap Dividing Bacterial Cells for Delivery to Lysosomes.

The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.

Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells.

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.

Septins recognize micron-scale membrane curvature.

How cells recognize membrane curvature is not fully understood. In this issue, Bridges et al. (2016.J. Cell Biol.http://dx.doi.org/10.1083/jcb.201512029) discover that septins, a component of the cytoskeleton, recognize membrane curvature at the micron scale, a common morphological hallmark of eukaryotic cellular processes.

Toxin-antitoxins and bacterial virulence.

Bacterial virulence relies on a delicate balance of signals interchanged between the invading microbe and the host. This communication has been extensively perceived as a battle involving harmful molecules produced by the pathogen and host defenses. In this review, we focus on a largely unexplored element of this dialogue, as are toxin-antitoxin (TA) systems of the pathogen. TA systems are reported to respond to stresses that are also found in the host and, as a consequence, could modulate the physiology of the intruder microbe. This view is consistent with recent studies that demonstrate a contribution of distinct TA systems to virulence since their absence alters the course of the infection. TA loci are stress response modules that, therefore, could readjust pathogen metabolism to favor the generation of slow-growing or quiescent cells 'before' host defenses irreversibly block essential pathogen activities. Some toxins of these TA modules have been proposed as potential weapons used by the pathogen to act on host targets. We discuss all these aspects based on studies that support some TA modules as important regulators in the pathogen-host interface.

Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test.

Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The "activation" of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity.