Contribución de los sistemas toxina-antitoxina de "Salmonella enterica serovar Typhimurium" en la adaptación a la vida intracelular

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-05-2015Los sistemas toxina-­‐antitoxina (TA) están compuestos por dos genes de pequeño tamaño que codifican una antitoxina inestable y una toxina químicamente más estable. Los sistemas TA son muy abundantes en el dominio Bacteria, y aparecen en el genoma de bacterias de vida libre, simbióticas y patógenas. Las toxinas tienen actividades comprendiendo desde ARNasas a inhibidores de ADN girasa, siendo su efecto en la célula principalmente bacteriostático. Los sistemas TA están implicados en distintos procesos como la supervivencia durante la escasez de nutrientes, la respuesta a estrés oxidativo, la formación de biopelículas o la tolerancia a antibióticos. Los sistemas TA se han relacionado también con el proceso de infección de diversas bacterias patógenas. Así, Escherichia coli uropatógena, Haemophilus influenzae y Salmonella enterica serovar Typhimurium (S. Typhimurium) emplean sistemas TA para colonizar y sobrevivir en órganos infectados. S. enterica es un patógeno intracelular que causa infecciones persistentes tanto en humanos como en ganado. En distintos modelos de ratón se ha comprobado que el serovar Typhimurium puede provocar tanto infección aguda como crónica. S. Typhimurium infecta preferencialmente macrófagos, aunque también puede infectar otros tipos celulares como fibroblastos, donde este patógeno atenúa su crecimiento. El objetivo de esta Tesis Doctoral fue determinar la posible implicación de los sistemas TA de S. Typhimurium durante la infección y supervivencia intracelular en células eucariotas. Empleando métodos informáticos, se detectaron 27 posibles sistemas TA en el genoma de la estirpe SL1344 de S. Typhimurium. Ensayos funcionales, basados en la inhibición de crecimiento por las toxinas y neutralización de esta inhibición por las antitoxinas, determinaron que únicamente 18 módulos TA son completamente funcionales. Se observó que la bacteria intracelular induce algunos sistemas preferentemente en fibroblastos pero no en células epiteliales. Además, se probaron en modelos de infección un total de 10 mutantes de deleción en sistemas TA, de los que cinco mostraron un descenso en la supervivencia intracelular en fibroblastos y uno en células epiteliales. Estos resultados indican una posible especialización de determinados sistemas TA involucrados en la supervivencia intracelular del patógeno en distintos tipos celularesToxin-­‐antitoxin modules (hereafter TA) are operons composed of two small genes encoding an unstable antitoxin and a stable toxin. TA loci abound in microbial genomes and are found in free-­‐ living, symbiotic and obligate intracellular bacteria. Toxins exhibit activities ranging from RNAses to DNA gyrase inhibitors, and mainly trigger bacteriostatic effects. TA modules are implicated in processes such as survival in response to nutrient starvation, response to oxidative damage, biofilm formation or tolerance to antibiotics. In addition, recent studies have focused in understanding whether TA modules contribute to pathogen survival during infection. Thus, uropathogenic Escherichia coli, Haemophilus influenzae and Salmonella enterica serovar Typhimurium (S. Typhimurium) use TA modules to colonize and survive in animal organs. S. enterica is an intracellular bacterial pathogen that can cause persistent infections in humans and livestock. The serovar Typhimurium has been extensively studied in murine models in which the pathogen causes either acute or chronic infections. In the animal, S. Typhimurium targets preferentially macrophages, but can also infect other cell types like fibroblasts, where this pathogen attenuates its intracellular growth rate. The aim of this work was to determine the possible role of S. Typhimurium TA modules during the infection and survival within eukaryotic cells. Twenty-­‐seven putative TA modules were identified using different bioinformatic approaches. Functional assays, based on the growth inhibition caused by the toxin and the neutralization of this effect by the antitoxin, showed that 18 TA modules were bona fide systems. It was observed that some TA modules are expressed by intracellular bacteria during fibroblast but not epithelial cell infection. Moreover, only five out of ten TA systems for which defective mutants were generated showed a clear reduction in the survival ability of intracellular S. Typhimurium in the fibroblast infection model, whereas only one showed such phenotype in epithelial cells. These data argue for a possible specialization of selected groups of toxins controlling bacterial survival in distinct host cell type

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 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

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.The work in RD and FG's laboratories is supported by grants BFU2011-25939 (RD), CSD2008-00013 (RD and FG), and BIO2013-46281-P/BIO2015-69085-REDC (FG) from the Spanish Ministry of Economy and Competitiveness.Peer reviewedPeer Reviewe

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

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

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

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.IMPORTANCEEnteroinvasive 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

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