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1.
PLoS Pathog ; 16(4): e1008446, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282860

RESUMO

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity.


Assuntos
Disenteria Bacilar/microbiologia , Enterócitos/microbiologia , Células Epiteliais/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Shigella flexneri/fisiologia , Células CACO-2 , Humanos , Listeria monocytogenes/genética , Shigella flexneri/genética
2.
Nat Microbiol ; 5(2): 354-367, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31873204

RESUMO

The cytosolic appearance and propagation of bacteria cause overwhelming cellular stress responses that induce apoptosis under normal conditions. Therefore, successful bacterial colonization depends on the ability of intracellular pathogens to block apoptosis and to safeguard bacterial replicative niches. Here, we show that the cytosolic Gram-negative bacterium Shigella flexneri stalls apoptosis by inhibiting effector caspase activity. Our data identified lipopolysaccharide (LPS) as a bona fide effector caspase inhibitor that directly binds caspases by involving its O-antigen (O Ag) moiety. Bacterial strains that lacked the O Ag or failed to replicate within the cytosol were incapable of blocking apoptosis and exhibited reduced virulence in a murine model of bacterial infection. Our findings demonstrate how Shigella inhibits pro-apoptotic caspase activity, effectively delays coordinated host-cell demise and supports its intracellular propagation. Next to the recently discovered pro-inflammatory role of cytosolic LPS, our data reveal a distinct mode of LPS action that, through the disruption of the early coordinated non-lytic cell death response, ultimately supports the inflammatory breakdown of infected cells at later time points.


Assuntos
Apoptose/fisiologia , Inibidores de Caspase/metabolismo , Caspases Efetoras/metabolismo , Bactérias Gram-Negativas/patogenicidade , Lipopolissacarídeos/metabolismo , Shigella flexneri/patogenicidade , Animais , Citosol/microbiologia , Feminino , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/fisiologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antígenos O/metabolismo , Shigella flexneri/genética , Shigella flexneri/fisiologia , Virulência
3.
Pathog Dis ; 77(7)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31578543

RESUMO

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.


Assuntos
Colo/microbiologia , Disenteria Bacilar/microbiologia , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/fisiologia , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Genes Reporter , Cobaias , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Especificidade de Órgãos , Distribuição Tecidual
4.
Microbiol Spectr ; 7(3)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31152523

RESUMO

Several pathogens have evolved to infect host cells from within, which requires subversion of many host intracellular processes. In the case of Gram-negative pathogenic bacteria, adaptation to an intracellular life cycle relies largely on the activity of type III secretion systems (T3SSs), an apparatus used to deliver effector proteins into the host cell, from where these effectors regulate important cellular functions such as vesicular trafficking, cytoskeleton reorganization, and the innate immune response. Each bacterium is equipped with a unique suite of these T3SS effectors, which aid in the development of an individual intracellular lifestyle for their respective pathogens. Some bacteria adapt to reside and propagate within a customized vacuole, while others establish a replicative niche in the host cytosol. In this article, we review the mechanisms by which T3SS effectors contribute to these different lifestyles. To illustrate the formation of a vacuolar and a cytosolic lifestyle, we discuss the intracellular habitats of the enteric pathogens Salmonella enterica serovar Typhimurium and Shigella flexneri, respectively. These represent well-characterized systems that function as informative models to contribute to our understanding of T3SS-dependent subversion of intracellular processes. Additionally, we present Vibrio parahaemolyticus, another enteric Gram-negative pathogen, as an emerging model for future studies of the cytosolic lifestyle.


Assuntos
Citoplasma/metabolismo , Citoplasma/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Sistemas de Secreção Tipo III/fisiologia , Citosol/microbiologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Negativas/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Transporte Proteico , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Shigella flexneri/patogenicidade , Shigella flexneri/fisiologia , Vacúolos/microbiologia , Vibrio parahaemolyticus/patogenicidade , Vibrio parahaemolyticus/fisiologia
5.
Cell Mol Life Sci ; 76(20): 4165-4178, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31076805

RESUMO

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.


Assuntos
Basigina/genética , Membrana Celular/microbiologia , Interações Hospedeiro-Patógeno/genética , Listeria monocytogenes/fisiologia , Shigella flexneri/fisiologia , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Basigina/antagonistas & inibidores , Basigina/metabolismo , Células CACO-2 , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ciclofilina A/deficiência , Ciclofilina A/genética , Endocitose , Fibroblastos/microbiologia , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Listeria monocytogenes/patogenicidade , Listeria monocytogenes/ultraestrutura , Camundongos , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shigella flexneri/patogenicidade , Shigella flexneri/ultraestrutura , Transdução de Sinais
6.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30988059

RESUMO

Shigella flexneri is an intracellular bacterial pathogen that invades epithelial cells in the colonic mucosa, leading to bloody diarrhea. A previous study showed that S. flexneri forms biofilms in the presence of bile salts, through an unknown mechanism. Here, we investigated the potential role of adhesin-like autotransporter proteins in S. flexneri biofilm formation. BLAST search analysis revealed that the S. flexneri 2457T genome harbors 4 genes, S1242, S1289, S2406, and icsA, encoding adhesin-like autotransporter proteins. Deletion mutants of the S1242, S1289, S2406 and icsA genes were generated and tested for biofilm formation. Phenotypic analysis of the mutant strains revealed that disruption of icsA abolished bile salt-induced biofilm formation. IcsA is an outer membrane protein secreted at the bacterial pole that is required for S. flexneri actin-based motility during intracellular infection. In extracellular biofilms, IcsA was also secreted at the bacterial pole and mediated bacterial cell-cell contacts and aggregative growth in the presence of bile salts. Dissecting individual roles of bile salts showed that deoxycholate is a robust biofilm inducer compared to cholate. The release of the extracellular domain of IcsA through IcsP-mediated cleavage was greater in the presence of cholate, suggesting that the robustness of biofilm formation was inversely correlated with IcsA processing. Accordingly, deletion of icsP abrogated IcsA processing in biofilms and enhanced biofilm formation.


Assuntos
Proteínas de Bactérias/fisiologia , Ácidos e Sais Biliares/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Proteínas de Ligação a DNA/fisiologia , Shigella flexneri/fisiologia , Fatores de Transcrição/fisiologia , Sistemas de Secreção Tipo V/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Ácidos e Sais Biliares/metabolismo , Proteínas de Ligação a DNA/genética , Shigella flexneri/efeitos dos fármacos , Shigella flexneri/metabolismo , Fatores de Transcrição/genética
7.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642900

RESUMO

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigella flexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.


Assuntos
Disenteria Bacilar/microbiologia , Intestinos/citologia , Organoides/microbiologia , Shigella flexneri/fisiologia , Células Cultivadas , Humanos , Intestinos/microbiologia , Modelos Biológicos , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Shigella flexneri/genética , Shigella flexneri/crescimento & desenvolvimento , Shigella flexneri/patogenicidade , Células-Tronco/citologia , Células-Tronco/metabolismo , Virulência
8.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642906

RESUMO

The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface.


Assuntos
Disenteria Bacilar/microbiologia , Mucosa Intestinal/microbiologia , Modelos Biológicos , Organoides/microbiologia , Shigella flexneri/fisiologia , Técnicas de Cultura de Células , Disenteria Bacilar/genética , Disenteria Bacilar/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Shigella flexneri/patogenicidade , Células-Tronco/citologia , Células-Tronco/microbiologia , Virulência
9.
Nanomedicine ; 18: 402-413, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30448527

RESUMO

We reported earlier about nano-formulation of tetracycline through its entrapment within calcium-phosphate nano-particle (CPNP) and about killing of pathogenic bacterium Shigella flexnari 2a, resistant to tetracycline (and 9 other antibiotics), by the nanonized antibiotic (Tet-CPNP). Here, we report on therapeutic role of Tet-CPNP against deadly diarrheal disease 'shigellosis' in mice, caused by Shigella infection. Our findings revealed that occurrence of mushy-stool excretion, colon-length shortening, weight-loss and bacterial colonization in gastrointestinal tract of mice due to shigellosis was significantly reduced by Tet-CPNP treatment. Histo- and immuno-logical studies showed that changes in morphology and level of inflammatory cytokines TNF-α, IL-1ß and IFN-γ in intestinal tissue of Shigella-infected mice were reverted to almost normal features by Tet-CPNP treatment. Bulk tetracycline had no anti-shigellosis action. Thus, nanonization of tetracycline rejuvenated the old, cheap, broad-spectrum antibiotic from obsolescence (due to resistance generation), making it highly beneficial for diarrhea-prone developing countries with limited health-care budgets.


Assuntos
Diarreia/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Disenteria Bacilar/tratamento farmacológico , Nanopartículas/química , Tamanho da Partícula , Shigella flexneri/fisiologia , Tetraciclina/uso terapêutico , Animais , Fosfatos de Cálcio/química , Colo/patologia , Contagem de Colônia Microbiana , Citocinas/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Shigella flexneri/efeitos dos fármacos , Tetraciclina/farmacologia
10.
J Immunol ; 202(1): 218-227, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510071

RESUMO

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-κB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-κB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/imunologia , Células Epiteliais/fisiologia , Inflamação/imunologia , Shigella flexneri/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células HEK293 , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Ligação Proteica , Estabilidade Proteica , Transdução de Sinais
11.
Cell Host Microbe ; 24(6): 866-874.e4, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543779

RESUMO

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.


Assuntos
Lisossomos/metabolismo , Pseudomonas aeruginosa/fisiologia , Septinas/metabolismo , Shigella flexneri/fisiologia , Staphylococcus aureus/fisiologia , Autofagia , Cardiolipinas/genética , Cardiolipinas/metabolismo , Divisão Celular , Proliferação de Células , Citoesqueleto/metabolismo , Células HeLa , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Septinas/genética , Shigella flexneri/genética , Shigella flexneri/patogenicidade , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade
12.
mSphere ; 3(3)2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29898979

RESUMO

Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.


Assuntos
Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue , Disenteria Bacilar/imunologia , Ensaios de Triagem em Larga Escala/métodos , Imunoensaio/métodos , Shigella flexneri/imunologia , Shigella sonnei/imunologia , Antígenos de Bactérias/imunologia , Humanos , Lipopolissacarídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soro/imunologia , Shigella flexneri/fisiologia , Shigella sonnei/fisiologia
13.
mBio ; 9(3)2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946048

RESUMO

Emergency granulopoiesis is a hematopoietic program of stem cell-driven neutrophil production used to counteract immune cell exhaustion following infection. Shigella flexneri is a Gram-negative enteroinvasive pathogen controlled by neutrophils. In this study, we use a Shigella-zebrafish (Danio rerio) infection model to investigate emergency granulopoiesis in vivo We show that stem cell-driven neutrophil production occurs in response to Shigella infection and requires macrophage-independent signaling by granulocyte colony-stimulating factor (Gcsf). To test whether emergency granulopoiesis can function beyond homoeostasis to enhance innate immunity, we developed a reinfection assay using zebrafish larvae that have not yet developed an adaptive immune system. Strikingly, larvae primed with a sublethal dose of Shigella are protected against a secondary lethal dose of Shigella in a type III secretion system (T3SS)-dependent manner. Collectively, these results highlight a new role for emergency granulopoiesis in boosting host defense and demonstrate that zebrafish larvae can be a valuable in vivo model to investigate innate immune memory.IMPORTANCEShigella is an important human pathogen of the gut. Emergency granulopoiesis is the enhanced production of neutrophils by hematopoietic stem and progenitor cells (HSPCs) upon infection and is widely considered a homoeostatic mechanism for replacing exhausted leukocytes. In this study, we developed a Shigella-zebrafish infection model to investigate stem cell-driven emergency granulopoiesis. We discovered that zebrafish initiate granulopoiesis in response to Shigella infection, via macrophage-independent signaling of granulocyte colony-stimulating factor (Gcsf). Strikingly, larvae primed with a sublethal dose of Shigella are protected against a secondary lethal dose of Shigella in a type III secretion system (T3SS)-dependent manner. Taken together, we show that zebrafish infection can be used to capture Shigella-mediated stem cell-driven granulopoiesis and provide a new model system to study stem cell biology in vivo Our results also highlight the potential of manipulating stem cell-driven granulopoiesis to boost innate immunity and combat infectious disease.


Assuntos
Coinfecção/imunologia , Modelos Animais de Doenças , Disenteria Bacilar/microbiologia , Leucopoese , Neutrófilos/imunologia , Shigella flexneri/fisiologia , Animais , Coinfecção/microbiologia , Coinfecção/fisiopatologia , Disenteria Bacilar/imunologia , Disenteria Bacilar/fisiopatologia , Feminino , Humanos , Larva/imunologia , Larva/microbiologia , Macrófagos/imunologia , Masculino , Neutrófilos/citologia , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia
14.
Microbiology ; 164(4): 540-550, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29488864

RESUMO

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Disenteria Bacilar/microbiologia , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Linhagem Celular , Citoplasma/metabolismo , Disenteria Bacilar/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Deleção de Genes , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Humanos , Interleucina-18/análise , Interleucina-18/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Ligação Proteica , Shigella flexneri/genética , Shigella flexneri/metabolismo , Sistemas de Secreção Tipo III/genética
15.
EMBO Rep ; 19(1): 29-42, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29141986

RESUMO

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase-activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host-pathogen interactions.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Proteínas Ativadoras de GTPase/genética , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/patogenicidade , Alvéolos Pulmonares/metabolismo , Células A549 , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/microbiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica , Humanos , Mycobacterium tuberculosis/fisiologia , Fosfolipase D/genética , Fosfolipase D/metabolismo , Polimerização , Alvéolos Pulmonares/microbiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Shigella flexneri/fisiologia , Transdução de Sinais , Especificidade da Espécie , Yersinia pseudotuberculosis/fisiologia
16.
mBio ; 8(6)2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29233899

RESUMO

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN-γ)-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri Rough lipopolysaccharide (LPS) mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6). GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis These studies reveal that human GBP1 uniquely functions as an intracellular "glue trap," inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.IMPORTANCE Several pathogenic bacterial species evolved to invade, reside in, and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to coopt the host's actin polymerization machinery in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria, such as Shigella species, to propel themselves into neighboring cells, thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic "glue trap," capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.


Assuntos
Actinas/metabolismo , Motivos de Aminoácidos , Arginina/química , Citosol/microbiologia , Proteínas de Ligação ao GTP/química , Shigella flexneri/fisiologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/fisiologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Lipopolissacarídeos/genética , Mutação , Antígenos O/metabolismo , Shigella flexneri/patogenicidade , Ubiquitinação , Fatores de Virulência
17.
Microb Pathog ; 113: 378-384, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29138083

RESUMO

Stomach acidity is an important barrier of the human body to protect itself from microbial pathogens entering the small intestine and causing infection. This study examined the survival adaptations of non-acid adapted diarrheal Shigella and Salmonella strains in an environment mimicking the human stomach. The bacterial responses to the challenge of acidic simulated gastric fluid were studied using flow cytometry physiological heterogeneity, membrane integrity and survival (culturability) respectively. Flow cytometry showed that bacterial cells, when exposed to gastric fluid, transformed distinctly, into physiologically heterogeneous sub-populations: intact, stressed and damaged cells, when stained with propidium iodide and thiazole orange. Shigella and Salmonella cells became membrane compromised during initial acid shock (0-30 min), and 80% of these cells shifted to the stressed state throughout gastric fluid exposure. Approximately 10-30% of bacterial strains remained culturable after 60 min of gastric fluid exposure at pH 2.5-4.5, with the percentage increasing with an inoculum size of 102 CFU/ml. This ability of non-acid adapted Shigella and Salmonella sp. to adapt and survive low pH gastric fluid, even though the bacterial numbers decreased or changed to a stressed state, further supports the possible risk of infection when consumed.


Assuntos
Adaptação Fisiológica/fisiologia , Ácido Gástrico , Viabilidade Microbiana , Salmonella typhimurium/fisiologia , Shigella dysenteriae/fisiologia , Shigella flexneri/fisiologia , Ácidos/efeitos adversos , Adaptação Fisiológica/efeitos dos fármacos , Membrana Celular/fisiologia , Contagem de Colônia Microbiana , Disenteria/microbiologia , Citometria de Fluxo , Microbiologia de Alimentos , Heterogeneidade Genética , Humanos , Concentração de Íons de Hidrogênio , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Shigella dysenteriae/efeitos dos fármacos , Shigella dysenteriae/crescimento & desenvolvimento , Shigella dysenteriae/patogenicidade , Shigella flexneri/efeitos dos fármacos , Shigella flexneri/crescimento & desenvolvimento , Shigella flexneri/patogenicidade , Fatores de Tempo
18.
EMBO J ; 36(17): 2567-2580, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701483

RESUMO

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca2+ increases are transient and oscillatory, defining the so-called Ca2+ code that links cell responses to specific Ca2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca2+ signals. Here, we show that by hydrolyzing phosphatidylinositol-(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol-(1,4,5)trisphosphate (InsP3) levels. By modifying InsP3 dynamics and diffusion, IpgD favors the elicitation of long-lasting local Ca2+ signals at Shigella invasion sites and converts Shigella-induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP3-dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca2+-dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.


Assuntos
Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Disenteria Bacilar/metabolismo , Interações Hospedeiro-Patógeno , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Shigella flexneri/fisiologia , Calpaína/metabolismo , Adesão Celular , Células HeLa , Humanos , Transdução de Sinais
19.
Nat Microbiol ; 2: 17063, 2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28481331

RESUMO

Cell-autonomous immunity relies on the ubiquitin coat surrounding cytosol-invading bacteria functioning as an 'eat-me' signal for xenophagy. The origin, composition and precise mode of action of the ubiquitin coat remain incompletely understood. Here, by studying Salmonella Typhimurium, we show that the E3 ligase LUBAC generates linear (M1-linked) polyubiquitin patches in the ubiquitin coat, which serve as antibacterial and pro-inflammatory signalling platforms. LUBAC is recruited via its subunit HOIP to bacterial surfaces that are no longer shielded by host membranes and are already displaying ubiquitin, suggesting that LUBAC amplifies and refashions the ubiquitin coat. LUBAC-synthesized polyubiquitin recruits Optineurin and Nemo for xenophagy and local activation of NF-κB, respectively, which independently restrict bacterial proliferation. In contrast, the professional cytosol-dwelling Shigella flexneri escapes from LUBAC-mediated restriction through the antagonizing effects of the effector E3 ligase IpaH1.4 on deposition of M1-linked polyubiquitin and subsequent recruitment of Nemo and Optineurin. We conclude that LUBAC-synthesized M1-linked ubiquitin transforms bacterial surfaces into signalling platforms for antibacterial immunity reminiscent of antiviral assemblies on mitochondria.


Assuntos
Autofagia , Citosol/microbiologia , NF-kappa B/metabolismo , Salmonella typhimurium/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Imunidade Inata , Camundongos , Poliubiquitina/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Shigella flexneri/imunologia , Shigella flexneri/fisiologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação
20.
Immunol Res ; 65(3): 609-621, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27838822

RESUMO

The c-Jun N-terminal kinase-mitogen-activated protein kinase (JNK-MAPK) pathway assists in modulating signals for growth, survival, and metabolism, thereby coordinating many cellular events during normal and stress conditions. To understand the role of the JNK-MAPK pathway during bacterial infection, an in vivo model organism Caenorhabditis elegans was used. In order to check the involvement of the JNK-MAPK pathway, the survival rate of C. elegans wild type (WT), and JNK-MAPK pathway mutant worms' upon exposure to selective Gram-positive and Gram-negative pathogenic bacteria, was studied. Among the pathogens, Shigella flexneri M9OT was found to efficiently colonize inside the WT and JNK-MAPK pathway mutant worms. qPCR studies had suggested that the above pathway-specific genes kgb-2 and jnk-1 were prominently responsible for the immune response elicited by the host during the M9OT infection. In addition, daf-16, which is a major transcription factor of the insulin/insulin growth factor-1 signaling (IIS) pathway, was also found to be involved during the host response. Crosstalk between IIS and JNK-MAPK pathways has probably been involved in the activation of the host immune system, which consequently leads to lifespan extension. Furthermore, it is also observed that daf-16 activation by JNK-MAPK pathway leads to antimicrobial response, by activating lys-7 expression. These findings suggest that JNK-MAPK is not the sole pathway that enhances the immunity of the host. Nonetheless, the IIS pathway bridges the JNK-MAPK pathway that influences in protecting the host in counter to the M9OT infection.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/imunologia , Disenteria Bacilar/imunologia , Fatores de Transcrição Forkhead/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Shigella flexneri/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Humanos , Imunidade Inata , Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Receptor Cross-Talk , Transdução de Sinais
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