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1.
Nat Commun ; 15(1): 3120, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600106

RESUMO

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.


Assuntos
Infecções por Salmonella , Salmonella typhimurium , Humanos , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Salmonella/microbiologia , Membrana Celular/metabolismo , Membranas/metabolismo , Células HeLa
2.
Nat Commun ; 12(1): 4707, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349110

RESUMO

Salmonella utilizes translocated virulence proteins (termed effectors) to promote host cell invasion. The effector SopD contributes to invasion by promoting scission of the plasma membrane, generating Salmonella-containing vacuoles. SopD is expressed in all Salmonella lineages and plays important roles in animal models of infection, but its host cell targets are unknown. Here we show that SopD can bind to and inhibit the small GTPase Rab10, through a C-terminal GTPase activating protein (GAP) domain. During infection, Rab10 and its effectors MICAL-L1 and EHBP1 are recruited to invasion sites. By inhibiting Rab10, SopD promotes removal of Rab10 and recruitment of Dynamin-2 to drive scission of the plasma membrane. Together, our study uncovers an important role for Rab10 in regulating plasma membrane scission and identifies the mechanism used by a bacterial pathogen to manipulate this function during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas de Bactérias/genética , Dinamina II , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Salmonella typhimurium/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Virulência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
3.
Nat Microbiol ; 4(12): 2511-2522, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31611645

RESUMO

Many bacterial pathogens express virulence proteins that are translocated into host cells (herein referred to as effectors), where they can interact with target proteins to manipulate host cell processes. These effector-host protein interactions are often dynamic and transient in nature, making them difficult to identify using traditional interaction-based methods. Here, we performed a systematic comparison between proximity-dependent biotin labelling (BioID) and immunoprecipitation coupled with mass spectrometry to investigate a series of Salmonella type 3 secreted effectors that manipulate host intracellular trafficking (SifA, PipB2, SseF, SseG and SopD2). Using BioID, we identified 632 candidate interactions with 381 unique human proteins, collectively enriched for roles in vesicular trafficking, cytoskeleton components and transport activities. From the subset of proteins exclusively identified by BioID, we report that SifA interacts with BLOC-2, a protein complex that regulates dynein motor activity. We demonstrate that the BLOC-2 complex is necessary for SifA-mediated positioning of Salmonella-containing vacuoles, and affects stability of the vacuoles during infection. Our study provides insight into the coordinated activities of Salmonella type 3 secreted effectors and demonstrates the utility of BioID as a powerful, complementary tool to characterize effector-host protein interactions.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Transporte Proteico/fisiologia , Salmonella/fisiologia , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Biotina , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Transporte Proteico/genética , Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Coloração e Rotulagem
4.
Cell Microbiol ; 20(10): e12938, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30010242

RESUMO

Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R-Ras1 also contributes to SopB-dependent Akt activation by promoting the localised production of PI(3,4)P2 /PI(3,4,5)P3 . Our studies reveal new signalling factors involved in SopB-dependent Salmonella infection.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Infecções por Salmonella/microbiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo
5.
Cell Microbiol ; 20(10): e12866, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29885024

RESUMO

Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells.


Assuntos
Actinas/metabolismo , Miosinas/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/patogenicidade , Septinas/metabolismo , Linhagem Celular Tumoral , Proteínas Fetais/metabolismo , Forminas , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Salmonella typhimurium/genética , Quinases Associadas a rho/metabolismo
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