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1.
PLoS Pathog ; 20(9): e1012568, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39298531

RESUMO

Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1ß and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Inflamassomos , Inflamação , Camundongos Endogâmicos C57BL , Animais , Camundongos , Clostridioides difficile/patogenicidade , Toxinas Bacterianas/metabolismo , Inflamação/metabolismo , Inflamassomos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/genética , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos Knockout , Enterotoxinas
2.
Mol Biol Cell ; 35(10): ar125, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39110530

RESUMO

Type IV P-type ATPases (P4-ATPases) are lipid flippases that generate an asymmetric membrane organization essential for cell viability. The five budding yeast P4-ATPases traffic between the Golgi complex, plasma membrane, and endosomes but how they are recycled from the endolysosomal system to the Golgi complex is poorly understood. In this study, we find that P4-ATPase endosomal recycling is primarily driven by the retromer complex and the F-box protein Rcy1. Defects in P4-ATPase recycling result in their mislocalization to the vacuole and a substantial loss of membrane asymmetry. The P4-ATPases contain multiple predicted retromer sorting signals, and the characterization of these signals in Dnf1 and Dnf2 led to the identification of a novel retromer-dependent signal, IPM[ST] that acts redundantly with predicted motifs. Together, these results emphasize the importance of endosomal recycling for the functional localization of P4-ATPases and membrane organization.


Assuntos
Adenosina Trifosfatases , Membrana Celular , Endossomos , Complexo de Golgi , Transporte Proteico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacúolos , Endossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexo de Golgi/metabolismo , Membrana Celular/metabolismo , Adenosina Trifosfatases/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sinais Direcionadores de Proteínas , ATPases do Tipo-P/metabolismo , Transportadores de Cassetes de Ligação de ATP
3.
JCI Insight ; 7(15)2022 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-35763345

RESUMO

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin ß1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin ß1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin ß1-mediated adhesion to ECM but are dependent on integrin ß1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin ß1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.


Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Adenocarcinoma/genética , Adenocarcinoma de Pulmão/genética , Animais , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Integrinas , Ligantes , Neoplasias Pulmonares/patologia , Camundongos
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