RESUMO
Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Feminino , Humanos , Animais , Camundongos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Receptor de Morte Celular Programada 1 , Interferon gama , Recidiva Local de Neoplasia , Modelos Animais de Doenças , Poli(ADP-Ribose) PolimerasesRESUMO
Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte de Cátions , Legionella pneumophila , Metais/metabolismo , Vacúolos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Doença dos Legionários/genética , Doença dos Legionários/metabolismo , Doença dos Legionários/patologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Células U937 , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ΔmavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Legionella pneumophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Proliferação de Células , Meios de Cultura/química , Citoplasma/metabolismo , Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Sistema Imunitário , Ferro/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Dados de Sequência Molecular , Fagossomos/metabolismo , Mutação Puntual , Estrutura Secundária de Proteína , Transporte Proteico , Células RAW 264.7 , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Células U937 , Vacúolos/metabolismo , VirulênciaRESUMO
Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular , Histoplasma/fisiologia , Histoplasmose/microbiologia , Macrófagos/microbiologia , Macrófagos/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Caspases/genética , Caspases/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Genes Fúngicos , Genoma Fúngico , Histoplasma/crescimento & desenvolvimento , Histoplasma/patogenicidade , Camundongos , Dados de Sequência Molecular , Mutação , Fatores de Virulência/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Macrophages are the front line of immune defense against invading microbes. Microbes, however, have evolved numerous and diverse mechanisms to thwart these host immune defenses and thrive intracellularly. Legionella pneumophila, a Gram-negative pathogen of amoebal and mammalian phagocytes, is one such microbe. In humans, it causes a potentially fatal pneumonia referred to as Legionnaires' disease. Armed with the Icm/Dot type IV secretion system, which is required for virulence, and approximately 300 translocated proteins, Legionella is able to enter host cells, direct the biogenesis of its own vacuolar compartment, and establish a replicative niche, where it grows to high levels before lysing the host cell. Efforts to understand the pathogenesis of this bacterium have focused on characterizing the molecular activities of its many effectors. In this article, we highlight recent strides that have been made in understanding how Legionella effectors mediate host-pathogen interactions.
Assuntos
Sistemas de Secreção Bacterianos , Interações Hospedeiro-Patógeno , Legionella pneumophila/imunologia , Legionella pneumophila/metabolismo , Doença dos Legionários/imunologia , Doença dos Legionários/microbiologia , Animais , Autofagia , Transporte Biológico , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/microbiologia , Humanos , Doença dos Legionários/genética , Doença dos Legionários/metabolismo , Lipídeos de Membrana/metabolismo , Fagócitos/imunologia , Fagócitos/microbiologia , TranscriptomaRESUMO
Histoplasma capsulatum is a fungal respiratory pathogen that survives and replicates within the phagolysosome of macrophages. The molecular factors it utilizes to subvert macrophage antimicrobial defenses are largely unknown. Although the ability of H. capsulatum to prevent acidification of the macrophage phagolysosome is thought to be critical for intracellular survival, this hypothesis has not been tested since H. capsulatum mutants that experience decreased phagosomal pH have not been identified. In a screen to identify H. capsulatum genes required for lysis of bone marrow-derived macrophages (BMDMs), we identified an insertion mutation disrupting the H. capsulatum homolog of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) lyase (HCL1). In addition to its inability to lyse macrophages, the hcl1 mutant had a severe growth defect in BMDMs, indicating that HMG CoA lyase gene function is critical for macrophage colonization. In other organisms, HMG CoA lyase catalyzes the last step in the leucine catabolism pathway. In addition, both fungi and humans deficient in HMG CoA lyase accumulate acidic intermediates as a consequence of their inability to catabolize leucine. Consistent with observations in other organisms, the H. capsulatum hcl1 mutant was unable to grow on leucine as the major carbon source, caused acidification of its growth medium in vitro, and resided in an acidified vacuole within macrophages. Mice infected with the hcl1 mutant took significantly longer to succumb to infection than mice infected with the wild-type strain. Taken together, these data indicate the importance of Hcl1 function in H. capsulatum replication in the harsh growth environment of the macrophage phagosome.