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1.
Proc Natl Acad Sci U S A ; 120(36): e2308752120, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37639588

RESUMO

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.


Assuntos
Antioxidantes , Coxiella , Humanos , Animais , Camundongos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Estresse Oxidativo , Morte Celular , Mamíferos
2.
PLoS Pathog ; 19(7): e1011491, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37399210

RESUMO

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.


Assuntos
Coxiella burnetii , Febre Q , Animais , Humanos , Peptidilprolil Isomerase/metabolismo , Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/metabolismo , Bactérias/metabolismo , Macrófagos/metabolismo
3.
PLoS Pathog ; 18(1): e1010166, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35007292

RESUMO

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.


Assuntos
Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Macrófagos/microbiologia , Vacúolos/microbiologia , Virulência/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
J Bacteriol ; 205(3): e0001323, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36847508

RESUMO

A recent study by S. Wachter, C. L. Larson, K. Virtaneva, K. Kanakabandi, et al. (J Bacteriol 205:e00416-22, 2023, https://doi.org/10.1128/JB.00416-22) utilizes new technologies to examine the role of two-component systems in Coxiella burnetii. This research demonstrates that the zoonotic pathogen C. burnetii mediates complex transcriptional control, throughout different bacterial phases and environmental conditions, with relatively few regulatory elements.


Assuntos
Coxiella burnetii , Febre Q , Humanos , Coxiella burnetii/genética , Regulação da Expressão Gênica , Febre Q/microbiologia
5.
Mol Microbiol ; 117(2): 235-251, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34874584

RESUMO

Anti-bacterial autophagy, known as xenophagy, is a host innate immune response that targets invading pathogens for degradation. Some intracellular bacteria, such as the enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilize effector proteins to interfere with autophagy. One such S. Typhimurium effector, SopF, inhibits recruitment of ATG16L1 to damaged Salmonella-containing vacuoles (SCVs), thereby inhibiting the host xenophagic response. SopF is also required to maintain the integrity of the SCV during the early stages of infection. Here we show disruption of the SopF-ATG16L1 interaction leads to an increased proportion of cytosolic S. Typhimurium. Furthermore, SopF was utilized as a molecular tool to examine the requirement for ATG16L1 in the intracellular lifestyle of Coxiella burnetii, a bacterium that requires a functional autophagy pathway to replicate efficiently and form a single, spacious vacuole called the Coxiella-containing vacuole (CCV). ATG16L1 is required for CCV expansion and fusion but does not influence C. burnetii replication. In contrast, SopF did not affect CCV formation or replication, demonstrating that the contribution of ATG16L1 to CCV biogenesis is via its role in autophagy, not xenophagy. This study highlights the diverse capabilities of bacterial effector proteins to dissect the molecular details of host-pathogen interactions.


Assuntos
Coxiella burnetii , Vacúolos , Proteínas Relacionadas à Autofagia/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coxiella/metabolismo , Coxiella burnetii/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Vacúolos/metabolismo
6.
Mol Cell Proteomics ; 20: 100005, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33177156

RESUMO

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.


Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/fisiologia , Interações Hospedeiro-Patógeno , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Células HEK293 , Células HeLa , Humanos , Proteoma , Proteômica , Febre Q , Células THP-1
7.
Proc Natl Acad Sci U S A ; 117(12): 6801-6810, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152125

RESUMO

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of the C. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical for C. burnetii virulence. Indeed, the C. burnetii PmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate the C. burnetii PmrA/B two-component system. This study has further enhanced our understanding of C. burnetii pathogenesis, the host-pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Coxiella burnetii/fisiologia , Interações Hospedeiro-Patógeno , Lisossomos/metabolismo , Febre Q/microbiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Lisossomos/microbiologia , Transporte Proteico , Tripeptidil-Peptidase 1 , Virulência
8.
PLoS Pathog ; 15(7): e1007959, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31339948

RESUMO

The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole.


Assuntos
Salmonella typhimurium/fisiologia , Sistemas de Secreção Tipo III/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Células HeLa , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Camundongos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Sistemas de Secreção Tipo III/química , Sistemas de Secreção Tipo III/genética , Vacúolos/metabolismo , Vacúolos/microbiologia
9.
Cell Microbiol ; 22(5): e13154, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31872956

RESUMO

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP+ binding site, suggesting it may facilitate NADP(H) regeneration by C. burnetii, a key process for surviving oxidative stress. Purified recombinant 6×His-SdrA was able to convert NADP+ to NADP(H) in vitro. Mutation to alanine of a conserved glycine residue at position 12 within the predicted NADP binding site abolished significant enzymatic activity. Complementation of the sdrA mutant (sdrA::Tn) with plasmid-expressed SdrA restored intracellular replication to wild-type levels, but expressing enzymatically inactive G12A_SdrA did not. The sdrA::Tn mutant was more susceptible in vitro to oxidative stress, and treating infected host cells with L-ascorbate, an anti-oxidant, partially rescued the intracellular growth defect of sdrA::Tn. Finally, stable isotope labelling studies demonstrated a shift in flux through metabolic pathways in sdrA::Tn consistent with the presence of increased oxidative stress, and host cells infected with sdrA::Tn had elevated levels of reactive oxygen species compared with C. burnetii NMII.


Assuntos
Coxiella burnetii/metabolismo , NADP/metabolismo , Estresse Oxidativo , Coxiella burnetii/crescimento & desenvolvimento , Citoplasma/metabolismo , Células HeLa , Humanos , Macrófagos/microbiologia , Mutação , NADP/genética , Febre Q/metabolismo , Febre Q/microbiologia , Regeneração , Vacúolos/microbiologia
10.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31818957

RESUMO

Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella-containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes. To characterize the host-pathogen interactions that occur during C. burnetii infection, stable-isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify changes in the host proteome during infection of a human-derived macrophage cell line. These data revealed that the abundances of many proteins involved in host cell autophagy and lysosome biogenesis were increased in infected cells. Thus, the role of the host transcription factors TFEB and TFE3, which regulate the expression of a network of genes involved in autophagy and lysosomal biogenesis, were examined in the context of C. burnetii infection. During infection with C. burnetii, both TFEB and TFE3 were activated, as demonstrated by the transport of these proteins from the cytoplasm into the nucleus. The nuclear translocation of these transcription factors was shown to be dependent on the T4SS, as a Dot/Icm mutant showed reduced nuclear translocation of TFEB and TFE3. This was supported by the observation that blocking bacterial translation with chloramphenicol resulted in the movement of TFEB and TFE3 back into the cytoplasm. Silencing of the TFEB and TFE3 genes, alone or in combination, significantly reduced the size of the CCV, which indicates that these host transcription factors facilitate the expansion and maintenance of the organelle that supports C. burnetii intracellular replication.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Coxiella burnetii/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Macrófagos/metabolismo , Proteoma/metabolismo
11.
Infect Immun ; 88(6)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32205404

RESUMO

The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.


Assuntos
Proteínas de Bactérias/genética , Coxiella burnetii/fisiologia , Interações Hospedeiro-Patógeno , Febre Q/microbiologia , Proteínas de Bactérias/metabolismo , Membrana Celular , Interações Hospedeiro-Patógeno/genética , Humanos , Metaboloma , Metabolômica/métodos , Viabilidade Microbiana , Modelos Biológicos , Mutação , Transporte Proteico , Vacúolos/microbiologia , Virulência/genética , Fatores de Virulência/genética
12.
Biochem J ; 476(19): 2851-2867, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31527117

RESUMO

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [13C]glucose or [13C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.


Assuntos
Coxiella burnetii/metabolismo , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Interações Hospedeiro-Patógeno , Febre Q/microbiologia , Virulência/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Transporte Biológico , Coxiella burnetii/patogenicidade , Gluconeogênese/fisiologia , Glicólise/fisiologia , Células HeLa , Humanos , Lepidópteros/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Células THP-1
13.
Biochem J ; 476(22): 3435-3453, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31675053

RESUMO

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.


Assuntos
Proteínas de Bactérias/metabolismo , N-Glicosil Hidrolases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , N-Glicosil Hidrolases/genética , Purina-Núcleosídeo Fosforilase/genética , S-Adenosil-Homocisteína/metabolismo , Salmonella typhimurium/genética , Virulência
14.
J Biol Chem ; 293(48): 18636-18645, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30315113

RESUMO

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.


Assuntos
Coxiella burnetii/crescimento & desenvolvimento , Coxiella burnetii/metabolismo , NAD/biossíntese , Febre Q/microbiologia , Cromatografia Gasosa , Cromatografia Líquida , Elementos de DNA Transponíveis , Células HeLa , Humanos , Espectrometria de Massas , Mutagênese Sítio-Dirigida
15.
J Biol Chem ; 293(24): 9506-9519, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29720401

RESUMO

Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and S-adenosylmethionine-mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.


Assuntos
Metionina/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Animais , Transporte Biológico , Vias Biossintéticas , Feminino , Células HeLa , Humanos , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium/metabolismo , Virulência
16.
Infect Immun ; 86(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29339460

RESUMO

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary for C. burnetii virulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identify C. burnetii Nine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies in Coxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations in cbu0414 (coxH1), cbu0513, cbu0978 (cem3), cbu1387 (cem6), cbu1524 (caeA), cbu1752, or cbu2028 resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. The cbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain of C. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by the cbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire of C. burnetii factors that contribute to CCV biogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/fisiologia , Febre Q/metabolismo , Febre Q/microbiologia , Autofagossomos/metabolismo , Sistemas de Secreção Bacterianos , Coxiella burnetii/genética , Coxiella burnetii/patogenicidade , Elementos de DNA Transponíveis , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mutação , Transporte Proteico , Vacúolos/metabolismo
17.
PLoS Pathog ; 12(12): e1006101, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28002452

RESUMO

Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.


Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Coxiella burnetii/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Febre Q/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Western Blotting , Coxiella burnetii/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/metabolismo
18.
Curr Top Microbiol Immunol ; 413: 243-268, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29536362

RESUMO

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which manifests in severe outbreaks and is associated with important health and economic burden. Moreover, C. burnetii belongs to the list of class B bioterrorism organisms, as it is an airborne and highly infective pathogen with remarkable resistance to environmental stresses. Detailed study of the host-pathogen interaction during C. burnetii infection has been hampered due to the obligate intracellular nature of this pathogen. However, the development of an axenic culture medium, together with the implementation of bioinformatics tools and high-content screening approaches, have significantly progressed C. burnetii research in the last decade. This has facilitated identification of the Dot/Icm type IV secretion system (T4SS) as an essential virulence factor. T4SS is used to deliver an arsenal of effector proteins into the cytoplasm of the host cell. These effectors mediate the survival of the host cell and the development of very large replicative compartments called Coxiella-containing vacuoles (CCVs). Biogenesis of the CCV relies on T4SS-dependent re-routing of numerous intracellular trafficking pathways to deliver membranes and nutrients that are essential for bacterial replication. This review aims to illustrate the key milestones that have contributed to ascribe C. burnetii as a model organism for the study of host/pathogen interactions as well as presenting an up-to-date description of our knowledge of the cell biology of C. burnetii infections.


Assuntos
Coxiella burnetii , Febre Q , Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Sistemas de Secreção Tipo IV
19.
J Infect Dis ; 215(3): 440-451, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27932612

RESUMO

BACKGROUND: Legionella longbeachae (Llo) and Legionella pneumophila (Lpn) are the most common pneumonia-causing agents of the genus. Although both species can be lethal to humans and are highly prevalent, little is known about the molecular pathogenesis of Llo infections. In murine models of infection, Lpn infection is self-limited, whereas Llo infection is lethal. METHODS: We used mouse macrophages, human macrophages, human epithelial cells, and mouse infections in vivo to evaluate multiple parameters of the infection. RESULTS: We determined that the Llo Dot/Icm secretion system is critical for virulence. Different than Lpn, Llo disseminates and the animals develop a severe pulmonary failure, as demonstrated by lung mechanics and blood oxygenation assays. As compared to Lpn, Llo is immunologically silent and fails to trigger the production of cytokines in human pulmonary epithelial cells and in mouse and human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice supported the participation of cytokines for the resistance phenotype. CONCLUSIONS: Both Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection outcome is strikingly different. Llo is immunologically silent, highly virulent, and lethal. The differences reported herein may reflect unappreciated clinical differences in patients infected with Lpn or Llo.


Assuntos
Legionella longbeachae/imunologia , Legionella longbeachae/patogenicidade , Legionelose/imunologia , Animais , Citocinas/metabolismo , Resistência à Doença/imunologia , Feminino , Humanos , Legionella pneumophila/imunologia , Legionelose/microbiologia , Legionelose/patologia , Legionelose/fisiopatologia , Leucócitos Mononucleares , Pulmão/fisiopatologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade da Espécie , Virulência
20.
Infect Immun ; 85(5)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28242621

RESUMO

Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.


Assuntos
Coxiella burnetii/crescimento & desenvolvimento , Coxiella burnetii/metabolismo , Interações Hospedeiro-Patógeno , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Febre Q/microbiologia , Fatores de Virulência/metabolismo , Linhagem Celular , Células Epiteliais/microbiologia , Humanos , Peso Molecular , Monócitos/microbiologia , Fatores de Virulência/química
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