RESUMO
BACKGROUND: Hereditary angioedema is a rare genetic disease that leads to severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1), with the goal of lifelong control of angioedema attacks after a single dose. METHODS: In this phase 1 dose-escalation portion of a combined phase 1-2 trial of NTLA-2002 in adults with hereditary angioedema, we administered NTLA-2002 at a single dose of 25 mg, 50 mg, or 75 mg. The primary end points were the safety and side-effect profile of NTLA-2002 therapy. Secondary and exploratory end points included pharmacokinetics, pharmacodynamics, and clinical efficacy determined on the basis of investigator-confirmed angioedema attacks. RESULTS: Three patients received 25 mg of NTLA-2002, four received 50 mg, and three received 75 mg. At all dose levels, the most common adverse events were infusion-related reactions and fatigue. No dose-limiting toxic effects, serious adverse events, grade 3 or higher adverse events, or clinically important laboratory findings were observed after the administration of NTLA-2002. Dose-dependent reductions in the total plasma kallikrein protein level were observed between baseline and the latest assessment, with a mean percentage change of -67% in the 25-mg group, -84% in the 50-mg group, and -95% in the 75-mg group. The mean percentage change in the number of angioedema attacks per month between baseline and weeks 1 through 16 (primary observation period) was -91% in the 25-mg group, -97% in the 50-mg group, and -80% in the 75-mg group. Among all the patients, the mean percentage change in the number of angioedema attacks per month from baseline through the latest assessment was -95%. CONCLUSIONS: In this small study, a single dose of NTLA-2002 led to robust, dose-dependent, and durable reductions in total plasma kallikrein levels, and no severe adverse events were observed. In exploratory analyses, reductions in the number of angioedema attacks per month were observed at all dose levels. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830.).
Assuntos
Angioedemas Hereditários , Sistemas CRISPR-Cas , Edição de Genes , Adulto , Humanos , Angioedema , Angioedemas Hereditários/sangue , Angioedemas Hereditários/tratamento farmacológico , Angioedemas Hereditários/genética , Proteína Inibidora do Complemento C1/uso terapêutico , Relação Dose-Resposta a Droga , Edição de Genes/métodos , Calicreína Plasmática/genética , Resultado do TratamentoRESUMO
Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Legionella pneumophila/metabolismo , Peptidoglicano/metabolismo , Vacúolos/metabolismo , Legionella/metabolismo , Lisossomos/metabolismo , Proteínas de Bactérias/metabolismo , Doença dos Legionários/microbiologiaRESUMO
Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.
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Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Escherichia coli/genética , Farmacorresistência Bacteriana , Plasmídeos , Biofilmes , Conjugação GenéticaRESUMO
The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.
RESUMO
The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Linhagem Celular , Cristalografia por Raios X , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Modelos Moleculares , Complexos Multiproteicos , Ligação Proteica , Transporte Proteico , Sistemas de Secreção Tipo IV/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria1-3. Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system4,5, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS-ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes.
Assuntos
Legionella pneumophila/química , Sistemas de Secreção Tipo II/química , Sistemas de Secreção Tipo II/ultraestrutura , Proteínas de Bactérias/química , Microscopia Crioeletrônica , Fímbrias Bacterianas/química , Fímbrias Bacterianas/ultraestrutura , Legionella pneumophila/ultraestrutura , Modelos Moleculares , Fatores de VirulênciaRESUMO
Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.
Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Desubiquitinantes/metabolismo , Legionella pneumophila/metabolismo , Diester Fosfórico Hidrolases/metabolismo , ADP-Ribosilação , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ubiquitina , Ubiquitinação , Vacúolos/microbiologiaRESUMO
Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.
Assuntos
Legionella pneumophila/química , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/ultraestrutura , Polaridade Celular , Tomografia com Microscopia Eletrônica , Legionella pneumophila/citologia , Legionella pneumophila/genética , Legionella pneumophila/ultraestrutura , Microscopia de Fluorescência , Mutação , Periplasma/química , Periplasma/ultraestrutura , Multimerização Proteica , Sistemas de Secreção Tipo IV/químicaRESUMO
A recurrent emerging theme is the targeting of proteins to subcellular microdomains within bacterial cells, particularly to the poles. In most cases, it has been assumed that this localization is critical to the protein's function. Legionella pneumophila uses a type IVB secretion system (T4BSS) to export a large number of protein substrates into the cytoplasm of host cells. Here we show that the Legionella export apparatus is localized to the bacterial poles, as is consistent with many T4SS substrates being retained on the phagosomal membrane adjacent to the poles of the bacterium. More significantly, we were able to demonstrate that polar secretion of substrates is critically required for Legionella's alteration of the host endocytic pathway, an activity required for this pathogen's virulence.
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Legionella pneumophila/fisiologia , Legionella pneumophila/patogenicidade , Sistemas de Secreção Tipo IV/metabolismo , Animais , Divisão Celular , Polaridade Celular , Feminino , CamundongosRESUMO
Although tetracyclines are an important class of antibiotics for use in agriculture and the clinic, their efficacy is threatened by increasing resistance. Resistance to tetracyclines can occur through efflux, ribosomal protection, or enzymatic inactivation. Surprisingly, tetracycline enzymatic inactivation has remained largely unexplored, despite providing the distinct advantage of antibiotic clearance. The tetracycline destructases are a recently discovered family of tetracycline-inactivating flavoenzymes from pathogens and soil metagenomes that have a high potential for broad dissemination. Here, we show that tetracycline destructases accommodate tetracycline-class antibiotics in diverse and novel orientations for catalysis, and antibiotic binding drives unprecedented structural dynamics facilitating tetracycline inactivation. We identify a key inhibitor binding mode that locks the flavin adenine dinucleotide cofactor in an inactive state, functionally rescuing tetracycline activity. Our results reveal the potential of a new tetracycline and tetracycline destructase inhibitor combination therapy strategy to overcome resistance by enzymatic inactivation and restore the use of an important class of antibiotics.
Assuntos
Antibacterianos/metabolismo , Inibidores Enzimáticos/farmacologia , Legionella longbeachae/efeitos dos fármacos , Legionella longbeachae/enzimologia , Resistência a Tetraciclina/efeitos dos fármacos , Tetraciclina/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Flavina-Adenina Dinucleotídeo/metabolismo , Legionella longbeachae/metabolismo , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Tetraciclina/química , Tetraciclina/farmacologiaRESUMO
OBJECTIVE: Pseudomonas aeruginosa, a known biofilm-forming organism, is an opportunistic pathogen that plays an important role in chronic otitis media, tracheitis, cholesteatoma, chronic wounds, and implant infections. Eradication of biofilm infections has been a challenge because the biofilm phenotype provides bacteria with a protective environment from the immune system and antibiotics; thus, there has been great interest in adjunctive molecules that may inhibit biofilm formation or cause biofilm dispersal. There are reports that D-amino acids may inhibit biofilms. In this study, we test the ability of various D-amino acids to inhibit P. aeruginosa biofilm formation in vitro. STUDY DESIGN: We evaluated the effect of D-alanine (10 mM), D-leucine (10 mM), D-methionine (10 mM), D-tryptophan (10 mM), and D-tyrosine (10 uM and 1 mM) on biofilm formation in two commonly studied laboratory strains of P. aeruginosa: PAO1 and PA14. METHODS: Biofilms were grown in 24-well and 96-well tissue culture plates, documented photographically and stained with 0.1% crystal violet and solubilized in 33% glacial acetic acid for quantification. RESULTS: In strains PAO1 and PA14, the addition of D-amino acids did not result in an inhibitory effect on biofilm growth in 24-well plates. Repeating the study in 96-well plates confirmed our findings that D-amino acids do not inhibit biofilm formation of P. aeruginosa. CONCLUSION: We conclude that D-amino acids only slow the production of biofilms rather than completely prevent biofilm formation; therefore, D-amino acids represent a poor option for potential clinically therapeutic interventions. LEVEL OF EVIDENCE: N/A.
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OBJECTIVE: Chronic, persistent infections complicate otologic procedures utilizing implantable devices such as cochlear implants or tympanostomy tubes. These infections are thought to be due to the establishment of microbial biofilms on implant surfaces. To address this issue, we hypothesized that surface charge modification may inhibit the formation of Pseudomonas aeruginosa biofilms on implant surfaces in vitro and in vivo. STUDY DESIGN: We evaluated the effect of surface charge modification on bacterial biofilm formation by assessing the effect of the surface charge on bacterial adhesion in vitro and bacterial persistence in vivo. METHODS: To study the effect of surface charge in vitro, the surface wells in culture plates were modified using a layer-by-layer polyelectrolyte assembly method. Bacterial adherence was measured at 30-, 60-, and 120-minute intervals. To study the effect of surface charge modification in vivo, the surface of titanium microscrews was similarly modified and then surgically implanted into the dorsal calvaria of adult rats and inoculated with bacteria. Two weeks after implantation and inoculation, the number of bacteria remaining in vivo was evaluated. RESULTS: Surface charge modification results in a significant decrease in adherence of bacteria in vitro. Surface charge modification of titanium microscrew implants also resulted in a significant decrease in P. aeruginosa recovered 2 weeks after surgical implantation. CONCLUSION: Charge modification decreases the number of bacteria adherent to a surface in vitro and decreases the risk and severity of implant infection in an in vivo rat infection model. These results have promising biomedical applications. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1655-1661, 2017.
Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Implantes Cocleares/microbiologia , Ventilação da Orelha Média/instrumentação , Infecções Relacionadas à Prótese/microbiologia , Pseudomonas aeruginosa/fisiologia , Eletricidade Estática , Propriedades de Superfície , Animais , Fenômenos Fisiológicos Bacterianos , Doença Crônica , Ensaio de Unidades Formadoras de Colônias , Técnicas In Vitro , Masculino , Ratos , TitânioRESUMO
Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Legionella pneumophila/química , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Legionella pneumophila/ultraestrutura , PlasmídeosRESUMO
Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.
Assuntos
Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/fisiologia , Ubiquitinação , ADP Ribose Transferases , Animais , Proteínas de Bactérias/genética , Células COS , Catálise , Chlorocebus aethiops , Rearranjo Gênico , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/patogenicidade , Proteínas Nogo/genética , Ubiquitina/metabolismoRESUMO
HYPOTHESIS: Bacterial biofilm formation within cholesteatomas is responsible for increased persistence and tissue destruction and Pseudomonas aeruginosa deficient in biofilm formation (PAO1 ΔfleQ) are less virulent than the parent bacteria. BACKGROUND: Infected aural cholesteatomas have been demonstrated to be more destructive than uninfected cholesteatomas and infections are more persistent. The chronicity and persistence of infections within cholesteatomas may be because of the presence of biofilm formation. METHODS: Twenty-seven mutant strains of PAO1 were screened for surface adherence. These strains were also screened for static biofilm formation. The biofilms were quantified by staining with crystal violet. Aural cholesteatomas were then induced in Mongolian gerbils by ligation of the ear canal. At the time of ligation, the ear canals were inoculated with wild-type PAO1 and a biofilm deficient PAO1 ΔfleQ strain of P. aeruginosa. A 7 weeks course of ciprofloxacin (20âmg/kg/day) was started on postoperative day 7. Eight weeks after induction of cholesteatomas, the cholesteatoma size, levels of bone destruction, and levels of bone remodeling were evaluated using microCT imaging. RESULTS: PAO1 ΔfleQ was identified as a poorly adherent and deficient biofilm forming mutant strain of P. aeruginosa. Infected cholesteatomas had more growth, bone destruction and bone remodeling than uninfected cholesteatomas. However, there was no difference observed between cholesteatomas infected with PAO1 (biofilm competent strain) and PAO1 ΔfleQ (biofilm deficient strain). CONCLUSION: We demonstrate that the biofilm phenotype is not an important virulence factor in cholesteatomas infected with P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Colesteatoma da Orelha Média/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Transativadores/metabolismo , Virulência/fisiologia , Animais , Modelos Animais de Doenças , Gerbillinae , Fatores de Virulência/metabolismoRESUMO
Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.
Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Membrana/metabolismo , Vacúolos/metabolismo , Fatores de Virulência/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Transporte Proteico/fisiologia , Especificidade por SubstratoRESUMO
The Legionella pneumophilaâ Dot/Icm T4SS injects â¼ 300 protein effector proteins into host cells. Dot/Icm substrates have been proposed to contain a carboxy-terminal signal sequence that is necessary and sufficient for export, although both traits have been demonstrated for only a small fraction of these proteins. In this study, we discovered that export of the substrate SidJ is mediated by dual signal sequences that include a conventional C-terminal domain and a novel internal motif. The C-terminal signal sequence facilitates secretion of SidJ into host cells at early points of infection, whereas the internal signal sequence mediates secretion at later time points. Interestingly, only the internal signal sequence is necessary for complementation of the intracellular growth defect of a ΔsidJ mutant. Although this is the first report of a Dot/Icm substrate being secreted by an internal signal sequence, many other substrates may be exported in a similar manner. In addition, efficient translocation of SidJ is dependent on the chaperone-like type IV adaptors IcmS/IcmW. Five IcmS/IcmW binding domains that are distinct from both signal sequences were elucidated and, interestingly, only secretion mediated by the internal signal sequence requires IcmS/IcmW. Thus, Legionella employs multiple sophisticated molecular mechanisms to regulate the export of SidJ.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/metabolismo , Sinais Direcionadores de Proteínas , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Chaperonas Moleculares , Transporte ProteicoRESUMO
HYPOTHESIS: When experimental cholesteatomas are infected with Pseudomonas aeruginosa (PA) mutants lacking factors associated with the formation of biofilms, host defenses are more effective against these strains when compared with wild-type strains (PAO1 and OPPA8) in preventing tissue destruction. BACKGROUND: Previous studies have identified biofilms within chronically infected aural cholesteatomas. These infected cholesteatomas are associated with increased tissue destruction. Because biofilms are highly resistant to host defenses leading to prolonged infection, we propose that the biofilm phenotype of P. aeruginosa may be a virulence factor leading to persistence of infection and increased tissue destruction. METHODS: Aural cholesteatomas were induced in Mongolian gerbils. At the time of induction, the ear canals were inoculated with wild-type (PAO1 and OPPA8) and biofilm-deficient (PAO1 ΔpilA, PAO1 algD::aacC1 and PAO1 galU::aacC1) strains of P. aeruginosa. After 8 weeks, the size of the cholesteatomas and levels of bone destruction and deposition were measured using microCT scanning and double fluorochrome bone labeling. RESULT: Infected cholesteatomas resulted in increased growth, bone destruction, and bone deposition when compared with vehicle-only controls. We observed no differences between the wild-type (biofilm forming) and the biofilm-deficient strains of P. aeruginosa. CONCLUSION: Our hypothesis that biofilm formation is a virulence factor in cholesteatomas infected with P. aeruginosa was not supported. A number of interpretations of these data are reasonable. It is possible that biofilms are not critical in infected cholesteatomas. Alternatively, the mutants that are deficient in generating biofilms in vitro may be able to form effective biofilms in vivo using alternative pathways.
Assuntos
Biofilmes , Colesteatoma da Orelha Média/microbiologia , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/toxicidade , Animais , Modelos Animais de Doenças , Gerbillinae , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , VirulênciaRESUMO
Legionella pneumophila, the causative agent of a severe pneumonia termed Legionnaires' Disease, survives and replicates within both protozoan hosts and human alveolar macrophages. Intracellular survival is dependent upon secretion of a plethora of protein effectors that function to form a replicative vacuole, evade the endocytic pathway and subvert host immune defenses. Export of these factors requires a type IV secretion system (T4SS) called Dot/Icm that is composed of twenty-seven proteins. This report focuses on the DotF protein, which was previously postulated to have several different functions, one of which centered on binding Dot/Icm substrates. In this report, we examined if DotF functions as the T4SS inner membrane receptor for Dot/Icm substrates. Although we were able to recapitulate the previously published bacterial two-hybrid interaction between DotF and several substrates, the interaction was not dependent on the Dot/Icm substrates' signal sequences as predicted for a substrate:receptor interaction. In addition, binding did not require the cytoplasmic domain of DotF, which was anticipated to be involved in recognizing substrates in the cytoplasm. Finally, inactivation of dotF did not abolish intracellular growth of L. pneumophila or translocation of substrates, two phenotypes dependent on the T4SS receptor. These data strongly suggest that DotF does not act as the major receptor for Dot/Icm substrates and therefore likely performs an accessory function within the core-transmembrane subcomplex of the L. pneumophila Dot/Icm type IV secretion system.