RESUMO
The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.
Assuntos
AMP Cíclico , Proteínas de Drosophila , Drosophila melanogaster , Estresse do Retículo Endoplasmático , Nucleotidiltransferases , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Animais , Camundongos , Alelos , AMP Cíclico/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Pâncreas/metabolismo , Pâncreas/fisiopatologia , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Bile acids are important for digestion of food and antimicrobial activity. Pathogenic Vibrio parahaemolyticus senses bile acids and induce pathogenesis. The bile acid taurodeoxycholate (TDC) was shown to activate the master regulator, VtrB, of this system, whereas other bile acids such as chenodeoxycholate (CDC) do not. Previously, VtrA-VtrC was discovered to be the co-component signal transduction system that binds bile acids and induces pathogenesis. TDC binds to the periplasmic domain of the VtrA-VtrC complex, activating a DNA-binding domain in VtrA that then activates VtrB. Here, we find that CDC and TDC compete for binding to the VtrA-VtrC periplasmic heterodimer. Our crystal structure of the VtrA-VtrC heterodimer bound to CDC revealed CDC binds in the same hydrophobic pocket as TDC but differently. Using isothermal titration calorimetry, we observed that most mutants in the binding pocket of VtrA-VtrC caused a decrease in bile acid binding affinity. Notably, two mutants in VtrC bound bile acids with a similar affinity as the WT protein but were attenuated for TDC-induced type III secretion system 2 activation. Collectively, these studies provide a molecular explanation for the selective pathogenic signaling by V. parahaemolyticus and reveal insight into a host's susceptibility to disease.
Assuntos
Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Ácidos e Sais Biliares/metabolismo , Transdução de Sinais , Ácido Quenodesoxicólico , Proteínas de Bactérias/metabolismoRESUMO
The Gram-negative bacterium Vibrio parahaemolyticus is an opportunistic human pathogen and the leading cause of seafood-borne acute gastroenteritis worldwide. Recently, this bacterium was implicated as the etiologic agent of a severe shrimp disease with consequent devastating outcomes to shrimp farming. In both cases, acquisition of genetic material via horizontal transfer provided V. parahaemolyticus with new virulence tools to cause disease. Dissecting the molecular mechanisms of V. parahaemolyticus pathogenesis often requires manipulating its genome. Classically, genetic deletions in V. parahaemolyticus are performed using a laborious, lengthy, multistep process. Here, we describe a fast and efficient method to edit this bacterium's genome based on V. parahaemolyticus natural competence. Although this method is similar to one previously described, V. parahaemolyticus requires counterselection for curing of acquired plasmids due to its recalcitrant nature of retaining extrachromosomal DNA. We believe this approach will be of use to the Vibrio community.IMPORTANCE Spreading of vibrios throughout the world correlates with increased global temperatures. As they spread, they find new niches in which to survive, proliferate, and invade. Therefore, genetic manipulation of vibrios is of the utmost importance for studying these species. Here, we have delineated and validated a rapid method to create genetic deletions in Vibrio parahaemolyticus This study provides insightful methodology for studies with other Vibrio species.
Assuntos
Proteínas de Bactérias/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Vibrio parahaemolyticus/genética , Proteínas de Bactérias/genética , Plasmídeos , Transformação Genética , Vibrio parahaemolyticus/fisiologiaRESUMO
Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae.
Assuntos
Proteínas de Bactérias/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Lipoproteínas/imunologia , Pneumonia Pneumocócica/imunologia , Streptococcus pneumoniae/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Células HEK293 , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Lipoproteínas/genética , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Pneumonia Pneumocócica/genética , Pneumonia Pneumocócica/patologia , Doenças da Imunodeficiência Primária , Streptococcus pneumoniae/genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.
Assuntos
Imunidade Inata , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Modelos Estatísticos , Pneumonia Pneumocócica/imunologia , Streptococcus pneumoniae/imunologia , Ácido 4-Aminobenzoico/metabolismo , Animais , Cápsulas Bacterianas/imunologia , Carga Bacteriana/imunologia , Feminino , Meia-Vida , Pulmão/microbiologia , Pulmão/patologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Mutação , Fagocitose , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/patologia , Índice de Gravidade de Doença , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , Fatores de TempoRESUMO
Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsule-switched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.
Assuntos
Cápsulas Bacterianas/imunologia , Complemento C3/imunologia , Complemento C3b/imunologia , Fator H do Complemento/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/imunologia , Adulto , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Criança , Complemento C3/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Humanos , Mutação/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Infecções Pneumocócicas/metabolismo , Ligação Proteica/imunologia , Sorotipagem/métodos , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadeRESUMO
It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB, located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals.
Assuntos
Vibrio parahaemolyticus , Membranas , Proteínas de Membrana , Proteínas de Bactérias , Ácidos e Sais BiliaresRESUMO
Zinc is an essential trace metal for living cells. The ABC transporter AdcABC was previously shown to be required for zinc uptake by Streptococcus pneumoniae. As we have recently described AdcAII as another zinc-binding lipoprotein, we have investigated the role of both AdcA and AdcAII in S. pneumoniae zinc metabolism. Deletion of either adcA or adcAII but not phtD reduced S. pneumoniae zinc uptake, with dual mutation of both adcA and adcAII further decreasing zinc import. For the Δ(adcA/adcAII) mutant, growth and intracellular concentrations of zinc were both greatly reduced in low zinc concentration. When grown in zinc-deficient medium, the Δ(adcA/adcAII) mutant displayed morphological defects related to aberrant septation. Growth and morphology of the Δ(adcA/adcAII) mutant recovered after supplementation with zinc. Dual deletion of adcA and adcAII strongly impaired growth of the pneumococcus in bronchoalveolar lavage fluid and human serum, and prevented S. pneumoniae establishing infection in mouse models of nasopharyngeal colonization, pneumonia and sepsis without altering the capsule. Taken together, our results show that AdcA and AdcAII play an essential and redundant role in specifically importing zinc into the pneumococcus, and that both zinc transporters are required for proper cell division and for S. pneumoniae survival during infection.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência/metabolismo , Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Divisão Celular , Meios de Cultura/química , Modelos Animais de Doenças , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Viabilidade Microbiana , Nasofaringe/microbiologia , Infecções Pneumocócicas/microbiologia , Sepse/microbiologia , Soro/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens. IMPORTANCE Many species of intracellular bacterial pathogens target host small Rho GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Escherichia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor 1 (CNF1) drives UPEC's internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed that the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. Moreover, they pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape and, more broadly, the regulation of successful pathogenesis.
Assuntos
Infecções Bacterianas , Proteínas de Escherichia coli , Escherichia coli Uropatogênica , Vibrio parahaemolyticus , Humanos , Sistemas de Secreção Tipo III/metabolismo , Escherichia coli Uropatogênica/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Fatores de Virulência , Proteínas rho de Ligação ao GTPRESUMO
Zinc homeostasis is critical for pathogen host colonization. Indeed, during invasion, Streptococcus pneumoniae has to finely regulate zinc transport to cope with a wide range of Zn(2+) concentrations within the various host niches. AdcAII was identified as a pneumococcal Zn(2+)-binding protein; its gene is present in an operon together with the phtD gene. PhtD belongs to the histidine triad protein family, but to date, its function has not been clarified. Using several complementary biochemical methods, we provide evidence that like AdcAII, PhtD is a metal-binding protein specific for zinc. When Zn(2+) binds (K(d) = 131 ± 10 nM), the protein displays substantial thermal stabilization. We also present the first direct evidence of a joint function of AdcAII and PhtD by demonstrating that their expression is corepressed by Zn(2+), that they interact directly in vitro, and that they are colocalized at the bacterial surface. These results suggest the common involvement of the AdcAII-PhtD system in pneumococcal zinc homeostasis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hidrolases/metabolismo , Streptococcus pneumoniae/metabolismo , Zinco/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Transporte de Cátions/química , Hidrolases/química , Lipoproteínas/química , Lipoproteínas/metabolismo , Proteínas Recombinantes/químicaRESUMO
Avirulent strains of a bacterial pathogen could be useful tools for investigating immunological responses to infection and potentially effective vaccines. We have therefore constructed an auxotrophic TIGR4 Δpab strain of Streptococcus pneumoniae by deleting the pabB gene Sp_0665. The TIGR4 Δpab strain grew well in complete medium but was unable to grow in serum unless it was supplemented with para-aminobenzoic acid (PABA). The TIGR4 Δpab strain was markedly attenuated in virulence in mouse models of S. pneumoniae nasopharyngeal colonization, pneumonia, and sepsis. Supplementing mouse drinking water with PABA largely restored the virulence of TIGR4 Δpab. An additional Δpab strain constructed in the D39 capsular serotype 2 background was also avirulent in a sepsis model. Systemic inoculation of mice with TIGR4 Δpab induced antibody responses to S. pneumoniae protein antigens, including PpmA, PsaA, pneumolysin, and CbpD, but not capsular polysaccharide. Flow cytometry demonstrated that IgG in sera from TIGR4 Δpab-vaccinated mice bound to the surface of TIGR4 and D39 bacteria but not to a capsular serotype 3 strain, strain 0100993. Mice vaccinated with the TIGR4 Δpab or D39 Δpab strain by intraperitoneal inoculation were protected from developing septicemia when challenged with the homologous S. pneumoniae strain. Vaccination with the TIGR4 Δpab strain provided only weak or no protection against heterologous challenge with the D39 or 0100993 strain but did strongly protect against a TIGR4 capsular-switch strain expressing a serotype 2 capsule. The failure of cross-protection after systemic vaccination with Δpab bacteria suggests that parenteral administration of a live attenuated vaccine is not an attractive approach for preventing S. pneumoniae infection.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Animais , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Feminino , Camundongos , Infecções Pneumocócicas/prevenção & controle , Streptococcus pneumoniae/imunologia , Fatores de Tempo , Vacinação , VirulênciaRESUMO
Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for bacterial culture, genetic manipulation, and analysis of virulence phenotypes. Detailed protocols for growth assessment, generation of mutants, and phenotype assessment are included in this article. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Assessment of growth of V. parahaemolyticus Alternate Protocol 1: Assessment of growth of V. parahaemolyticus using a plate reader Basic Protocol 2: Swimming/swarming motility assay Basic Protocol 3: Genetic manipulation Alternate Protocol 2: Natural transformation Basic Protocol 4: Secretion assay and sample preparation for mass spectrometry analysis Basic Protocol 5: Invasion assay (gentamicin protection assay) Basic Protocol 6: Immunofluorescence detection of intracellular V. parahaemolyticus Basic Protocol 7: Cytotoxicity assay for T3SS2.
Assuntos
Técnicas Bacteriológicas/métodos , Vibrioses/microbiologia , Vibrio parahaemolyticus/crescimento & desenvolvimento , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Gentamicinas/farmacologia , Células HeLa , Humanos , Coloração e Rotulagem , Natação , Vibrioses/tratamento farmacológico , Vibrio parahaemolyticus/efeitos dos fármacos , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade , Virulência/genéticaRESUMO
The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of adcAII in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 adcAII mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated ΔadcAII mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the ΔadcAII mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated ΔadcAII strains. However, transformation of ΔadcAII with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated ΔadcAII strains contained the same single nucleotide polymorphism in the capsule locus gene cps2E, which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII Further investigation will be needed to characterize how mutation of adcAII affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype.IMPORTANCE The Streptococcus pneumoniae capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of adcAII, a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the ΔadcAII strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to affect capsule thickness. Our data provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identify an unexpected link between capsule thickness and ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation.
Assuntos
Alelos , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Enzimas de Restrição-Modificação do DNA/genética , Deleção de Genes , Lipoproteínas/genética , Streptococcus pneumoniae/fisiologia , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Sistema Complemento/imunologia , Enzimas de Restrição-Modificação do DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genômica/métodos , Lipoproteínas/metabolismo , Mutação , Fagocitose , Transcriptoma , VirulênciaRESUMO
Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. Vibrio parahaemolyticus is a facultative intracellular pathogen that uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. Analysis of the T3SS2 pathogenicity island encoding the T3SS2 appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of molecular tools, we found that VPA0226, a constitutively secreted lipase, is required for escape of V. parahaemolyticus from the host cells. This lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems and host cell resources to evolve an ingenious scheme for survival and escape.
Assuntos
Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Lipase/metabolismo , Vibrio parahaemolyticus/metabolismo , Esterificação , Ilhas Genômicas , Sistemas de Secreção Tipo III , Vibrio parahaemolyticus/enzimologiaRESUMO
Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae, largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis.IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae, but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.
Assuntos
Proteínas de Bactérias/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Inflamação/fisiopatologia , Macrófagos/fisiologia , Transdução de Sinais/fisiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , HumanosRESUMO
Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter).
Assuntos
Transformação Celular Neoplásica/patologia , Exossomos/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Exossomos/química , Genômica , Humanos , Camundongos , Células NIH 3T3 , Transplante de Neoplasias , ProteômicaRESUMO
The Gram-negative marine bacterium Vibrio parahaemolyticus is a common cause of infectious gastroenteritis due to the ingestion of contaminated seafood. Most virulent V. parahaemolyticus strains encode two type III secretion systems (T3SS1 and T3SS2); however, the roles they and their translocated effectors play in causing intestinal disease remain unclear. While studies have identified T3SS1 effectors as responsible for killing epithelial cells in culture, the T3SS2 effectors caused massive epithelial cell disruption in a rabbit ileal loop model. Additional models are thus needed to clarify the pathogen-host interactions that drive V. parahaemolyticus-associated gastroenteritis. Germfree mice were infected with a pathogenic clinical isolate of V. parahaemolyticus, RIMD2210633 (RIMD). The pathogen was found to adhere to as well as invade the cecal mucosa, accompanied by severe inflammation and dramatic mucosal damage, including widespread sloughing of infected epithelial cells. Mice infected with a V. parahaemolyticus strain lacking the T3SS1 (POR2) also developed severe pathology, similar to that seen with RIMD. In contrast, the ΔT3SS2 strain (POR3) appeared unable to invade the intestinal mucosa or cause any mucosal pathology. Confirming a role for TS332 effectors, a strain expressing the T3SS2 but lacking VopC (POR2ΔvopC), a T3SS2 effector implicated in epithelial cell invasion in culture, was strongly attenuated in invading the intestinal mucosa and in causing gastroenteritis, although infection with this mutant resulted in more pathology than the ΔT3SS2 strain. We thus present an experimental system that enables further characterization of T3SS effectors as well as the corresponding host inflammatory response involved in the gastroenteritis caused by invasive V. parahaemolyticusIMPORTANCEVibrio parahaemolyticus causes severe gastroenteritis following consumption of contaminated seafood. Global warming has allowed this pathogen to spread worldwide, contributing to recent outbreaks. Clinical isolates are known to harbor an array of virulence factors, including T3SS1 and T3SS2; however, the precise role these systems play in intestinal disease remains unclear. There is an urgent need to improve our understanding of how V. parahaemolyticus infects hosts and causes disease. We present a novel mouse model for this facultative intracellular pathogen and observe that the T3SS2 is essential to pathogenicity. Moreover, we show that the T3SS2 effector VopC, previously shown to be a Rac and Cdc42 deamidase that facilitates bacterial uptake by nonphagocytic cells, also plays a key role in the ability of V. parahaemolyticus to invade the intestinal mucosa and cause gastroenteritis. This experimental model thus provides a valuable tool for future elucidation of virulence mechanisms used by this facultative intracellular pathogen during in vivo infection.
Assuntos
Gastroenterite/microbiologia , Sistemas de Secreção Tipo III , Vibrioses/microbiologia , Vibrio parahaemolyticus/fisiologia , Fatores de Virulência/genética , Animais , Antibacterianos/farmacologia , Morte Celular , Proliferação de Células , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Gastroenterite/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Estreptomicina/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , VirulênciaRESUMO
Although squamous cell carcinomas (SqCCs) of the lungs, head and neck, oesophagus, and cervix account for up to 30% of cancer deaths, the mechanisms that regulate disease progression remain incompletely understood. Here, we use gene transduction and human tumor xenograft assays to establish that the tumour suppressor Cell adhesion molecule 1 (CADM1) inhibits SqCC proliferation and invasion, processes fundamental to disease progression. We determine that the extracellular domain of CADM1 mediates these effects by forming a complex with HER2 and integrin α6ß4 at the cell surface that disrupts downstream STAT3 activity. We subsequently show that treating CADM1 null tumours with the JAK/STAT inhibitor ruxolitinib mimics CADM1 gene restoration in preventing SqCC growth and metastases. Overall, this study identifies a novel mechanism by which CADM1 prevents SqCC progression and suggests that screening tumours for loss of CADM1 expression will help identify those patients most likely to benefit from JAK/STAT targeted chemotherapies.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Moléculas de Adesão Celular/metabolismo , Imunoglobulinas/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Imunoglobulinas/genética , Integrina alfa6beta4/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Nitrilas , Pirazóis/química , Pirimidinas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a K(D) of â¼25 nM, and a ΔmetQ strain had reduced uptake of C(14)-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/ΔmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 - Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.
Assuntos
Metionina/biossíntese , Metionina/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/patogenicidade , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Transporte Biológico , Loci Gênicos/genética , Lipoproteínas/deficiência , Lipoproteínas/genética , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Camundongos , Fenótipo , Alinhamento de Sequência , Deleção de Sequência , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , VirulênciaRESUMO
Live attenuated vaccines have been proposed as a strategy to induce protective immunity against infectious diseases. Recent data have demonstrated that nasopharyngeal colonisation with Streptococcus pneumoniae induces protective immunity against subsequent invasive infection, suggesting nasal vaccination with live attenuated bacteria could be a preventative strategy. However the bacterial factors affecting the strength of this adaptive immune response remain unclear. In a direct comparison with the parent wild-type strain, we found that colonisation with bacteria lacking either capsule or surface lipoproteins led to significantly diminished protection. Immunity after colonisation was not dependent on serum IgG to capsular antigens. Colonisation density and duration was reduced for all the non-protective strains, suggesting that protective immunity maybe related to the extent of nasopharyngeal bacterial exposure. To investigate this hypothesis, we utilised an auxotrophic bacterial Δpab strain where duration of colonisation could be controlled by supply and removal of para-amino-benzoic acid (PABA) to mouse drinking water. Supporting colonisation with the Δpab strain for 5 days with PABA led to a faster serum antibody response compared to colonisation for less than 48 h. This enhanced immunogenicity was associated with a trend towards protection. The data presented here aid our understanding of why only certain live attenuated strains are able to function as effective vaccines, and may be valuable in informing the constituents of future live attenuated vaccines.