RESUMO
Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.
Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias , Fímbrias Bacterianas , Francisella , Tularemia , Fatores de Virulência , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Francisella/genética , Francisella/metabolismo , Francisella/patogenicidade , Francisella/ultraestrutura , Francisella tularensis/genética , Francisella tularensis/metabolismo , Francisella tularensis/patogenicidade , Francisella tularensis/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Tularemia/genética , Tularemia/metabolismo , Tularemia/patologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.
RESUMO
Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.
RESUMO
The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an ΔiglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, ΔiglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-α, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first â¼ 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.
Assuntos
Aminoácidos/química , Proteínas da Membrana Bacteriana Externa/química , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Lipoproteínas/química , Mutagênese , Sistemas de Secreção Tipo VI , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Francisella tularensis/química , Ilhas Genômicas/genética , Inflamassomos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Macrófagos/microbiologia , Camundongos , Fagossomos/microbiologia , Tularemia/microbiologia , Sistemas de Secreção Tipo VI/genética , Fatores de Virulência/metabolismoRESUMO
Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal lcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/patogenicidade , Animais , Macrófagos , Camundongos , Fagocitose , Transporte Proteico , VirulênciaRESUMO
The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/genética , Ilhas Genômicas/genética , Tularemia/microbiologia , Sistemas de Secreção Tipo VI/genética , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Francisella tularensis/imunologia , Francisella tularensis/patogenicidade , Regulação Bacteriana da Expressão Gênica , Macrófagos/metabolismo , Modelos Moleculares , Alinhamento de Sequência , Deleção de Sequência , Tularemia/imunologia , Sistemas de Secreção Tipo VI/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ΔiglA, ΔiglÇ ΔiglG, ΔiglI, and ΔpdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, ΔpdpE and ΔiglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ΔiglA and ΔiglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.
Assuntos
Replicação do DNA , Francisella tularensis/crescimento & desenvolvimento , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/microbiologia , Tularemia/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Citosol/metabolismo , Citosol/microbiologia , Francisella tularensis/genética , Francisella tularensis/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeriose/genética , Listeriose/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Microinjeções , Tularemia/genética , Tularemia/metabolismoRESUMO
The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A1 activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/efeitos dos fármacos , Proteínas Hemolisinas/metabolismo , Fosfolipases A1/antagonistas & inibidores , Vibrio cholerae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fosfolipases A1/metabolismo , Vibrio cholerae/patogenicidade , Fatores de Virulência/antagonistas & inibidoresRESUMO
BACKGROUND: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. RESULTS: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. CONCLUSIONS: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Mapeamento de Interação de Proteínas , Vibrio cholerae O1/metabolismo , Proteínas de Bactérias/genética , Análise Mutacional de DNA , Escherichia coli/crescimento & desenvolvimento , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Vibrio cholerae O1/genética , Vibrio cholerae O1/crescimento & desenvolvimentoRESUMO
Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ΔiglI or ΔiglC mutants, although the former strain replicated. The ΔiglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ΔpdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/fisiologia , Ilhas Genômicas/fisiologia , Macrófagos/microbiologia , Animais , Anexina A5/metabolismo , Proteínas de Bactérias/genética , Caspase 9/metabolismo , Morte Celular/fisiologia , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Ilhas Genômicas/genética , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Mutação , VirulênciaRESUMO
BACKGROUND: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system. RESULTS: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1ß, albeit lower than LVS, and markedly induced LDH release. CONCLUSIONS: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.
Assuntos
Francisella tularensis/genética , Francisella tularensis/patogenicidade , Macrófagos/microbiologia , Fatores de Virulência/deficiência , Animais , Vacinas Bacterianas/genética , Modelos Animais de Doenças , Feminino , Francisella tularensis/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Tularemia/microbiologia , Tularemia/patologia , Vacinas Atenuadas/genética , Fatores de Virulência/genéticaRESUMO
Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM ß-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native ß-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.
Assuntos
Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/farmacologia , Francisella tularensis/fisiologia , Macrófagos/microbiologia , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Linhagem Celular , Francisella tularensis/patogenicidade , Genes Reporter , Ilhas Genômicas/genética , Macrófagos/imunologia , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Tularemia/prevenção & controle , Vacinas Atenuadas , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both ΔvgrG and ΔdotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella/metabolismo , Francisella/patogenicidade , Proteínas de Bactérias/genética , Ilhas Genômicas/genética , Ilhas Genômicas/fisiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The most recently discovered secretion pathway in gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Via Secretória/fisiologia , Vibrio cholerae/classificação , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Técnicas Bacteriológicas , Meio Ambiente , Escherichia coli K12 , Concentração Osmolar , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temperatura , Vibrio cholerae/patogenicidade , VirulênciaRESUMO
Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5' mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5' end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Linhagem Celular , Mutação da Fase de Leitura , Humanos , Biossíntese de Proteínas , Transporte Proteico , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/genéticaRESUMO
The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.
Assuntos
Francisella tularensis/genética , Francisella tularensis/patogenicidade , Ilhas Genômicas , Fatores de Virulência/genética , Animais , Apoptose , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Inflamassomos/fisiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fagocitose/genética , Fagossomos/genética , Fagossomos/metabolismo , Fagossomos/microbiologia , Reação em Cadeia da Polimerase , Deleção de Sequência , Tularemia/microbiologia , Tularemia/patologia , Fatores de Virulência/biossínteseRESUMO
YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Yersinia pseudotuberculosis/metabolismo , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Proteínas Citotóxicas Formadoras de Poros/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/crescimento & desenvolvimentoRESUMO
Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them.
RESUMO
Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same alpha-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Francisella tularensis/patogenicidade , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Animais , Linhagem Celular , Sequência Conservada , Francisella tularensis/genética , Teste de Complementação Genética , Macrófagos/microbiologia , Camundongos , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética , Saccharomyces cerevisiae , Salmonella typhimurium/genética , Técnicas do Sistema de Duplo-Híbrido , Vibrio cholerae/genética , Virulência , Yersinia pseudotuberculosis/genéticaRESUMO
Bacteria utilise Twin arginine translocation (Tat) to deliver folded proteins across the cytoplasmic membrane. Disruption of Tat typically results in pleiotropic effects on e.g. growth, stress resistance, bacterial membrane biogenesis, motility and cell morphology. Further, Tat is coupled to virulence in a range of pathogenic bacteria, including species of Pseudomonas, Legionella, Agrobacterium and Mycobacterium. We have investigated this, for Yersinia, previously unexplored system, and have shown that the Tat pathway is functional and absolutely required for virulence of Yersinia pseudotuberculosis. A range of putative Yersinia Tat substrates have been predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments. Here we present a brief review of bacterial Tat and discuss our results concerning this system in Yersinia.