RESUMO
Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.
Assuntos
Aldose-Cetose Isomerases/análise , Dicroísmo Circular/métodos , Ensaios Enzimáticos/métodos , Proteínas de Bactérias/metabolismo , Catálise , Francisella tularensis/metabolismo , Lipopolissacarídeos/metabolismo , Pentosefosfatos/metabolismo , Ribulosefosfatos/análise , Ribulosefosfatos/metabolismo , Especificidade por Substrato , Açúcares Ácidos/metabolismo , Fosfatos Açúcares/metabolismoRESUMO
Low molecular mass penicillin binding proteins (LMM PBP) are bacterial enzymes involved in the final steps of peptidoglycan biosynthesis. In Escherichia coli, most LMM PBP exhibit dd-carboxypeptidase activity, are not essential for growth in routine laboratory media, and contributions to virulent phenotypes remain largely unknown. The Francisella tularensis Schu S4 genome harbors the dacD gene (FTT_1029), which encodes a LMM PBP with homology to PBP6b of E. coli. Disruption of this locus in the fully virulent Schu S4 strain resulted in a mutant that could not grow in Chamberlain's Defined Medium and exhibited severe morphological defects. Further characterization studies demonstrated that the growth defects of the dacD mutant were pH-dependent, and could be partially restored by growth at neutral pH or fully restored by genetic complementation. Infection of murine macrophage-like cells showed that the Schu S4 dacD mutant is capable of intracellular replication. However, this mutant was attenuated in BALB/c mice following intranasal challenge (LD50â¯=â¯603â¯CFU) as compared to mice challenged with the parent (LD50â¯=â¯1â¯CFU) or complemented strain (LD50â¯=â¯1â¯CFU). Additionally, mice that survived infection with the dacD mutant showed significant protection against subsequent challenge with the parent strain. Collectively, these results indicate that the DacD protein of F. tularensis is essential for growth in low pH environments and virulence in vivo. These results also suggest that a PBP mutant could serve as the basis of a novel, live attenuated vaccine strain.
Assuntos
Francisella tularensis/enzimologia , Francisella tularensis/patogenicidade , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Tularemia/imunologia , Animais , Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Linhagem Celular , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Francisella tularensis/genética , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas de Ligação às Penicilinas , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Tularemia/microbiologia , Vacinas Atenuadas/imunologia , Virulência , Fatores de Virulência/genéticaRESUMO
Pantothenate, commonly referred to as vitamin B(5), is an essential molecule in the metabolism of living organisms and forms the core of coenzyme A. Unlike humans, some bacteria and plants are capable of de novo biosynthesis of pantothenate, making this pathway a potential target for drug development. Francisella tularensis subsp. tularensis Schu S4 is a zoonotic bacterial pathogen that is able to synthesize pantothenate but is lacking the known ketopantoate reductase (KPR) genes, panE and ilvC, found in the canonical Escherichia coli pathway. Described herein is a gene encoding a novel KPR, for which we propose the name panG (FTT1388), which is conserved in all sequenced Francisella species and is the sole KPR in Schu S4. Homologs of this KPR are present in other pathogenic bacteria such as Enterococcus faecalis, Coxiella burnetii, and Clostridium difficile. Both the homologous gene from E. faecalis V583 (EF1861) and E. coli panE functionally complemented Francisella novicida lacking any KPR. Furthermore, panG from F. novicida can complement an E. coli KPR double mutant. A Schu S4 ΔpanG strain is a pantothenate auxotroph and was genetically and chemically complemented with panG in trans or with the addition of pantolactone. There was no virulence defect in the Schu S4 ΔpanG strain compared to the wild type in a mouse model of pneumonic tularemia. In summary, we characterized the pantothenate pathway in Francisella novicida and F. tularensis and identified an unknown and previously uncharacterized KPR that can convert 2-dehydropantoate to pantoate, PanG.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Francisella tularensis/enzimologia , Ácido Pantotênico/biossíntese , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animais , Clostridioides difficile/enzimologia , Coenzima A/biossíntese , Coxiella burnetii/enzimologia , Enterococcus faecalis/enzimologia , Escherichia coli/enzimologia , Francisella tularensis/genética , Francisella tularensis/metabolismo , Camundongos , Tularemia/microbiologiaRESUMO
Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.
Assuntos
Francisella tularensis/patogenicidade , Genes Bacterianos/imunologia , Inflamação/genética , Macrófagos/imunologia , Proteínas Quinases Ativadas por Mitógeno/genética , Transdução de Sinais/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Western Blotting , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Francisella tularensis/genética , Francisella tularensis/imunologia , Genes Bacterianos/genética , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/imunologia , Transdução de Sinais/imunologia , Tularemia/genética , Tularemia/imunologiaRESUMO
Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 ΔiclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔiclR were equally cytotoxic and induced equivalent levels of interleukin-1ß expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and ΔiclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.
Assuntos
Francisella tularensis/patogenicidade , Fatores de Transcrição/fisiologia , Tularemia/microbiologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Francisella tularensis/genética , Francisella tularensis/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Interleucina-1beta/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência/fisiologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved among Francisella species and is required for intracellular growth. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm. RESULTS: Further analysis of ripA with respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR, ripA-lacZ transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both ripA transcription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, including iglA, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantified ripA and iglA expression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns of ripA and iglA and that MglA and SspA are positive regulators of iglA we tested the impact of mglA and sspA deletions on ripA and iglA expression. In the deletion mutant strains iglA expression was reduced dramatically as expected, however ripA expression was increased over 2-fold. CONCLUSION: Expression of ripA is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of ripA coincided with iglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on iglA expression, MglA and SspA negatively impacted ripA expression in vitro.
Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Francisella tularensis/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , RNA Bacteriano/genética , Análise de Sequência de DNA , Deleção de Sequência , Transcrição GênicaRESUMO
Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.
Assuntos
Proteínas de Bactérias/genética , Francisella tularensis/genética , Francisella tularensis/patogenicidade , Proteínas de Membrana/genética , Tularemia/genética , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Proliferação de Células , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Reação em Cadeia da PolimeraseRESUMO
Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.