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1.
J Bacteriol ; 203(2)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33077633

RESUMO

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.


Assuntos
Adesinas Bacterianas/metabolismo , Matriz Extracelular/metabolismo , Lipoproteínas/metabolismo , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Western Blotting/veterinária , Bovinos , Biologia Computacional , Eletroforese em Gel de Poliacrilamida/veterinária , Matriz Extracelular/química , Fibronectinas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Modelos Estruturais , Infecções por Mycoplasma/microbiologia , Mycoplasma bovis/genética , Proteólise , Ratos , Ratos Sprague-Dawley , Ruminantes , Alinhamento de Sequência/veterinária
2.
Cell Microbiol ; 22(5): e13154, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31872956

RESUMO

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


Assuntos
Coxiella burnetii/metabolismo , NADP/metabolismo , Estresse Oxidativo , Coxiella burnetii/crescimento & desenvolvimento , Citoplasma/metabolismo , Células HeLa , Humanos , Macrófagos/microbiologia , Mutação , NADP/genética , Febre Q/metabolismo , Febre Q/microbiologia , Regeneração , Vacúolos/microbiologia
3.
Pathog Dis ; 77(8)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31845968

RESUMO

The zoonotic disease Q fever caused by the intracellular bacterium Coxiella burnetii remains a global health threat due to its high infectivity, environmental stability, the debilitating nature and the long duration of treatment. Designing new and potent drugs that target previously unexplored pathways is essential to shorten treatment time and minimise antibiotic resistance. Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor in all living organisms. NadB, an L-aspartate oxidase catalysing the first step of the prokaryotic-specific NAD de novo biosynthetic pathway, is required for C. burnetii growth and replication inside host cells. In this study, in vitro enzyme assays utilising recombinant glutathione S-transferase tagged NadB (GST-NadB) demonstrated inhibition of the L-aspartate oxidase activity of NadB by meso-tartrate. Furthermore, meso-tartrate inhibits intracellular growth and replication of C. burnetii inside host cells in a dose-dependent manner, and has no effect on the viability of mammalian cells. Unexpectedly, meso-tartrate also inhibited growth of C. burnetii in axenic medium, and further reduces replication of the nadB mutant inside host cells, suggesting it is acting more widely than simple inhibition of NadB. Overall, these results suggest that the antibacterial activity of meso-tartrate warrants further study, including investigation of its additional target(s).


Assuntos
Antibacterianos/farmacologia , Coxiella burnetii/efeitos dos fármacos , Coxiella burnetii/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Tartaratos/farmacologia , Aminoácido Oxirredutases/antagonistas & inibidores , Coxiella burnetii/enzimologia , Coxiella burnetii/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Viabilidade Microbiana/efeitos dos fármacos , NAD/metabolismo , Células THP-1
4.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30567986

RESUMO

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, Phascolarctid gammaherpesvirus 1 (PhaHV1) and Vombatid gammaherpesvirus 1 (VoHV1), which infect koalas (Phascolarctos cinereus) and wombats (Vombatus ursinus), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch, which may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted open reading frames (ORFs) homologous to those in eutherian herpesviruses and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired prespeciation, approximately 30 to 40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher-order eukaryotes, with a very small number being found in bacteria. This is the first time that an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic data sets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated in vitro, suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined.IMPORTANCE The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae Their genomes contain several new ORFs, including ORFs encoding a ß-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.


Assuntos
Gammaherpesvirinae/genética , Marsupiais/virologia , Phascolarctidae/virologia , Pirofosfatases/genética , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Genoma Viral/genética , Fases de Leitura Aberta/genética , Filogenia , Transcriptoma/genética
5.
J Med Microbiol ; 66(2): 236-244, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28266284

RESUMO

PURPOSE: Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas. METHODOLOGY: Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status. RESULTS: The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and 'wet bottom' (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98). CONCLUSION: Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.


Assuntos
Phascolarctidae/virologia , Infecções por Retroviridae/veterinária , Retroviridae/isolamento & purificação , Animais , DNA Viral/genética , Genótipo , Técnicas de Genotipagem , Modelos Logísticos , Análise Multivariada , New South Wales/epidemiologia , Prevalência , Queensland/epidemiologia , Retroviridae/genética , Infecções por Retroviridae/epidemiologia
6.
PLoS Negl Trop Dis ; 8(12): e3402, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25521752

RESUMO

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host.


Assuntos
Antígenos CD/fisiologia , Apirase/fisiologia , Glicoesfingolipídeos/metabolismo , Complexo de Golgi/enzimologia , Leishmania major/patogenicidade , Animais , Antígenos CD/genética , Apirase/genética , Proteínas do Sistema Complemento/imunologia , Feminino , Leishmania major/metabolismo , Leishmaniose Cutânea/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Virulência
7.
Biochem J ; 462(2): 279-89, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24957128

RESUMO

Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment.


Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Proteínas de Bactérias/genética , Cálcio/farmacologia , Cátions Bivalentes , Linhagem Celular , Feminino , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Legionella pneumophila/fisiologia , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Macrófagos/microbiologia , Magnésio/farmacologia , Camundongos , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virulência , Zinco/farmacologia
8.
PLoS One ; 8(2): e56064, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437085

RESUMO

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H(2)O(2) compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H(2)O(2) and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.


Assuntos
Leishmania major/enzimologia , Leishmania major/crescimento & desenvolvimento , Macrófagos/parasitologia , Metionina Sulfóxido Redutases/metabolismo , Estresse Oxidativo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Citoplasma/efeitos dos fármacos , Citoplasma/parasitologia , Modelos Animais de Doenças , Deleção de Genes , Genes de Protozoários/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/farmacologia , Leishmania major/citologia , Leishmania major/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/efeitos dos fármacos , Espectrometria de Massas , Metionina/análogos & derivados , Metionina/metabolismo , Metionina Sulfóxido Redutases/genética , Camundongos , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Parasitos/citologia , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Transporte Proteico/efeitos dos fármacos , Alinhamento de Sequência , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo
9.
Structure ; 18(2): 228-38, 2010 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-20159467

RESUMO

Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.


Assuntos
Proteínas de Bactérias/química , Eucariotos/enzimologia , Legionella pneumophila/enzimologia , Estrutura Terciária de Proteína , Pirofosfatases/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Pirofosfatases/genética , Pirofosfatases/metabolismo , Ratos , Alinhamento de Sequência
10.
Infect Immun ; 76(7): 3075-85, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18426879

RESUMO

Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC(N430A/R434A), encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC(N430A/R434A) resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.


Assuntos
Acanthamoeba castellanii/microbiologia , Adenilil Ciclases/metabolismo , Células Epiteliais/microbiologia , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Monócitos/microbiologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Adenilil Ciclases/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Feminino , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Virulência
11.
J Biol Chem ; 283(19): 12909-18, 2008 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-18337253

RESUMO

Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.


Assuntos
Apirase/metabolismo , Legionella pneumophila/enzimologia , Legionella pneumophila/patogenicidade , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apirase/genética , Catálise , Linhagem Celular , Citidina/metabolismo , Ativação Enzimática , Guanosina Difosfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Metais/farmacologia , Camundongos , Complexos Multienzimáticos/metabolismo , Mutação/genética , Especificidade por Substrato , Uridina/metabolismo
12.
Infect Immun ; 75(12): 5575-85, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17893138

RESUMO

The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.


Assuntos
Proteínas de Bactérias/fisiologia , Legionella pneumophila/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismo , Fatores de Virulência/fisiologia , Acanthamoeba castellanii/microbiologia , Motivos de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Células HeLa , Humanos , Imunoglobulinas/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos A , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
13.
Cell Microbiol ; 9(8): 1922-35, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17388784

RESUMO

As part of its pathogenesis, Legionella pneumophila persists within human alveolar macrophages in non-acidified organelles that do not mature into phagolysosomes. Two L. pneumophila genes, lpg0971 and lpg1905, are predicted to encode ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) that share sequence similarity with human CD39/NTPDase1. The predicted products possess five apyrase conserved domains that are typical of eukaryotic ecto-NTPDases. In this study, we found that an lpg1905 mutant was recovered in lower numbers from macrophages, alveolar epithelial cells and the amoeba, Hartmannella vermiformis compared with wild-type L. pneumophila and an lpg0971 mutant. Similar to human CD39, recombinant purified Lpg1905 exhibited ATPase and ADPase activity and possessed the ability to inhibit platelet aggregation. Mutation of a conserved Glu159 residue that is essential for CD39 activity inhibited ATPase and ADPase activity of Lpg1905. In addition, enzyme activity was inhibited in the presence of the specific ecto-NTPDase inhibitor, ARL67156. The entry and replication defect of the lpg1905 mutant was reversed upon transcomplementation with lpg1905 but not lpg1905E159A encoding an enzymatically inactive form of the protein. Although several protozoan parasites exhibit ecto-NTPDase activity, including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi, this is the first time a bacterial ecto-NTPDase has been implicated in virulence.


Assuntos
Adenosina Trifosfatases/fisiologia , Antígenos CD/genética , Apirase/genética , Proteínas de Bactérias/fisiologia , Legionella pneumophila/fisiologia , Pirofosfatases/fisiologia , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Transporte Biológico Ativo , Linhagem Celular , Biologia Computacional , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Hartmannella/microbiologia , Humanos , Técnicas In Vitro , Legionella pneumophila/enzimologia , Legionella pneumophila/isolamento & purificação , Dados de Sequência Molecular , Monócitos/metabolismo , Monócitos/microbiologia , Mutação , Agregação Plaquetária , Pirofosfatases/genética , Pirofosfatases/farmacologia , Proteínas Recombinantes/farmacologia , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/fisiologia
14.
Infect Immun ; 74(3): 1683-91, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16495539

RESUMO

Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5' end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.


Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/análise , Genoma Bacteriano , Legionella pneumophila/genética , Alvéolos Pulmonares/microbiologia , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Linhagem Celular , Células Epiteliais/microbiologia , Humanos , Legionella pneumophila/patogenicidade , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , Alvéolos Pulmonares/citologia
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