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1.
J Biol Chem ; 288(1): 132-40, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23150671

RESUMO

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to ß-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/ß-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 µm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/ß-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.


Assuntos
Proteínas de Bactérias/metabolismo , Metiltransferases/química , RNA Ribossômico/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Cinética , Metiltransferases/genética , Modelos Genéticos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
2.
J Clin Microbiol ; 50(2): 469-71, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22116150

RESUMO

Among 23 patients carrying methicillin-resistant Staphylococcus aureus (MRSA) in their anterior nares, 6 (26%) also carried methicillin-susceptible S. aureus (MSSA) as less prevalent flora. In 4 of the 6 patients, the MSSA was unrelated to prevalent MRSA, as determined by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing. However, in two patients, the strains were identical except for the absence of spontaneous staphylococcal cassette chromosome mec (SCCmec). We consider this evidence of spontaneous SCCmec excision in vivo.


Assuntos
Portador Sadio/microbiologia , DNA Bacteriano/genética , Deleção de Genes , Resistência a Meticilina , Mucosa Nasal/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Cromossomos Bacterianos , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Tipagem Molecular , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação
3.
Microb Genom ; 7(12)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34898422

RESUMO

The composition of the human vaginal microbiome has been extensively studied and is known to influence reproductive health. However, the functional roles of individual taxa and their contributions to negative health outcomes have yet to be well characterized. Here, we examine two vaginal bacterial taxa grouped within the genus Megasphaera that have been previously associated with bacterial vaginosis (BV) and pregnancy complications. Phylogenetic analyses support the classification of these taxa as two distinct species. These two phylotypes, Megasphaera phylotype 1 (MP1) and Megasphaera phylotype 2 (MP2), differ in genomic structure and metabolic potential, suggestive of differential roles within the vaginal environment. Further, these vaginal taxa show evidence of genome reduction and changes in DNA base composition, which may be common features of host dependence and/or adaptation to the vaginal environment. In a cohort of 3870 women, we observed that MP1 has a stronger positive association with bacterial vaginosis whereas MP2 was positively associated with trichomoniasis. MP1, in contrast to MP2 and other common BV-associated organisms, was not significantly excluded in pregnancy. In a cohort of 52 pregnant women, MP1 was both present and transcriptionally active in 75.4 % of vaginal samples. Conversely, MP2 was largely absent in the pregnant cohort. This study provides insight into the evolutionary history, genomic potential and predicted functional role of two clinically relevant vaginal microbial taxa.


Assuntos
Proteínas de Bactérias/genética , Megasphaera/classificação , Análise de Sequência de DNA/métodos , Vagina/microbiologia , Vaginose Bacteriana/epidemiologia , Composição de Bases , Estudos de Casos e Controles , Evolução Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Tamanho do Genoma , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Megasphaera/genética , Megasphaera/isolamento & purificação , Megasphaera/metabolismo , Filogenia , Gravidez , RNA Ribossômico 16S/genética , Saúde Reprodutiva , Vaginose Bacteriana/microbiologia
4.
Infect Immun ; 76(11): 5127-32, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18794284

RESUMO

Staphylococcus epidermidis is one of the most common causes of infections of prosthetic heart valves (prosthetic valve endocarditis [PVE]) and an increasingly common cause of infections of native heart valves (native valve endocarditis [NVE]). While S. epidermidis typically causes indolent infections of prosthetic devices, including prosthetic valves and intravascular catheters, S. epidermidis NVE is a virulent infection associated with valve destruction and high mortality. In order to see if the differences in the course of infection were due to characteristics of the infecting organisms, we examined 31 S. epidermidis NVE and 65 PVE isolates, as well as 21 isolates from blood cultures (representing bloodstream infections [BSI]) and 28 isolates from nasal specimens or cultures considered to indicate skin carriage. Multilocus sequence typing showed both NVE and PVE isolates to have more unique sequence types (types not shared by the other groups; 74 and 71%, respectively) than either BSI isolates (10%) or skin isolates (42%). Thirty NVE, 16 PVE, and a total of 9 of the nasal, skin, and BSI isolates were tested for virulence in Caenorhabditis elegans. Twenty-one (70%) of the 30 NVE isolates killed at least 50% of the worms by day 5, compared to 1 (6%) of 16 PVE isolates and 1 (11%) of 9 nasal, skin, or BSI isolates. In addition, the C. elegans survival rate as assessed by log rank analyses of Kaplan-Meier survival curves was significantly lower for NVE isolates than for each other group of isolates (P < 0.0001). There was no correlation between the production of poly-beta(1-6)-N-acetylglucosamine exopolysaccharide and virulence in worms. This study is the first analysis suggesting that S. epidermidis isolates from patients with NVE constitute a more virulent subset within this species.


Assuntos
Biofilmes , Endocardite Bacteriana/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Staphylococcus epidermidis/patogenicidade , Animais , Caenorhabditis elegans , Genes Bacterianos , Genótipo , Humanos , Reação em Cadeia da Polimerase , Staphylococcus epidermidis/imunologia
5.
Trends Microbiol ; 25(2): 153-166, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27751626

RESUMO

A deluge of whole-genome sequencing has begun to give insights into the patterns and processes of microbial evolution, but genome sequences have accrued in a haphazard manner, with biased sampling of natural variation that is driven largely by medical and epidemiological priorities. For instance, there is a strong bias for sequencing epidemic lineages of methicillin-resistant Staphylococcus aureus (MRSA) over sensitive isolates (methicillin-sensitive S. aureus: MSSA). As more diverse genomes are sequenced the emerging picture is of a highly subdivided species with a handful of relatively clonal groups (complexes) that, at any given moment, dominate in particular geographical regions. The establishment of hegemony of particular clones appears to be a dynamic process of successive waves of replacement of the previously dominant clone. Here we review the phylogenomic structure of a diverse range of S. aureus, including both MRSA and MSSA. We consider the utility of the concept of the 'core' genome and the impact of recombination and horizontal transfer. We argue that whole-genome surveillance of S. aureus populations could lead to better forecasting of antibiotic resistance and virulence of emerging clones, and a better understanding of the elusive biological factors that determine repeated strain replacement.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Transferência Genética Horizontal/fisiologia , Genoma Bacteriano/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Antibacterianos/farmacologia , Sequência de Bases , Evolução Molecular , Humanos , Staphylococcus aureus Resistente à Meticilina/classificação , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Fatores de Virulência/genética
6.
Insect Biochem Mol Biol ; 36(8): 674-82, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16876710

RESUMO

The effect of xenobiotics (phenobarbital and atrazine) on the expression of Drosophila melanogaster CYP genes encoding cytochromes P450, a gene family generally associated with detoxification, was analyzed by DNA microarray hybridization and verified by real-time RT-PCR in adults of both sexes. Only a small subset of the 86 CYP genes was significantly induced by the xenobiotics. Eleven CYP genes and three glutathione S-transferases (GST) genes were significantly induced by phenobarbital, seven CYP and one GST gene were induced by atrazine. Cyp6d5, Cyp6w1, Cyp12d1 and the ecdysone-inducible Cyp6a2 were induced by both chemicals. The constitutive expression of several of the inducible genes (Cyp6a2, Cyp6a8, Cyp6d5, Cyp12d1) was higher in males than in females, and the induced level similar in both sexes. Thus, the level of induction was consistently higher in females than in males. The female-specific and hormonally regulated yolk protein genes were significantly induced by phenobarbital in males and repressed by atrazine in females. Our results suggest that the numerous CYP genes of Drosophila respond selectively to xenobiotics, providing the fly with an adaptive response to chemically adverse environments. The xenobiotic inducibility of some CYP genes previously associated with insecticide resistance in laboratory-selected strains (Cyp6a2, Cyp6a8, Cyp12d1) suggests that deregulation of P450 gene expression may be a facile way to achieve resistance. Our study also suggests that xenobiotic-induced changes in P450 levels can affect insect fitness by interfering with hormonally regulated networks.


Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/efeitos dos fármacos , Xenobióticos/farmacologia , Animais , Atrazina , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Masculino , Fenobarbital , Fatores Sexuais , Ativação Transcricional
7.
Bioelectrochemistry ; 68(1): 67-71, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15936991

RESUMO

This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).


Assuntos
Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Oxazinas/química , Animais , Monóxido de Carbono/farmacologia , Sistema Enzimático do Citocromo P-450/química , Drosophila melanogaster/classificação , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Eletroquímica , Metilação , Microssomos/química , Microssomos/metabolismo , Estrutura Molecular , NADP/metabolismo , Espectrometria de Fluorescência , Especificidade por Substrato , Fatores de Tempo
8.
FEMS Microbiol Rev ; 26(5): 433-56, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12586390

RESUMO

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.


Assuntos
Genoma Bacteriano , Photorhabdus , Simbiose/genética , Animais , Bacteriocinas/genética , Bacteriocinas/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , Insetos/metabolismo , Insetos/microbiologia , Insetos/ultraestrutura , Estágios do Ciclo de Vida , Modelos Genéticos , Nematoides/genética , Nematoides/microbiologia , Nematoides/fisiologia , Photorhabdus/genética , Photorhabdus/isolamento & purificação , Photorhabdus/patogenicidade , Photorhabdus/fisiologia , Fatores de Virulência/biossíntese , Fatores de Virulência/genética
9.
mBio ; 4(6): e00889-13, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24345744

RESUMO

UNLABELLED: The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone. IMPORTANCE: Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.


Assuntos
Evolução Molecular , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/fisiologia , Viabilidade Microbiana , Poliaminas/metabolismo , Pele/microbiologia , Staphylococcus epidermidis/genética , Antibacterianos/metabolismo , Biotransformação , DNA Bacteriano/genética , Transferência Genética Horizontal , Humanos , Sequências Repetitivas Dispersas , Filogenia
10.
Toxicon ; 57(2): 199-207, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21112350

RESUMO

Pierisin-like proteins comprise a growing family of ADP-ribosyltransferases expressed in various species of white butterflies. The prototype pierisin-1 from the cabbage butterfly, Pieris rapae, was identified as a potent apoptosis-inducing agent, acting on various types of carcinoma cell lines by mono-ADP-ribosylation of DNA. The characterization of pierisin-like proteins is hampered by its potent toxicity, which prevents its expression as a recombinant protein in Escherichia coli. Here we characterized a new member of the pierisin protein family named pierisin-1b, which was cloned from P. rapae. Pierisin-1b consists of 849 amino acids residues and shares 63%-91% identity with already described pierisins. For expression of pierisin-1b a novel in vitro translation system was utilized. Obtained protein exhibits specific ADP-ribosyltransferase activity on deoxyguanosine residues of DNA leading to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/química , Apoptose/efeitos dos fármacos , Borboletas/química , Citotoxinas/química , Proteínas de Insetos/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/farmacologia , Sequência de Aminoácidos , Animais , Sistema Livre de Células , Clonagem Molecular , Citotoxinas/genética , Citotoxinas/farmacologia , Células HeLa , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/farmacologia , Dados de Sequência Molecular , Alinhamento de Sequência
11.
Pest Manag Sci ; 66(10): 1106-15, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20583201

RESUMO

BACKGROUND: Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. RESULTS: Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. CONCLUSION: The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Proteínas de Drosophila/química , Drosophila/enzimologia , Resistência a Inseticidas , Animais , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 6 do Citocromo P450 , DDT/metabolismo , DDT/farmacologia , Drosophila/química , Drosophila/efeitos dos fármacos , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Inseticidas/metabolismo , Inseticidas/farmacologia , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Especificidade por Substrato
12.
FEMS Microbiol Lett ; 293(2): 170-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19243439

RESUMO

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.


Assuntos
Manduca/enzimologia , Manduca/microbiologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Photorhabdus/genética , Photorhabdus/patogenicidade , Animais , Elementos de DNA Transponíveis , Escherichia coli/genética , Técnicas de Inativação de Genes , Biblioteca Gênica , Mutagênese Insercional , Análise de Sobrevida , Virulência
13.
Proc Natl Acad Sci U S A ; 104(7): 2419-24, 2007 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-17284598

RESUMO

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA- mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA- mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA- mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.


Assuntos
Antibacterianos/farmacologia , Insetos/microbiologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Animais , Antibacterianos/biossíntese , Bactérias/patogenicidade , Imunidade , Mutação , Photorhabdus/genética , Photorhabdus/patogenicidade , Virulência/genética
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