RESUMO
The GAD system is widely present in several types of organisms and is known to play an important role in bacterial acid tolerance. There is only one account of this system playing a role in oxidative stress in bacteria and one in yeasts. Here we show for first time that it affects the oxidative stress resistance of a Gram-positive bacterium, (L. monocytogenes, tested in three strains; 10403S, EGD-e, and LO28). We found a statistically significant reduction in survival after H2O2 exposure in ΔgadD3 and ΔgadD2 of EGD-e and in ΔgadD1 of LO28. Furthermore, we observed a lag phase prolongation in ΔgadD3 of 10403S and EGD-e and a larger inhibition zone in disk diffusion assay for ΔgadD1 and ΔgadD3 of EGD-e upon H2O2 exposure. All GAD genes playing a role in oxidative stress resistance are part of GADi system and this occurs partly through catalase activity, while the most potent GADe system plays no role. The latter effects could occur through the GABA shunt, but we show here that mutants in succinate semialdehyde dehydrogenase do not show a phenotype suggesting that either effects are through the GABA transaminase or, this pathway is not involved. Our study highlights for first time the role of the GAD system in oxidative stress resistance of a Gram-positive bacterium, which could be used in Food Hurdle Technology to eliminate pathogens such as L. monocytogenes, while it gives an insight on the general mechanism.
Assuntos
Glutamato Descarboxilase/metabolismo , Peróxido de Hidrogênio/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/enzimologia , Estresse Oxidativo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glutamato Descarboxilase/genética , Concentração de Íons de Hidrogênio , Listeria monocytogenes/genéticaRESUMO
BACKGROUND: Although vitamin B-12 (B-12) is known to contribute to the structural and functional development of the brain, it is unclear if B-12 supplementation has any beneficial effect in healthy populations in terms of enhanced neurologic status of the brain or improved cognitive function. OBJECTIVES: We investigated the effect of dietary supplementation of B-12 on the cortical neural activity of well-nourished young adult rats and tested the hypothesis that B-12 supplementation in healthy rats may reduce sensory-evoked neural activity due to enhanced inhibition. METHODS: Female Lister Hooded rats weighing 190-265 g (2-4 mo old) were included in the study. The experimental group was fed with B-12 (cyanocobalamin)-enriched water at a concentration of 1 mg/L, and the control (CON) group with tap water for 3 wk. Animals were then anesthetized and cortical neural responses to whisker stimulation were recorded in vivo through the use of a multichannel microelectrode, from which local field potentials (LFPs) were extracted. RESULTS: Somatosensory-evoked LFP was 25% larger in the B-12 group (4.13 ± 0.24 mV) than in the CON group (3.30 ± 0.21 mV) (P = 0.02). Spontaneous neural activity did not differ between groups; frequency spectra at each frequency bin of interest did not pass the cluster-forming threshold at the 5% significance level. CONCLUSIONS: These findings do not provide evidence supporting the hypothesis of decreased neural activity due to B-12 supplementation. As the spontaneous neural activity was unaffected, the increase in somatosensory-evoked LFP may be due to enhanced afferent signal reaching the barrel cortex from the whisker pad, indicating that B-12-supplemented rats may have enhanced sensitivity to sensory stimulation compared with the CON group. We suggest that this enhancement might be the result of lowered sensory threshold, although the underlying mechanism has yet to be elucidated.
Assuntos
Encéfalo/efeitos dos fármacos , Suplementos Nutricionais , Sensação/efeitos dos fármacos , Limiar Sensorial/efeitos dos fármacos , Vibrissas , Vitamina B 12/farmacologia , Complexo Vitamínico B/farmacologia , Animais , Feminino , RatosRESUMO
UNLABELLED: SigB is the main stress gene regulator in Listeria monocytogenes affecting the expression of more than 150 genes and thus contributing to multiple-stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain, as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that unlike for all other stresses, loss of sigB results in hyperresistance to H2O2 (more than 8 log CFU ml(-1) compared to the wild type) in aerobically grown stationary-phase cultures of L. monocytogenes strains 10403S and EGD-e. Furthermore, growth at 30°C resulted in higher resistance to oxidative stress than that at 37°C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, the loss of SigB in 10403S did not affect survival against H2O2, while in EGD-e, it resulted in a sensitive phenotype. During exponential phase, minor differences occurred, and this result was expected due to the absence of sigB transcription. Catalase tests were performed under all conditions, and stronger catalase results corresponded well with a higher survival rate, underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates, which corresponded with the catalase tests and survival. In addition, reverse transcription-PCR (RT-PCR) showed no differences in transcription between the wild type and the ΔsigB mutant in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are under way. IMPORTANCE: SigB is the most important stress gene regulator in L. monocytogenes and other Gram-positive bacteria. Its increased expression during stationary phase results in resistance to multiple stresses. However, despite its important role in general stress resistance, its expression is detrimental for the cell in the presence of oxidative stress, as it promotes hypersensitivity against hydrogen peroxide. This peculiar phenotype is an important element of the physiology of L. monocytogenes, and it might help us explain the behavior of this organism in environments where oxidative stress is present.
Assuntos
Proteínas de Bactérias/genética , Peróxido de Hidrogênio/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Estresse Oxidativo , Fator sigma/metabolismoRESUMO
The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.
RESUMO
The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.
Assuntos
Bacteriemia , Infecções Estafilocócicas , Animais , Humanos , Camundongos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Parede Celular/metabolismo , Antibacterianos/farmacologia , Ácidos Teicoicos/metabolismoRESUMO
OBJECTIVES: To determine the antibiotic phenotype and MAST-genotype distribution of Neisseria gonorrhoeae isolates in Portugal between 2004 and 2009, and to evaluate specific associations between MAST-genotypes and sexual orientation, age and antibiotic resistance. METHODS: A total of 236 N gonorrhoeae isolates were typed through N gonorrhoeae multiantigen sequence typing (NG-MAST). The degree of polymorphism and the phylogenetic relatedness among NG-MAST sequence types (STs) were evaluated with MEGA4 software on concatenated sequences of por and tbpb alleles. Etest was used to determine the susceptibility to ceftriaxone, ciprofloxacin, penicillin and spectinomycin. RESULTS: No isolates displayed resistance to spectinomycin and ceftriaxone, whereas 79.1% and 37.4% were resistant to penicillin and ciprofloxacin, respectively. A total of 104 different STs (one per 2.3 isolates) were found; the most common were ST210 (8.1%) and ST225 (7.6%). STs formed two major groups separated by 159.8 (SE 8.9) nucleotide differences, yielding several subgroups, one of them including the worldwide-prevalent ST225. The probability of ciprofloxacin resistance among isolates within this subgroup was 73.5-fold higher than for the remaining isolates. Indeed, for the genetically closest subgroup, which includes the most prevalent ST (ST210), only 8.0% of isolates were resistant to ciprofloxacin. There was a non-homogeneous distribution per year for ST225 (p<0.001), ST210 (p=0.011) and ST2 (p=0.007). CONCLUSIONS: The heterogeneous ST scenario may represent the 'tip of the iceberg', reflecting a high number of undiagnosed and unreported gonorrhoea cases. A laboratory-based national surveillance of N gonorrhoeae infections is necessary to provide a broader spectrum of isolates that will allow the sexual network situation in Portugal to be established.
Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Gonorreia/tratamento farmacológico , Neisseria gonorrhoeae/genética , Adolescente , Adulto , Antígenos de Bactérias/genética , Técnicas de Tipagem Bacteriana , Criança , Pré-Escolar , Feminino , Gonorreia/epidemiologia , Heterossexualidade , Homossexualidade Masculina , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neisseria gonorrhoeae/efeitos dos fármacos , Fenótipo , Polimorfismo Genético , Portugal/epidemiologia , Adulto JovemRESUMO
Neurocysticercosis (NCC) is the most common cause of acquired epilepsy in resource-poor countries. We report the case of a 24-year-old woman born and residing in Guinea-Bissau, who was transferred to Portugal two months after the onset of a possible meningitis (fever, headache, seizures, and coma) that did not respond to antibiotic treatment. The diagnosis of NCC was confirmed by MR imaging, which showed multiple lesions compatible with cysticercus, and by polymerase chain reaction (PCR) of the cerebrospinal fluid. After 28 days on albendazole and dexamethasone without improvement, she was started on praziquantel, which she completed in six weeks with progressive recovery.
Assuntos
Neurocisticercose/diagnóstico , Neurocisticercose/patologia , Anti-Helmínticos/uso terapêutico , Líquido Cefalorraquidiano/parasitologia , Feminino , Guiné-Bissau , Cabeça/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética , Neurocisticercose/tratamento farmacológico , Reação em Cadeia da Polimerase , Portugal , Praziquantel/uso terapêutico , Radiografia , Resultado do Tratamento , Adulto JovemRESUMO
INTRODUCTION: Recent data indicates that low vitamin D (25(OH)D) levels can lead to a worst prognosis in HIV-infected individuals, even in those on successful antiretroviral therapy (ART) [1]. Portugal is the European country that has the largest average sun exposure time but prevalence of hypovitaminosis D is mostly unknown. Our aim was to determine the prevalence of hypovitaminosis D in HIV patients in Lisbon and the possible association with ART. METHODS: From 2012 to January 2014, plasma samples from 518 HIV-infected patients were collected to 25(OH)D levels determination. Data on demographic features (age, ethnicity, country of origin) and clinical/laboratory parameters were collected from clinical files (HIV subtype, CD4+ cell count, CD4+ nadir, viral load (VL), HBV/HCV co-infection and ART). 25(OH)D status was defined as: deficiency <20 ng/mL, insufficiency 20-30 ng/mL, optimal >30 ng/mL. RESULTS: Median age was 46 years old (±11); 62.0% (321/518) were male; 81.3% (421/518) were Caucasian and 78.6% (407/518) were Portuguese. Most patients (96.1%; 498/518), were HIV-1 infected, 22.9% (114/498) and 4.0% (20/498) of them were HCV and/or HBV co-infected, respectively. Mean CD4+ cell count was 648 cells/µL (±333) and nadir was 219 cells/µL (±179). On treated patients VL was <40 HIV RNA/mL in 86.7% (417/481). The median levels of 25(OH)D was 20.0 ng/mL (range 4.1-99.7) and we found differences between values observed during Winter (median 16.7 ng/mL) and Summer (median 24.9 ng/mL) (p<0.0001). Low 25(OH)D levels were not correlated to ethnicity (p=0.066). 25(OH)D level was <30 ng/mL in 80.1% (415/518) of the patients, from which 30.9% (160/518) and 49.2% (255/518) had insufficiency and deficiency levels, respectively. Most (92.9%; 481/518) were on ART: regimens containing PI (47.5%), NNRTI (40.3%; 41.3% on NVP and 58.7% on EFV), II (1.2%), PI+NNRTI (3.9%). Comparing the 25(OH)D level along the different ART regimens (PI vs NVP; PI vs EFV; PI vs no ART) there were differences between PI and EFV (p=0.044). CONCLUSIONS: In this study, 80.1% of the HIV-infected patients had hypovitaminosis D and ART regimens with EFV were more often associated with low 25(OH)D levels. Understanding the impact of the different antiretroviral drugs on 25(OH)D status could help to decide in clinical practice whether 25(OH)D supplementation or drug switch are the best options for each patient.
RESUMO
In malaria, hemozoin (Hz) is released during erythrocyte rupture, and subsequent phagocytosis appears to cause important immune-modulatory effects. Hz obtained from Plasmodium falciparum cultures or synthesized from heme is used to study this modulation in vitro. Immune-activating and suppressive effects have been reported, and these discrepant results are often attributed to the different types of Hz that were used. However, it remains unclear which type of Hz accurately reproduces what happens in vivo. Importantly, Hz remains in the body for long periods and appears to be actively redistributed. Thus, phagocytosis of Hz in the body is not static but probably happens more than once, and the characteristics of Hz may change over time, eventually causing different immune-modulatory effects.