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3.
Methods Mol Biol ; 1921: 45-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30694484

RESUMO

Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry.


Assuntos
Citometria de Fluxo , Legionella pneumophila/fisiologia , Viabilidade Microbiana , Citometria de Fluxo/métodos , Indicadores e Reagentes , Propídio/metabolismo , Coloração e Rotulagem
4.
Autophagy ; 15(5): 900-907, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30563411

RESUMO

Macroautophagy/autophagy, a pathway by which cellular components are sequestered and degraded in response to homeostatic and cell stress-related signals, is required to preserve hematopoietic stem and progenitor cell function. Loss of chromosomal regions carrying autophagy genes and decreased autophagy gene expression are characteristic of acute myeloid leukemia (AML) cells. Deficiency of autophagy proteins is also linked to an altered AML metabolic profile; altered metabolism has recently emerged as a potential druggable target in AML. Here, we sought to understand the mitochondria-specific changes that occur in leukemia cells after knockdown of BNIP3L/Nix or SQSTM1/p62, which are two autophagy genes involved in mitochondrial clearance and are downregulated in primary AML cells. Mitochondrial function, as measured by changes in endogenous levels of reactive oxygen species (ROS) and mitochondrial membrane potential, was altered in leukemia cells deficient in these autophagy genes. Further, these AML cells were increasingly sensitive to mitochondria-targeting drugs while displaying little change in sensitivity to DNA-targeting agents. These findings suggest that BNIP3L or SQSTM1 may be useful prognostic markers to identify AML patients suitable for mitochondria-targeted therapies. Abbreviations: AML: acute myeloid leukemia; DHE: dihydroethidium; mtDNA: mitochondrial DNA; NAO: 10-N-nonyl acridine orange; PD: population doubling; R123: rhodamine 123; ROS: reactive oxygen species; TRC: transduced scramble controls.


Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/farmacologia , Proteína Sequestossoma-1/genética , Proteínas Supressoras de Tumor/genética , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/antagonistas & inibidores
5.
Artigo em Inglês | MEDLINE | ID: mdl-30280092

RESUMO

Citrobacter rodentium is a murine pathogen used to model the intestinal infection caused by Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC and EHEC), two diarrheal pathogens responsible for morbidity and mortality in developing and developed countries, respectively. During infection, these bacteria must sense and adapt to the gut environment of the host. In order to adapt to changing environmental cues and modulate expression of specific genes, bacteria can use two-component signal transduction systems (TCS). We have shown that the deletion of the Cpx TCS in C. rodentium leads to a marked attenuation in virulence in C3H/HeJ mice. In E. coli, the Cpx TCS is reportedly activated in response to signals from the outer-membrane lipoprotein NlpE. We therefore investigated the role of NlpE in C. rodentium virulence. We also assessed the role of the reported negative regulator of CpxRA, CpxP. We found that as opposed to the ΔcpxRA strain, neither the ΔnlpE, ΔcpxP nor the ΔnlpEΔcpxP strains were significantly attenuated, and had similar in vivo localization to wild-type C. rodentium. The in vitro adherence of the Cpx auxiliary protein mutants, ΔnlpE, ΔcpxP, ΔnlpEΔcpxP, was comparable to wild-type C. rodentium, whereas the ΔcpxRA strain showed significantly decreased adherence. To further elucidate the mechanisms behind the contrasting virulence phenotypes, we performed microarrays in order to define the regulon of the Cpx TCS. We detected 393 genes differentially regulated in the ΔcpxRA strain. The gene expression profile of the ΔnlpE strain is strikingly different than the profile of ΔcpxRA with regards to the genes activated by CpxRA. Further, there is no clear inverse correlation in the expression pattern of the ΔcpxP strain in comparison to ΔcpxRA. Taken together, these data suggest that in these conditions, CpxRA activates gene expression in a largely NlpE- and CpxP-independent manner. Compared to wildtype, 161 genes were downregulated in the ΔcpxRA strain, while being upregulated or unchanged in the Cpx auxiliary protein deletion strains. This group of genes, which we hypothesize may contribute to the loss of virulence of ΔcpxRA, includes T6SS components, ompF, the regulator for colanic acid synthesis, and several genes involved in maltose metabolism.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Citrobacter rodentium/crescimento & desenvolvimento , Citrobacter rodentium/patogenicidade , Regulação Bacteriana da Expressão Gênica , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C3H , Análise em Microsséries , Proteínas Quinases/genética , Virulência
6.
Can J Microbiol ; 64(12): 1030-1041, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30212639

RESUMO

The intracellular pathogen Legionella pneumophila (Lp) is a strict aerobe, surviving and replicating in environments where it frequently encounters reactive oxygen species (ROS), such as the nutrient-poor water environment and its replicative niche inside host cells. In many proteobacteria, the LysR-type regulator OxyR controls the oxidative stress response; however, the importance of the OxyR homologue in Lp is still unclear. Therefore, we undertook the characterization of phenotypes associated with the deletion of oxyR in Lp. Contrary to the wild type, the oxyR deletion mutant exhibits a severe growth defect on charcoal - yeast extract (CYE) agar lacking α-ketoglutarate supplementation. Growth in AYE broth (CYE without agar and charcoal), in amoeba and in human cultured macrophages, and survival in water is unaffected by the deletion. Supplementing CYE agar with antioxidants that neutralize ROS or introducing the oxyR gene in trans rescues the observed growth defect. Moreover, the mutant grows as well as the wild type on CYE plates made with agarose instead of agar, suggesting that a compound present in the latter is responsible for the growth defect phenotype.


Assuntos
Proteínas de Bactérias/fisiologia , Legionella pneumophila/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Ágar , Humanos , Legionella pneumophila/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência
7.
Sci Rep ; 8(1): 6764, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712912

RESUMO

Surviving the nutrient-poor aquatic environment for extended periods of time is important for the transmission of various water-borne pathogens, including Legionella pneumophila (Lp). Previous work concluded that the stringent response and the sigma factor RpoS are essential for the survival of Lp in water. In the present study, we investigated the role of the LetA/S two-component signal transduction system in the successful survival of Lp in water. In addition to cell size reduction in the post-exponential phase, LetS also contributes to cell size reduction when Lp is exposed to water. Importantly, absence of the sensor kinase results in a significantly lower survival as measured by CFUs in water at various temperatures and an increased sensitivity to heat shock. According to the transcriptomic analysis, LetA/S orchestrates a general transcriptomic downshift of major metabolic pathways upon exposure to water leading to better culturability, and likely survival, suggesting a potential link with the stringent response. However, the expression of the LetA/S regulated small regulatory RNAs, RsmY and RsmZ, is not changed in a relAspoT mutant, which indicates that the stringent response and the LetA/S response are two distinct regulatory systems contributing to the survival of Lp in water.


Assuntos
Proteínas de Bactérias/genética , Legionella pneumophila/genética , Doença dos Legionários/genética , Transcriptoma/genética , Fibronectinas/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Legionella pneumophila/crescimento & desenvolvimento , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Fator sigma/genética , Virulência , Microbiologia da Água
8.
Front Microbiol ; 8: 1781, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28979243

RESUMO

Campylobacter jejuni is the leading cause of campylobacteriosis in the developed world. Although most cases are caused by consumption of contaminated meat, a significant proportion is linked to ingestion of contaminated water. The differences between C. jejuni strains originating from food products and those isolated from water are poorly understood. Working under the hypothesis that water-borne C. jejuni strains are better equipped at surviving the nutrient-poor aquatic environment than food-borne strains, the present study aims to characterize these differences using outbreak strains 81116 and 81-176. Strain 81116 caused a campylobacteriosis outbreak linked to consumption of water, while strain 81-176 was linked to consumption of raw milk. CFU counts and viability assays showed that 81116 survives better than 81-176 at 4°C in a defined freshwater medium (Fraquil). Moreover, 81116 was significantly more resistant to oxidative stress and bile salt than strain 81-176 in Fraquil. To better understand the genetic response of 81116 to water, a transcriptomic profiling study was undertaken using microarrays. Compared to rich broth, strain 81116 represses genes involved in amino acid uptake and metabolism, as well as genes involved in costly biosynthetic processes such as replication, translation, flagellum synthesis and virulence in response to Fraquil. In accordance with the observed increase in stress resistance in Fraquil, 81116 induces genes involved in resistance to oxidative stress and bile salt. Interestingly, genes responsible for cell wall synthesis were also induced upon Fraquil exposure. Finally, twelve unique genes were expressed in Fraquil; however, analysis of their distribution in animal and water isolates showed that they are not uniquely and ubiquitously present in water isolates, and thus, unlikely to play a major role in adaptation to water. Our results show that some C. jejuni strains are more resilient than others, thereby challenging current water management practices. The response of 81116 to Fraquil serves as a starting point to understand the adaptation of C. jejuni to water and its subsequent transmission.

9.
PLoS One ; 10(9): e0139277, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26406895

RESUMO

Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires' disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil.


Assuntos
Água Doce/microbiologia , Legionella pneumophila/patogenicidade , Viabilidade Microbiana , Concentração de Íons de Hidrogênio , Espaço Intracelular/microbiologia , Legionella pneumophila/crescimento & desenvolvimento , Metais/toxicidade , Viabilidade Microbiana/efeitos dos fármacos , Temperatura , Fatores de Tempo , Virulência/efeitos dos fármacos
10.
BMC Genomics ; 16: 637, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26306795

RESUMO

BACKGROUND: Legionella pneumophila (Lp) is a water-borne opportunistic pathogen. In water, Lp can survive for an extended period of time until it encounters a permissive host. Therefore, identifying genes that are required for survival in water may help develop strategies to prevent Legionella outbreaks. RESULTS: We compared the global transcriptomic response of Lp grown in a rich medium to that of Lp exposed to an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours. We uncovered successive changes in gene expression required for the successful adaptation to a nutrient-limited water environment. The repression of major pathways involved in cell division, transcription and translation, suggests that Lp enters a quiescent state in water. The induction of flagella associated genes (flg, fli and mot), enhanced-entry genes (enh) and some Icm/Dot effector genes suggests that Lp is primed to invade a suitable host in response to water exposure. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to these stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than Lp cultured in rich medium. In addition, the bdhA gene, involved in the degradation pathway of the intracellular energy storage compound polyhydroxybutyrate, is also highly expressed in water. Further characterization show that expression of bdhA during short-term water exposure is dependent upon RpoS, which is required for the survival of Lp in water. Deletion of bdhA reduces the survival of Lp in water at 37 °C. CONCLUSIONS: The increase of antibiotic resistance and the importance of bdhA to the survival of Lp in water seem consistent with the observed induction of these genes when Lp is exposed to water. Other genes that are highly induced upon exposure to water could also be necessary for Lp to maintain viability in the water environment.


Assuntos
Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/genética , Transcriptoma , Microbiologia da Água , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Análise por Conglomerados , Farmacorresistência Bacteriana , Perfilação da Expressão Gênica , Genes Bacterianos , Legionella pneumophila/efeitos dos fármacos , Viabilidade Microbiana/genética , Mutação , Reprodutibilidade dos Testes
11.
Anal Bioanal Chem ; 407(18): 5541-5, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25935681

RESUMO

Legionellosis is a very devastating disease worldwide mainly due to unpredictable outbreaks in man-made water systems. Developing a highly specific and sensitive rapid detection system that detects only metabolically active bacteria is a main priority for water quality assessment. We previously developed a versatile technique for sensitive and specific detection of synthetic RNA. In the present work, we further investigated the performance of the developed biosensor for detection of Legionella pneumophila in complex environmental samples, particularly those containing protozoa. The specificity and sensitivity of the detection system were verified using total RNA extracted from L. pneumophila in spiked water co-cultured with amoebae. We demonstrated that the expression level of ribosomal RNA (rRNA) is extremely dependent on the environmental conditions. The presence of amoebae with L. pneumophila, especially in nutrition-deprived samples, increased the amount of L. pneumophila 15-fold after 1 week as measured through the expression of 16s rRNA. Using the developed surface plasmon resonance imaging (SPRi) detection method, we were also able to successfully detect L. pneumophila within 3 h, both in the presence and absence of amoebae in the complex environmental samples obtained from a cooling water tower. These findings suggest that the developed biosensing system is a viable method for rapid, real-time and effective detection not only for L. pneumophila in environmental samples but also to assess the risk associated with the use of water contaminated with other pathogens.


Assuntos
Legionella pneumophila/isolamento & purificação , Doença dos Legionários/microbiologia , Ressonância de Plasmônio de Superfície/métodos , Microbiologia da Água , Amoeba/isolamento & purificação , Desenho de Equipamento , Humanos , Legionella pneumophila/genética , Limite de Detecção , RNA Ribossômico 16S/genética , Ressonância de Plasmônio de Superfície/economia , Ressonância de Plasmônio de Superfície/instrumentação , Fatores de Tempo
12.
Can J Microbiol ; 60(11): 777-81, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25352257

RESUMO

Pseudomonas aeruginosa is an opportunistic pathogen that can infect susceptible patients suffering from cystic fibrosis, immunosuppression, and severe burns. Nosocomial- and community-acquired infection is likely due to contact with water sources contaminated with P. aeruginosa. Most of what is known about the virulence properties of P. aeruginosa was derived from studies using fairly rich broths, which do not represent conditions found in water, such as low nutrient concentrations. Here, we compare biofilm production, invasion of epithelial cells, cytotoxicity, and pyocyanin production of P. aeruginosa in water with P. aeruginosa grown in rich broth. Since tap water is variable, we used a defined water medium, Fraquil, to ensure reproducibility of the results. We found that P. aeruginosa does not readily form biofilm in Fraquil. Pseudomonas aeruginosa is equally able to attach to and invade epithelial cells but is more cytotoxic after incubation in water for 30 days than when it is grown in rich broth. Moreover, P. aeruginosa produces less pyocyanin when exposed to water. Our results show that P. aeruginosa seems to have different properties when exposed to water than when grown in rich broth.


Assuntos
Células Epiteliais/microbiologia , Água Doce/microbiologia , Pseudomonas aeruginosa/patogenicidade , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Sobrevivência Celular , Meios de Cultura , Fibrose Cística , Humanos , Pulmão , Viabilidade Microbiana , Piocianina/biossíntese , Reprodutibilidade dos Testes , Virulência
13.
PLoS One ; 9(8): e103852, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25105285

RESUMO

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.


Assuntos
Surtos de Doenças , Genoma Bacteriano/genética , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , Filogenia , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Análise por Conglomerados , Biologia Computacional , Eletroforese em Gel de Campo Pulsado , História do Século XXI , Humanos , Dados de Sequência Molecular , Quebeque/epidemiologia , Análise de Sequência de DNA , Estatísticas não Paramétricas
14.
Front Microbiol ; 5: 258, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24917854

RESUMO

Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.

15.
Curr Top Microbiol Immunol ; 376: 53-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23918178

RESUMO

Legionella pneumophila is a water-borne pathogen that causes a severe lung infection in humans. It is able to replicate inside amoeba in the water environment, and inside lung macrophages in humans. Efficient regulation of gene expression is critical for responding to the conditions that L. pneumophila encounters and for intracellular multiplication in host cells. In the last two decades, many reports have contributed to our understanding of the critical importance of small regulatory RNAs (sRNAs) in the regulatory network of bacterial species. This report presents the current state of knowledge about the sRNAs expressed by L. pneumophila and discusses a few regulatory pathways in which sRNAs should be involved in this pathogen.


Assuntos
Legionella pneumophila/genética , Pequeno RNA não Traduzido/fisiologia , Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/fisiologia , Ferro/metabolismo , Estresse Oxidativo , Fator sigma/fisiologia
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