RESUMO
BACKGROUND: For most pathogens, iron (Fe) homeostasis is crucial for maintenance within the host and the ability to cause disease. The primary transcriptional regulator that controls intracellular Fe levels is the Fur (ferric uptake regulator) protein, which exerts its action on transcription by binding to a promoter-proximal sequence termed the Fur box. Fur-regulated transcriptional responses are often fine-tuned at the post-transcriptional level through the action of small regulatory RNAs (sRNAs). Consequently, identifying sRNAs contributing to the control of Fe homeostasis is important for understanding the Fur-controlled bacterial Fe-response network. RESULTS: In this study, we sequenced size-selected directional libraries representing sRNA samples from Neisseria gonorrhoeae strain FA 1090, and examined the Fe- and temporal regulation of these sRNAs. RNA-seq data for all time points identified a pool of at least 340 potential sRNAs. Differential analysis demonstrated that expression appeared to be regulated by Fe availability for at least fifteen of these sRNAs. Fourteen sRNAs were induced in high Fe conditions, consisting of both cis and trans sRNAs, some of which are predicted to control expression of a known virulence factor, and one SAM riboswitch. An additional putative cis-acting sRNA was repressed by Fe availability. In the pathogenic Neisseria species, one sRNA that contributes to Fe-regulated post-transcriptional control is the Fur-repressible sRNA NrrF. The expression of five Fe-induced sRNAs appeared to be at least partially controlled by NrrF, while the remainder was expressed independently of NrrF. The expression of the 14 Fe-induced sRNAs also exhibited temporal control, as their expression levels increased dramatically as the bacteria entered stationary phase. CONCLUSIONS: Here we report the temporal expression of Fe-regulated sRNAs in N. gonorrhoeae FA 1090 with several appearing to be controlled by the Fe-repressible sRNA NrrF. Temporal regulation of these sRNAs suggests a regulatory role in controlling functions necessary for survival, and may be important for phenotypes often associated with altered growth rates, such as biofilm formation or intracellular survival. Future functional studies will be needed to understand how these regulatory sRNAs contribute to gonococcal biology and pathogenesis.
Assuntos
Ferro/farmacologia , Neisseria gonorrhoeae/genética , RNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/crescimento & desenvolvimento , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , Riboswitch/efeitos dos fármacos , Riboswitch/genética , Análise de Sequência de RNA , Transcriptoma/efeitos dos fármacos , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Regulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA half-lives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Análise em Microsséries , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Succinato Desidrogenase/biossíntese , Transcrição GênicaRESUMO
To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies--temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)--to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.
Assuntos
Proteínas de Bactérias/genética , Neisseria gonorrhoeae/genética , Regulon/genética , Regulon/fisiologia , Proteínas Repressoras/genética , Biologia Computacional , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/farmacologia , Neisseria gonorrhoeae/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neonatal bacteremia Escherichia coli strains commonly belong to the K1 capsular type. Their ability to cause invasive neonatal disease appears to be determined by other virulence factors that have yet to be identified. We report here the genome sequences of four E. coli neonatal bacteremia isolates, including that of the archetypal strain RS218.
RESUMO
Gene expression profiling using DNA microarrays has become commonplace in current molecular biology practices, and has dramatically enhanced our understanding of the biology of Neisseria spp., and the interaction of these organisms with the host. With the choice of microarray platforms offered for gene expression profiling and commercially available arrays, investigators must ask several central questions to make decisions based on their research focus. Are arrays on hand for their organism and if not then would it be cost-effective to design custom arrays. Other important considerations; what types of specialized equipment for array hybridization and signal detection are required and is the specificity and sensitivity of the array adequate for your application. Here, we describe the use of a custom 12K CombiMatrix ElectraSense™ oligonucleotide microarray format for assessing global gene expression profiles in Neisseria spp.
Assuntos
Perfilação da Expressão Gênica/métodos , Neisseria gonorrhoeae/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Complementar/metabolismo , Neisseria gonorrhoeae/crescimento & desenvolvimento , Hibridização de Ácido Nucleico , Fenol/química , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Transcrição Reversa , Coloração e RotulagemRESUMO
Patients with disseminated cryptococcosis typically have measurable levels of cryptococcal polysaccharide in serum samples but minimal leukocyte infiltration into infected tissues. In vitro data have shown that cryptococcal polysaccharide induces L-selectin (CD62L) shedding from leukocytes. To assess shedding in vivo, we compared leukocyte L-selectin levels in human immunodeficiency virus (HIV) type 1-negative and -positive subjects with and without circulating cryptococcal polysaccharide. Results showed that subjects with cryptococcal polysaccharide in serum samples have significantly lower percentages of neutrophils, monocytes, and CD3+ T cells with L-selectin on their surfaces than do healthy subjects, regardless of HIV status. There was significantly more soluble L-selectin in serum samples from subjects with cryptococcosis than in those from uninfected subjects. Reduced L-selectin levels on leukocytes in subjects with circulating cryptococcal polysaccharide and increased serum levels of soluble L-selectin indicates that surface L-selectin shedding is a mechanism that likely explains reduced leukocyte extravasation into infected tissues of patients with disseminated cryptococcosis.