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Proteome analysis of Streptococcus pneumoniae EF3030

ABSTRACT: Proteome analysis of pellet and supernatant samples of Streptococcus pneumoniae cultivated in minimal medium.

INSTRUMENT(S):

ORGANISM(S): Bacteria Streptococcus Pneumoniae

SUBMITTER: Leif Steil

LAB HEAD: Uwe Völker

PROVIDER: PXD062379 | Pride | 2025-06-11

REPOSITORIES: Pride

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			Action	DRS
	20230802_Exp_LS_GEN_19F_04_01_p.raw	Raw
	20230802_Exp_LS_GEN_19F_04_02_p.raw	Raw
	20230802_Exp_LS_GEN_19F_04_03_p.raw	Raw
	20230802_Exp_LS_GEN_19F_06_01_p.raw	Raw
	20230802_Exp_LS_GEN_19F_06_02_p.raw	Raw

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Project description:The Gram-positive pathogenic bacterium Staphylococcus aureus permanently colonizes the nasal mucosa in about 30% of the healthy population. However, as an opportunistic pathogen, S. aureus can also cause a variety of diseases, the treatment of which is becoming increasingly difficult due to antibiotic resistance. Adaptation to changing environmental conditions during colonization and infection of the host requires precise regulation of the expression of genes for virulence factors and metabolic functions. The host conditions essentially include low iron availability, with the majority of iron in the human organism being present intracellularly in the form of haemoglobin. Therefore, S. aureus has various adaptive mechanisms, in particular iron uptake systems, which are regulated by the global iron uptake regulator Fur (ferric-uptake regulator). Iron deficiency conditions lead to the derepression of Fur-dependent genes. In many bacteria, these genes also include regulatory RNAs (small regulatory RNAs, sRNAs), which mediate an iron-sparing response by inhibiting the synthesis of non-essential iron-containing proteins. A Fur-dependent sRNA (S596) was identified in a transcriptome analysis of S. aureus. In a study by Coronel-Tellez et al. (2022) it was shown that S596/IsrR (“iron sparing response regulator”) is necessary for the virulence of S. aureus. In the presented secretome analysis, the S. aureus HG001 wild type (WT) was compared with an isogenic isrR mutant (ΔisrR) under iron-limiting conditions and a constitutively isrR-expressing strain (HG001 pJLisrR) with an empty vector control (HG001 pJLctrl) under iron-rich conditions as well as corresponding sae mutant (Δsae) strains. Culture supernatants were collected in the exponential and stationary growth phases and extracellular proteins were subsequently analyzed by mass spectrometry. Statistical data analysis was performed to identify proteins with significantly altered amounts between the respective strains. We demonstrate that IsrR positively influences the protein levels of numerous virulence factors which in particular belong to the Sae regulon.

Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.

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Proteome analysis of Streptococcus pneumoniae EF3030

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