A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa

Rao, Jayasimha; DiGiandomenico, Antonio; Unger, Jason; Bao, Yongde; Polanowska-Grabowska, Renata; Goldberg, Joanna B.

doi:10.1099/mic.0.2007/011429-0

Cited by 34 publications

(55 citation statements)

References 65 publications

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“…Proteins were transferred by electroblotting onto 0.2-mm nitrocellulose membranes (Bio-Rad) at 100 V for 45 min. Membranes were blocked with 5% (w/v) fat-free milk in PBS-T [10-mM phosphate buffered saline (PBS) (pH 7.4) with 0.05% Tween-20] for 1 h at room temperature after which blots were probed with anti-recombinant-RahU (PA0122) mouse serum (52) diluted 1:2000 in PBS-T, and incubated overnight at 4°C. Immunodetection was performed with peroxidase-conjugated rabbit anti-mouse immunoglobulin G secondary antibody (Sigma) at a dilution of 1:5000 in PBS-T.…”

Section: Methodsmentioning

confidence: 99%

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

Kulkarni

Jia

Kuehne

et al. 2014

Nucleic Acids Research

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CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.

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Section: Methodsmentioning

confidence: 99%

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

Kulkarni

Jia

Kuehne

et al. 2014

Nucleic Acids Research

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“…Standard curves were produced for each primer-probe set listed in Table S1 in the supplemental material by analyzing cDNA generated from serial dilutions of RNA and used to determine relative amounts of the RNAs, as described previously (22). Relative RNA levels were then normalized to the levels of the oprF mRNA as previously described (22,59,60) for aerobic expression studies or to the omlA level as previously described (21) for microaerobic expression studies. The expression of each of these genes was constant under these respective conditions (Fig.…”

Section: Methodsmentioning

confidence: 99%

The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

et al. 2017

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Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrH IG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrH IG sequence [prrF(ΔH IG )]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.KEYWORDS Pseudomonas aeruginosa, sRNA, PrrF, PrrH, iron regulation, PQS, small RNA P seudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in a variety of patient populations, including acute blood and lung infections in hospitalized patients and chronic lung infections in individuals with cystic fibrosis (CF) (1-4). Iron is an essential nutrient for virulence in P. aeruginosa (5-9), but it is sequestered by mammalian host proteins such as lactoferrin and transferrin (10). To overcome this barrier to infection, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, which scavenge ferric iron (Fe 3ϩ ) from host proteins and are required for acute infections (5,(7)(8)(9). P. aeruginosa also obtains iron from host heme using outer membrane heme transporters and a cytosolic HemO heme oxygenase that degrades heme (11,12). Ferrous iron (Fe 2ϩ ) can also be obtained through the Feo system in microaerobic environments, such as those found within biofilms and the CF lung (13-15).

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“…For the bacterium Ps. aeruginosa, it was observed that expression of its haemolysin, PA0122, is highest in the stationary phase of growth (Rao et al, 2008). In contrast, limited information is available for the expression of aegerolysins in ascomycetes.…”

Section: Discussionmentioning

confidence: 99%

“…Expression of haemolysin in relation to growth has been studied previously for Pleurotus ostreatus and Pseudomonas aeruginosa (Berne et al, 2002(Berne et al, , 2007Rao et al, 2008). For P. ostreatus, a basidiomycete, the greatest expression of ostreolysin was observed during the initial stages of fungal fruiting.…”

Section: Discussionmentioning

confidence: 99%

Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

Nayak

Green

Friend

et al. 2012

Journal of Medical Microbiology

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Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and five strains of A. terreus, showed that three mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 6C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest that terrelysin may be a candidate biomarker for A. terreus infection.

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A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa

Cited by 34 publications

References 65 publications

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

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