scholarly journals Proteomic analysis of thePseudomonas aeruginosairon starvation response reveals PrrF sRNA-dependent regulation of amino acid metabolism, iron-sulfur cluster biogenesis, motility, and zinc homeostasis

2018 ◽  
Author(s):  
Cassandra E. Nelson ◽  
Weiliang Huang ◽  
Luke K. Brewer ◽  
Angela T. Nguyen ◽  
Maureen A. Kane ◽  
...  
Keyword(s):  
Amino Acid ◽  
Opportunistic Pathogen ◽  
Zinc Homeostasis ◽  
Microbial Pathogens ◽  
Label Free ◽  
Iron Starvation ◽  
Iron Sulfur Cluster ◽  
Starvation Response ◽  
Iron Sulfur ◽  
Small Regulatory Rnas

ABSTRACTIron is a critical nutrient for most microbial pathogens, and the innate immune system exploits this requirement by sequestering iron and other metals through a process termed nutritional immunity. The opportunistic pathogenPseudomonas aeruginosaprovides a model system for understanding the microbial response to host iron depletion, as this organism exhibits a high requirement for iron as well as an exquisite ability to overcome iron deprivation during infection. Hallmarks ofP. aeruginosa’siron starvation response include the induction of multiple high affinity iron acquisition systems and an “iron sparing response” that is post-transcriptionally mediated by the PrrF small regulatory RNAs (sRNAs). Here, we used liquid chromatography-tandem mass spectrometry to conduct label-free proteomics of theP. aeruginosairon starvation response, revealing several iron-regulated processes that have not been previously described. Iron starvation induced multiple proteins involved in branched chain and aromatic amino acid catabolism, providing the capacity for iron-independent entry of carbons into the TCA cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were paradoxically increased, highlighting the central role of iron inP. aeruginosametabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins, as well as the first experimental evidence for increased levels of twitching motility proteins upon iron starvation. Subsequent proteomics analyses demonstrated that the PrrF sRNAs were required for iron regulation of many of these newly-identified proteins, and we identified PrrF complementarity with mRNAs encoding key iron-regulated proteins involved in amino acid metabolism, iron-sulfur biogenesis, and zinc homeostasis. Combined, these results provide the most comprehensive view of theP. aeruginosairon starvation response to date and outline novel roles for the PrrF sRNAs in theP. aeruginosairon sparing response and pathogenesis.AUTHOR SUMMARYIron is central for the metabolism of almost all microbial pathogens, and as such this element is sequestered by the host innate immune system to restrict microbial growth. Defining the response of microbial pathogens to iron starvation is therefore critical for understanding how pathogens colonize and propagate within the host. The opportunistic pathogenPseudomonas aeruginosa, which causes significant morbidity and mortality in compromised individuals, provides an excellent model for studying this response due to its high requirement for iron yet well-documented ability to overcome iron starvation. Here we used label-free proteomics to investigate theP. aeruginosairon starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes are regulated through the iron responsive PrrF small regulatory RNAs, which are integral toP. aeruginosairon homeostasis and virulence. These results demonstrate the power of proteomics for defining stress responses of microbial pathogens, and they provide the most comprehensive analysis to date of theP. aeruginosairon starvation response.

scholarly journals Proteomic Analysis of thePseudomonas aeruginosaIron Starvation Response Reveals PrrF Small Regulatory RNA-Dependent Iron Regulation of Twitching Motility, Amino Acid Metabolism, and Zinc Homeostasis Proteins

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Cassandra E. Nelson ◽  
Weiliang Huang ◽  
Luke K. Brewer ◽  
Angela T. Nguyen ◽  
Maureen A. Kane ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune System ◽  
Amino Acid ◽  
Microbial Pathogens ◽  
Twitching Motility ◽  
Iron Starvation ◽  
Starvation Response ◽  
Content Type ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas

ABSTRACTIron is a critical nutrient for most microbial pathogens, and the immune system exploits this requirement by sequestering iron. The opportunistic pathogenPseudomonas aeruginosaexhibits a high requirement for iron yet an exquisite ability to overcome iron deprivation during infection. Upon iron starvation,P. aeruginosainduces the expression of several high-affinity iron acquisition systems, as well as the PrrF small regulatory RNAs (sRNAs) that mediate an iron-sparing response. Here, we used liquid chromatography-tandem mass spectrometry to conduct proteomics of the iron starvation response ofP. aeruginosa. Iron starvation increased levels of multiple proteins involved in amino acid catabolism, providing the capacity for iron-independent entry of carbons into the tricarboxylic acid (TCA) cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were increased, highlighting the central role of iron inP. aeruginosametabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins and increased levels of twitching motility proteins. Subsequent analyses provided evidence for the regulation of many of these proteins via posttranscriptional regulatory events, some of which are dependent upon the PrrF sRNAs. Moreover, we showed that iron-regulated twitching motility is partially dependent upon theprrFlocus, highlighting a novel link between the PrrF sRNAs and motility. These findings add to the known impacts of iron starvation inP. aeruginosaand outline potentially novel roles for the PrrF sRNAs in iron homeostasis and pathogenesis.IMPORTANCEIron is central for growth and metabolism of almost all microbial pathogens, and as such, this element is sequestered by the host innate immune system to restrict microbial growth. Here, we used label-free proteomics to investigate thePseudomonas aeruginosairon starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes, including twitching motility, are regulated through the iron-responsive PrrF small regulatory RNAs. As such, this study demonstrates the power of proteomics for defining stress responses of microbial pathogens.

scholarly journals Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

2013 ◽  
Vol 195 (20) ◽  
pp. 4556-4568 ◽  
Author(s):  
D. K. C. Fung ◽  
W. Y. Lau ◽  
W. T. Chan ◽  
A. Yan
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

Mitochondrial-type iron–sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3519-3530 ◽  
Author(s):  
Michael J. LaGier ◽  
Jan Tachezy ◽  
Frantisek Stejskal ◽  
Katerina Kutisova ◽  
Janet S. Keithly
Keyword(s):  
Cryptosporidium Parvum ◽  
Fluorescent Protein ◽  
Opportunistic Pathogen ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Primary Sequence Analysis ◽  
Genes Encoding ◽  
Green Fluorescent

Several reports have indicated that the iron–sulfur cluster [Fe–S] assembly machinery in most eukaryotes is confined to the mitochondria and chloroplasts. The best-characterized and most highly conserved [Fe–S] assembly proteins are a pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS), and IscU, a protein functioning as a scaffold for the assembly of [Fe–S] prior to their incorporation into apoproteins. In this work, genes encoding IscS and IscU homologues have been isolated and characterized from the apicomplexan parasite Cryptosporidium parvum, an opportunistic pathogen in AIDS patients, for which no effective treatment is available. Primary sequence analysis (CpIscS and CpIscU) and phylogenetic studies (CpIscS) indicate that both genes are most closely related to mitochondrial homologues from other organisms. Moreover, the N-terminal signal sequences of CpIscS and CpIscU predicted in silico specifically target green fluorescent protein to the mitochondrial network of the yeast Saccharomyces cerevisiae. Overall, these findings suggest that the previously identified mitochondrial relict of C. parvum may have been retained by the parasite as an intracellular site for [Fe–S] assembly.

scholarly journals Natively Oxidized Amino Acid Residues in the Spinach Cytochrome b6f Complex

2017 ◽  
Author(s):  
Ryan M. Taylor ◽  
Larry Sallans ◽  
Laurie K. Frankel ◽  
Terry M. Bricker
Keyword(s):  
Amino Acid ◽  
Oxidative Modification ◽  
Oxygenic Photosynthesis ◽  
Ros Generation ◽  
Ros Production ◽  
Amino Acid Residues ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
The Subject ◽  
Oxidatively Modified

AbstractThe cytochrome b6f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b6f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc1 complex. The types of ROS produced (O2•−,1O2, and, possibly, H2O2) and the site(s) of ROS production within the b6f complex has been the subject of some debate. Proposed sources of ROS have include the heme bp, PQp•− (possible sources for O2•−), the Rieske iron-sulfur cluster (possible source of O2•− and/or H2O2), Chl a (possible source of 1O2) and heme Cn (possible source of O2•− and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b6f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b6f complex.

Peroxynitrite and Nitric Oxide Differently Target the Iron−Sulfur Cluster and Amino Acid Residues of Human Iron Regulatory Protein 1†

Biochemistry ◽  
2003 ◽  
Vol 42 (25) ◽  
pp. 7648-7654 ◽  
Author(s):  
Emmanuelle Soum ◽  
Xavier Brazzolotto ◽  
Charilaos Goussias ◽  
Cécile Bouton ◽  
Jean-Marc Moulis ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Iron Regulatory Protein ◽  
Amino Acid Residues ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Regulatory Protein 1

Amino acid induced iron-sulfur cluster interconversions studied by 1H NMR and Mössbauer spectroscopies

1996 ◽  
Vol 247 (1) ◽  
pp. 113-118 ◽  
Author(s):  
J.Elaine Barclay ◽  
David J. Evans
Keyword(s):  
Amino Acid ◽  
1H Nmr ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Cluster Interconversions

scholarly journals Cryptococcus neoformans Iron-Sulfur Protein Biogenesis Machinery Is a Novel Layer of Protection against Cu Stress

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Sarela Garcia-Santamarina ◽  
Marta A. Uzarska ◽  
Richard A. Festa ◽  
Roland Lill ◽  
Dennis J. Thiele
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Defense Mechanism ◽  
Opportunistic Pathogen ◽  
Microbial Pathogens ◽  
S Protein ◽  
Cu Toxicity ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
Underlying Mechanisms

ABSTRACT Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans , host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen. IMPORTANCE C. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson’s disease.

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