ABSTRACT: This project tested if siderophores are publig goods in Pseudomonas, and involved resequencing a well used siderophore mutant made by non-specific mutagenesis techniques
Project description:Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membranes of P. aeruginosa by the two-outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-culture under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles heel of P. aeruginosa in communities with bacteria producing ENT.
Project description:Iron is an essential nutrient for the opportunistic pathogen Pseudomonas aeruginosa, as for almost all living organisms. To access this element, the pathogen is able to express at least 15 different iron-uptake pathways, the vast majority involving small iron chelators called siderophores. Indeed, P. aeruginosa produces two siderophores, pyoverdine and pyochelin, but can also use many produced by other microorganisms. This implies that the bacterium expresses appropriate TonB-dependent transporters (TBDTs) at the outer membrane to import the ferric form of each of the siderophores used. These transporters are highly selective for a given ferri-siderophore complex or for siderophores with similar chemical structures. Here, we show that P. aeruginosa can also use rhizoferrin, staphyloferrin A, aerobactin, and schizokinen as siderophores to access iron. Growth assays in iron-restricted conditions and 55Fe uptake assays showed that the two alpha-carboxylate type siderophores rhizoferrin-Fe and staphyloferrin A-Fe are transported into P. aeruginosa cells by the TBDT ActA (PA3268). Among the mixed alpha-carboxylate/hydroxamate type siderophores, we found aerobactin-Fe to be transported by ChtA (as previously described) and schizokinen-Fe by ChtA and another unidentified TBDT.
Project description:Pseudomonas aeruginosa is a common nosocomial pathogen which produces siderophores to solubilize and transport chelated Fe3+ to aid its survival in both the environment and the host. However, there is a lack of comprehensive understanding regarding the molecular mechanisms underlying siderophore synthesis, uptake, and regulation within various ecological niches. In this study, we demonstrated that the BfmRS two-component system, part of the core genome of P. aeruginosa, plays a crucial role in siderophore metabolism. We have identified BfmS as an osmosensing histidine kinase that responds to external osmolytes, then modulates the activation of the response regulator BfmR. Under high osmolality, BfmR could directly bind to the promoters of pvd, fpv, and femARI gene clusters, thereby enhancing their expression and promoting siderophore metabolism. The proteomic and phenotypic analyses confirmed that deletion of bfmRS results in reduced expression levels of siderophore-related proteins as well as siderophore production. Importantly, loss of bfmR or bfmS significantly impaired bacterial survival in both iron deficiency medium and mouse lung infection models. Furthermore, phylogenetic analysis revealed that BfmRS is highly conserved and widely distributed across Pseudomonas species, evidences also proved that the BfmR of P. putida KT2440 and P. sp. MRSN12121 activated siderophore genes in response to high osmolality. Overall, this study sheds light on the previously unexplored signal transduction pathway, BfmRS, which governs the siderophore regulation in Pseudomonas species through perceiving an osmotic upshift. Considering that siderophores serve as unique social mediators, our findings contribute to a better understanding of how siderophores facilitate bacterial interactions with their eukaryotic hosts and contribute to the establishment of stable communities.
Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in regulating miRNA expression profiling in neutrophils derived from mouse bone marrow, we performed microarray analysis of miRNAs in wild type (N=2) and lcn2 knockout (N=2) neutrophils.
Project description:Iron is an essential nutrient for bacterial growth but poorly bioavailable. To scavenge ferric iron present in their environment, bacteria synthesize and secrete siderophores, small compounds with a high affinity for iron. Pyochelin (PCH) is one of the two siderophores produced by the opportunistic pathogen Pseudomonas aeruginosa. Once having captured a ferric iron, PCH-Fe is imported back into bacteria first by the outer membrane transporter FptA and afterwards by the inner membrane permease FptX. Here using molecular biology, 55Fe uptake assays and LC-MS/MS quantification of PCH in the different bacterial cell fractions, we show that (i) PCH (probably under its PCH-Fe form) is able to rich bacterial periplasm and cytoplasm when both FptA and FptX are expressed, and (ii) that PchHI (a heterodimeric ABC transporter) plays a role in the translocation of siderophore-free iron siderophore-free iron across the inner membrane into the cytoplasm. Consequently, probably the first fraction of PCH-Fe internalized by FptA may be transported further by FptX in the bacterial cytoplasm to activate the transcriptional regulator PchR, regulating the transcription of all genes of the PCH pathway. The further fractions of PCH-Fe transported by FptA may dissociate in the bacterial periplasm by an unknown mechanism, with the siderophore-free iron being transported into the cytoplasm by PchHI.
Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p
Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p
Project description:Lipocalin 24p3 (24p3) is a neutrophil secondary granule protein. 24p3 is also a siderocalin, which binds several bacterial siderophores. It was therefore proposed that synthesis and secretion of 24p3 by stimulated macrophages or release of 24p3 upon neutrophil degranulation sequesters iron-laden siderophores to attenuate bacterial growth. Accordingly, 24p3-deficient mice are susceptible to bacterial pathogens whose siderophores would normally be chelated by 24p3. Specific granule deficiency (SGD) is a rare congenital disorder characterized by complete absence of proteins in secondary granules. Neutrophils from SGD patients, who are prone to bacterial infections, lack normal functions but the potential role of 24p3 in neutrophil dysfunction in SGD is not known. Here we show that neutrophils from 24p3-deficient mice are defective in many neutrophil functions. Specifically, neutrophils in 24p3-deficient mice do not extravasate to sites of infection and are defective for chemotaxis. A transcriptome analysis revealed that genes that control cytoskeletal reorganization are selectively suppressed in 24p3-deficient neutrophils. Additionally, small regulatory RNAs (miRNAs) that control upstream regulators of cytoskeletal proteins are also increased in 24p3-deficient neutrophils. Further, 24p3-deficient neutrophils failed to phagocytose bacteria, which may account for the enhanced sensitivity of 24p3-deficient mice to both intracellular (Listeria monocytogenes) and extracellular (Candida albicans, Staphylococcus aureus) pathogens. Interestingly, Listeria does not secrete siderophores and additionally, the siderophore secreted by Candida is not sequestered by 24p3. Therefore, the heightened sensitivity of 24p3-deficient mice to these pathogens is not due to sequestration of siderophores limiting iron availability, but is a consequence of impaired neutrophil function. Key words: Lipocalin, 24p3, neutrophils, cell motility, chemotaxis, MIRNA-362-3p To address the role of lipocalin 2 in unstimulated and fMLP-stimulated neutrophils derived from mouse bone marrow, we performed micorarray analysis of gene expression in unstimulated wild type (N=3), unstimulated lcn2 knockout (N=3), fMLP-stimulated wild type (N=2) and fMLP-stimulated lcn2 knockout (N=2) neutrophils. Upon stimulation, neutrophils were treated by fMLP at 10 micromolar for 20 minutes at 37 centigrade.
Project description:Siderophores are specialized molecules with different chemical structures, produced by bacteria and fungi to scavenge iron from the environment, a crucial nutrient for their growth and metabolism. These iron-chelating compounds enable bacteria to overcome iron limitation, a key factor in microbial survival and pathogenesis. Catecholate-type siderophores are primarily produced by bacteria, while hydroxamates are predominantly produced by fungi. The capacity of nine hydroxamate-type siderophores produced by fungi to serve as siderophores for iron acquisition by Pseudomonas aeruginosa, a human pathogen, has been investigated. Growth assays under iron limitation and 55Fe incorporation tests clearly highlighted that all nine siderophores promoted bacterial growth and facilitated iron transport. Additionally, the study aimed to identify the TonB-dependent transporters (TBDTs) responsible for iron import mediated by the tested siderophores. Mutant strains lacking genes encoding TBDTs were employed, revealing that iron is imported into P. aeruginosa cells solely by FpvB for the siderophores coprogen, triacetylfusarinine C, fusigen, ferrirhodin, and ferrirubin siderophores. Iron complexed by desferioxamine G is imported by two TBDTs, FpvB and FoxA. Ferricrocin-Fe and ferrichrycin-Fe complexes are imported by FpvB and FiuA. Lastly, rhodotorulic acid-Fe complexes are imported by FpvB, FiuA, and another unidentified TBDT. In conclusion, the data illustrate the effectiveness of hydroxamate-type siderophores in transporting iron into P. aeruginosa cells and provide insights into the intricate molecular mechanisms involved in iron acquisition, which have implications for understanding bacterial pathogenesis and developing potential therapeutic strategies.
Project description:Competition for limited iron resources is a key driver of microbial community structure in many regions of the surface ocean. The bacterial siderophores ferrioxamine and amphibactin have been identified in marine surface waters, suggesting that they may represent an important bacterial strategy for obtaining iron from a scarcely populated pool. We screened several strains of marine Vibrio for the presence of putative amphibactin biosynthesis gene homologues and amphibactin production. Whole cell proteomics, siderophore isolation, and isotopically labeled iron uptake experiments were performed. Here, we show that an amphibactin-producing marine bacterium, Vibrio cyclitrophicus str. 1F-53, harbors an independently regulated uptake pathway for ferrioxamines. Proteomic analyses identified upregulation of the amphibactin NRPS system and a putative amphibactin siderophore transporter in response to low iron concentrations. In addition, multiple other transporters were upregulated, however when desferrioxamine was present, amphibactin production decreased and the ferrioxamine receptor increased in abundance. Such cheating phenotypes, which appear widespread among marine amphibactin producers, highlight the strategies that contribute to the fitness of marine bacteria in the face of iron stress. These results demonstrate siderophore producer and cheater phenotypes and highlight the cellular restructuring which is involved due to competition for iron, that shapes the community structure of marine ecosystems.