Investigation of Pseudomonas aeruginosa responses to zinc starvation reveals the existence of a pyochelin Zurregulated transport system

Previous studies have suggested that P. aeruginosa possesses redundant zinc uptake systems. In addition to the already characterized metal transport systems, genome wide expression studies have revealed other possible contributors to zinc uptake. The main goal of this study was to investigate the role of two uncharacterized Zur-regulated operons encoding putative metal import systems: PA4063-4065 and PA2911-2914. A structural characterization of the protein encoded by the PA4063 gene has shown that it has characteristics typical of zinc-binding proteins, in line with the hypothesis that it may play a role in the management of zinc in the periplasmic space. Deletion of genes of these two operons encoding components of transport systems located in the inner membrane (PA4065 or PA2914) reduces the ability of a strain lacking a functional ZnuABC transporter and unable to synthesize the metallophore pseudopaline to grow in a medium poor of zinc, confirming the assumption that these operons are involved in the response to zinc shortage. A series of experiments aimed at measuring the effect of these mutations on the intracellular zinc status revealed that the two systems are functionally interconnected but contradicted the initial hypothesis that both transport systems are directly involved in zinc uptake. Whereas the proteins encoded by PA2911-2914 have the typical features of a metal-chelated metal-import system involving an outer membrane TonB-dependent protein and an inner membrane ABC transporter, a bioinformatic analysis of the proteins PA4064 and PA4065 unexpectedly revealed that these can form a MacB transporter. Since this family of transporters is usually involved in the export of large molecules, we hypothesized that it is involved in the export of the metallophore that enters through PA2911-2914 and which, if kept inside the cell, may interfere with the intracellular zinc status. Spectrophotometric analyses of the supernatants, RT-PCR experiments and feeding assays have revealed that the double mutant znuAzrmB produces low levels of pyochelin, and that the supernatants from the wild type strain, but not those from a pchE mutant, are able to induce zinc-starvation in a strain lacking PA4065. All these observations converge in indicating that the two systems under investigation are involved in the import (PA2911-PA2914) and in the export of pyochelin (PA4063-PA4065). Our results support this hypothesis, but intracellular metal measurements unexpectedly showed that these two systems are involved in the import and export of the pyochelin-cobalt complex, suggesting that they constitute a backup system aimed at favoring the incorporation of cobalt in proteins under condition of zinc unavailability. At the same time, the observation that purified exogenous pyochelin, when complexed with zinc, is able to reduce the intracellular zinc requirement more efficiently than free zinc suggests that pyochelin is also able to transport zinc through these systems. This metals absorption pathway appears as a double-edged sword strategy to cope with zinc deficiency since, in the absence of zinc, an excess of extracellular pyochelin reduces the intracellular availability of zinc. Taken together, these studies reveal an unexpected role for pyochelin in cobalt and zinc transport and change the paradigm that pyochelin is just an iron transporting molecule. Although many aspects of pyochelin role in zinc/cobalt transport have yet to be elucidated, this study suggests that this molecule has a key role in connecting iron and zinc homeostasis. We think that the identification of this additional role of pyochelin should lead to a reinterpretation of many previous studies and may foster a better understanding of the interaction between P. aeruginosa and its host. A further aim of this study was to investigate the role of Zur-regulated genes in the infectious capacity of Pseudomonas. For this purpose, we have developed an infection protocol in Danio rerio (zebra fish) larvae. Competition experiments revealed that mutants that have a reduced ability to import zinc, such as the single mutant znuA or the double mutant znuAzrmA, are outcompeted by the wild type strain at 24 and 48 hours after infection. However, these mutants were not disadvantaged at short times after infection, and they even showed an advantage over the wt. This suggests that in the early stages of the infection the host is able to cope with the infection through mechanisms of zinc intoxication operated by phagocytes, while during the advanced stages of the infection, the P. aeruginosa cells that escape phagocytic engulfment colonize niches of the host characterized by a scarce availability of zinc. This hypothesis is confirmed by analyses of the expression of host zinc transporters and bacterial genes coding for zinc extrusion pumps. These results confirm the importance of zinc for P. aeruginosa ability to infect its host and indicate that zebrafish is an interesting model for assessing the contribution of metal ions to the hostpathogen interaction.