Involvement of the Pseudomonas aeruginosa MexAB-OprM efflux pump in the secretion of the metallophore pseudopaline.

To overcome the metal restriction imposed by the host's nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB-OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa.

The ancient roots of nicotianamine: diversity, role, regulation and evolution of nicotianamine-like metallophores.

Nicotianamine (NA) is a metabolite synthesized by all plants, in which it is involved in the homeostasis of different micronutrients such as iron, nickel or zinc. In some plants it also serves as a precursor of phytosiderophores, which are used for extracellular iron scavenging. Previous studies have also established the presence of NA in filamentous fungi and some mosses, whereas an analogue of NA was inferred in an archaeon. More recently, opine-type metallophores with homology to NA were uncovered in bacteria, especially in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersinia pestis, synthesizing respectively staphylopine, pseudopaline and yersinopine. Here, we review the current state of knowledge regarding the discovery, biosynthesis, function and regulation of these metallophores. We also discuss the genomic environment of the cntL gene, which is homologous to the plant NA synthase (NAS) gene, and plays a central role in the synthesis of NA-like metallophores. This reveals a large diversity of biosynthetic, export and import pathways. Using sequence similarity networks, we uncovered that these metallophores are widespread in numerous bacteria thriving in very different environments, such as those living at the host-pathogen interface, but also in the soil. We additionally established a phylogeny of the NAS/cntL gene and, as a result, we propose that this gene is an ancient gene and NA, or its derivatives, is an ancient metallophore that played a prominent role in metal acquisition or metal resistance. Indeed, our phylogenetic analysis suggests an evolutionary model where the possibility to synthesize this metallophore was present early in the appearance of life, although it was later lost by most living microorganisms, unless facing metal starvation such as at the host-pathogen interface or in some soils. According to our model, NA then re-emerged as a central metabolite for metal homeostasis in fungi, mosses and all known higher plants.

The nature of the arena surface affects the outcome of host-finding behavior bioassays in Varroa destructor (Anderson & Trueman).

Varroa destructor, an acarian parasite of the Western honey bee Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. The parasite lifecycle occurs in close synchrony with its host development. The females have to discriminate between different developmental stages of the host and trigger an appropriate behavioral response. Many studies have focused on these behavioral aspects, whether it is the choice of a precise host stage or the reproduction of female mites. Behavioral tests often require laboratory settings that are very different from the mite's environment. Our first experiment was designed to study the impact of the surface of test arena on the mite behavior. We found that plastic from Petri dishes commonly used as test arenas disturbs the female mites and can cause death. We searched for a substrate that does not harm mites and found that gelatin-coated plastic Petri dishes responded to these expectations. We then investigated the host choice behavior of phoretic mites confronted with larval stages of the bee on gelatin-coated arenas to watch if the well-documented orientation towards 5th instar larva was observable in our conditions. Pupal stages were included in the host choice experiments, initially to act as neutral stimuli. As white-eyed pupae were revealed attractive to the mite, several pupal stages were then included in a series of host choice bioassays. These additional experiments tend to show that the positive response to the white-eyed pupa stage depends on cues only delivered by living pupae. Further investigation on the nature and impact of these cues are needed as they could shed light on key signals involved in the parasite lifecycle.

Simple rules govern the diversity of bacterial nicotianamine-like metallophores.

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis, respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity toward α-ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore is governed by the absence/presence of a cntK gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.