Magnesium homeostasis in Staphylococcus aureus

Staphylococcus aureus, an opportunistic pathogen present in the nasal cavities of approximatively 25% of the healthy population, causes thousands of deaths per year in the United States alone. This bacterium supports hundreds of millimolar of magnesium (up to 770mM), a concentration much higher than many other bacteria. To maintain intracellular magnesium concentrations within a physiological range bacteria make use of active importers and probably exporters. Here, we study the magnesium homeostasis in Staphylococcus aureus and more precisely the roles of the putative magnesium transporters: mpfA, mpfB, corA1, corA2 and mgtE. We focus mainly on the mutations that appeared to compensate the magnesium sensitivity due to the loss of MpfA, a putative magnesium exporter. We also investigate the question of which importer(s) is/are fundamental for the growth and show that magnesium import in S. aureus relies on the dual activity of both CorA2 and MgtE

Less is more: Loss of outer membrane constituents in hyperpermeable <i>Pseudomonas aeruginosa</i> mutants improves resistance to certain antibiotics

Gram-negative bacteria have an envelope composed of three different layers: an inner membrane (IM), a peptidoglycan layer, and an outer membrane (OM). The inner leaflet of the OM is composed of the same types of phospholipids that compose both leaflets of the IM, while the outer leaflet is composed of lipopolysaccharides (LPS). The barrier function of the outer membrane is important to restrict the entry of detergents, hydrophobic antimicrobial agents and charged antibiotics that are capable of crossing a phospholipid bilayer. In E. coli, the biosynthesis of the outer membrane requires the Lpt machinery, which is composed of seven essential proteins (LptABCDEFG). LptD and LptE form a heterodimeric OM translocon with a plug-and barrel architecture, which recieves LPS from LptA and transports it to the OM surface. LptD is a b-barrel OM protein, and its insertion in the OM requires the b-barrel assembly machinery (BAM). The BAM complex is composed of the essential b-barrel OM protein BamA, and five OM lipoproteins, BamB to BamE, of which only BamD is essential. Sampson et al. discovered that in Escherichia coli K12, a specific small in frame deletion in lptD, called lptD4213, confers increased sensitivity to various antibiotics, detergents, and dyes. Although cell envelope biosynthesis is well studied in the Gram-negative model bacterium Escherichia coli K12, far less is known about this process in Pseudomonas aeruginosa, a pathogen with a particularly impermeable outer membrane. Therefore, we used three different approaches to dissect the OM biosynthesis in P. aeruginosa. In the first project, we engineered a hypomorphic lptD4213 allele in P. aeruginosa PA14. The lptD4213 mutation not only made PA14 more sensitive to large soluble antibiotics, such as vancomycin, but also unexpectedly made it more sensitive to high concentrations of various inorganic salts. To dissect P. aeruginosa PA14 envelope homeostasis, we selected salt and vancomycin suppressors of the lptD4213 mutant. Analysis of a NaCl suppressor mutant of P. aeruginosa PA14 lptD4213 highlighted the role of the Bam complex, while the vancomycin suppressor mutants pointed out the interaction between the LPS O-antigen and the peptidoglycan biosynthesis pathway. In the second project, we decided to start from the WT PA14 strain, and we selected genes whose disruption impaired the barrier function of the OM. In this respect, we used two transposon mutant libraries to identify strains with outer membrane permeability barrier defects by searching for mutants that are sensitive to vancomycin. We identified bamB, which codes for one of the non-essential subunits of the BAM complex and showed that the loss of BamB was sufficient to selectively increase the permeability of the OM to several large antibiotics. Further analyses revealed that this increase in permeability was dependent on the presence of porins in the OM of P. aeruginosa PA14. Finally, in the last project, we performed competition between the transposon mutagenized library of PA14 and Staphylococcus aureus. We identified a few genes in which transposon insertions consistently decreased after exposure to S. aureus. However, while Tn-mutants in several genes were underrepresented in the Tn-seq analysis of P. aeruginosa cultivated with S. aureus, we could not reproduce this effect with clean deletion of two of the identified genes.</p

Genetic screens reveal novel major and minor players in magnesium homeostasis of Staphylococcus aureus

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA

Identification and Characterization of Mediators of Fluconazole Tolerance in Candida albicans

International audienceCandida albicans is an important human pathogen and a major concern in intensive care units around the world. C. albicans infections are associated with a high mortality despite the use of antifungal treatments. One of the causes of therapeutic failures is the acquisition of antifungal resistance by mutations in the C. albicans genome. Fluconazole (FLC) is one of the most widely used antifungal and mechanisms of FLC resistance occurring by mutations have been extensively investigated. However, some clinical isolates are known to be able to survive at high FLC concentrations without acquiring resistance mutations, a phenotype known as tolerance. Mechanisms behind FLC tolerance are not well studied, mainly due to the lack of a proper way to identify and quantify tolerance in clinical isolates. We proposed here culture conditions to investigate FLC tolerance as well as an easy and efficient method to identity and quantify tolerance to FLC. The screening of C. albicans strain collections revealed that FLC tolerance is pH- and strain-dependent, suggesting the involvement of multiple mechanisms. Here, we addressed the identification of FLC tolerance mediators in C. albicans by an overexpression strategy focusing on 572 C. albicans genes. This strategy led to the identification of two transcription factors, CRZ1 and GZF3. CRZ1 is a C2H2-type transcription factor that is part of the calcineurin-dependent pathway in C. albicans, while GZF3 is a GATA-type transcription factor of unknown function in C. albicans. Overexpression of each gene resulted in an increase of FLC tolerance, however, only the deletion of CRZ1 in clinical FLC-tolerant strains consistently decreased their FLC tolerance. Transcription profiling of clinical isolates with variable levels of FLC tolerance confirmed a calcineurin-dependent signature in these isolates when exposed to FLC