Membrane remodeling and ion homeostasis in non-actively growing Salmonella

Une stratégie utilisée par de nombreuses bactéries pour survivre en conditions de stress ou en présence d'antibiotiques consiste à entrer en quiescence. Cet arrêt temporaire de croissance leur permet de résister à de nombreux stress et à persister dans des environnements défavorables. Cet état de multi-résistance a un impact environnemental et médical et il est crucial d'en comprendre les déterminants moléculaires et physiologiques. En utilisant des approches globales et analytiques chez le pathogène humain Salmonella, nous étudions un nouvel aspect de ce remodelage, le contrôle de l'homéostasie de certains ions métalliques.One strategy used by many bacteria to survive under stressful conditions or in the presence of antibiotics is to enter in a quiescent state. This temporary arrest of growth allows them to resist many stresses and to persist in adverse environments. This state of multi-resistance has an environmental and medical impact and it is crucial to understand its molecular and physiological determinants. Using global and analytical approaches in the human pathogen Salmonella, we study a new aspect of this remodeling, the control of the homeostasis of certain metal ions

The stress sigma factor σS/RpoS counteracts Fur repression of genes involved in iron and manganese metabolism and modulates the ionome of Salmonella enterica serovar Typhimurium

International audienceIn many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σ S /RpoS, remodels global gene expression to reshape the physiology of quiescent cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σ S is also required for biofilm formation and virulence. We have previously identified sRNAs genes positively controlled by σ S in Salmonella , including the two paralogous sRNA genes, ryhB1 and ryhB2 / isrE . Expression of ryhB1 and ryhB2 is repressed by the ferric uptake regulator Fur when iron is available. In this study, we show that σ S alleviates Fur-mediated repression of the ryhB genes and of additional Fur target genes. Moreover, σ S induces transcription of the manganese transporter genes mntH and sitABCD and prevents their repression, not only by Fur, but also by the manganese-responsive regulator MntR. These findings prompted us to evaluate the impact of a Δ rpoS mutation on the Salmonella ionome. Inductively coupled plasma mass spectrometry analyses revealed a significant effect of the Δ rpoS mutation on the cellular concentration of manganese, magnesium, cobalt and potassium. In addition, transcriptional fusions in several genes involved in the transport of these ions were regulated by σ S . This study suggests that σ S controls fluxes of ions that might be important for the fitness of quiescent cells. Consistent with this hypothesis, the Δ rpoS mutation extended the lag phase of Salmonella grown in rich medium supplemented with the metal ion chelator EDTA, and this effect was abolished when magnesium, but not manganese or iron, was added back. These findings unravel the importance of σ S and magnesium in the regrowth potential of quiescent cells

Corrélation génotype-phénotype dans la neurofibromatose de type 1 Évaluation des corrélations phénotype-génotype dans la neurofibromatose de type 1

La neurofibromatose de type 1 (NF1 ; numéro OMIM 162200), ou maladie de Von Recklinghausen, est l'une des maladies génétiques rares les plus fréquentes. Elle a une répartition mondiale homogène et une incidence estimée à 1 cas pour 2500 naissances. Il s'agit d'une maladie à transmission autosomique dominante dont la pénétrance est quasi-complète à l'âge de huit ans. Le gène NF1 responsable de la maladie est localisé sur le bras long du chromosome 17, en position 17q11.2. Il s'agit d'un gène suppresseur de tumeur de grande taille qui code la neurofibromine 1 (OMIM 613113), une protéine cytoplasmique. La neurofibromatose est caractérisée par une très grande variabilité de son expression phénotypique, notamment au sein de la même famille. Certains patients sont susceptibles de développer une forme plus sévère de la maladie tout en ayant la même mutation qu'un patient dont les symptômes sont moins importants. La grande variabilité phénotypique pourrait être liée à l’importante diversité des mutations causales à l’origine de la très grande hétérogénéité allélique observée dans la neurofibromatose. Les mutations mises en cause sont les mutations faux-sens, non-sens, d'épissage et les délétions. Les grandes délétions du gène NF1 se caractérisent par une délétion du gène entier NF1 mais également d'une douzaine de gènes situés à proximité. La délétion simultanée de tous les gènes peut expliquer le phénotype un peu particulier des patients porteurs d'une grande délétion. Nous nous proposons ici d'étudier l'influence dutype d'altération génétique sur la variabilité de l'expression de la maladie, afin de mettre en lumière certaines corrélations génotype-phénotype et d'en déduire de possibles mécanismes physiopathologiques à l'origine des divers symptômes caractéristiques

Loss of CorA, the primary magnesium transporter of Salmonella, is alleviated by MgtA and PhoP-dependent compensatory mechanisms.

In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms

S11 Raw images -

In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms.</div

Protein-protein interaction network of the proteins significantly less abundant in the Δ<i>corA</i>Δ<i>phoP</i> mutant, but not found less abundant in the Δ<i>corA</i> and Δ<i>phoP</i> strains, when comparing to the wild-type strain.

Top enriched functional categories of proteins are colored: flagellum-dependent motility in blue, chemotaxis in red, T3SS-dependent secretion in pink and cobalamin biosynthetic pathway in green. See also S2 Dataset for details.</p

Schematic illustration of the synthetic effects of the Δ<i>corA</i> and Δ<i>phoP</i> mutations on <i>Salmonella</i> physiology.

When magnesium is sufficient, the housekeeping CorA protein is the main Mg2+ transporter. PhoP is a global transcriptional regulator that promotes expression of the other two Mg2+-transporters, MgtA and MgtB, under magnesium starvation. Vertical arrows indicate positive (green) or negative (red) effects of the ΔcorA and ΔphoP mutations on the indicated phenotypes of Salmonella. The line thickness is indicative of the magnitude of the effect. When the corA gene is deleted, MgtA production increased in a PhoP-independent fashion, which alleviates the negative effect of the ΔcorA mutation on magnesium homeostasis (a). The exact mechanisms by which the ΔcorA mutation promotes MgtA production is unknown (see text). When the ΔcorA mutation is combined with the ΔphoP mutation, synthetic phenotypes are observed, including reduction in growth in monoculture, competitive fitness, and motility (b). It is unknown so far whether the synthetic growth phenotype of the ΔcorAΔphoP mutant and the phenotype of the ΔcorA mutant in competitive fitness to exit from stationary phase rely on the same mechanism. A synthetic growth phenotype is also observed when the ΔcorA mutation is combined with the ΔmgtA mutation, unless the phoQ*R16S allele is present (see text for details). This phenomenon may be due to a reduced level of PhoP activated molecules in the ΔcorAΔmgtA mutant, alleviated somehow by the phoQ*R16S mutation.</p

Competition experiments between the wild-type strain and the Δ<i>corA</i> mutant of <i>Salmonella</i>.

Competition assays were performed between the wild-type strain ATCC14028 (WT) and the ΔcorA mutant (panel A) or the control strain ATCC14028 2922K (panel B) which carries the same kanamycin resistance cartridge as the ΔcorA mutant. The control competition assay showing similar fitness of ATCC14028 and 2922K is consistent with our previous data [5, 29]. Equal cell numbers of stationary phase LB cultures of the wild-type strain and the mutant strain were mixed in fresh LB medium to give a total of about 3000 cells ml-1 (Day 0) and the mixtures were incubated at 37°C with shaking. Aliquots of bacteria were removed at timed intervals and numbers of viable cells of each strain were determined. For each time point, cells number of each strain is reported as a percentage of the total number of viable cells in the culture. The error bars represent standard errors for three independent measurements. At day 0, the ΔcorA mutant represented about 50% of a total population of 3000 CFU/ml. At day 1, the ΔcorA mutant represented about 34% of a total population of 5 109 CFU/ml, which corresponds to 1.7 109 ΔcorA cells and 3.3 109 wild-type cells. * Statistically significant competitive disadvantage of the mutant compared to the wild-type (p-value corA mutant were monitored immediately after inoculation of the bacterial mixture into fresh LB medium. The black curve represents the growth of the total bacterial population estimated by CFU counts (left y axis) at regular intervals after inoculation of the bacterial mixture into fresh LB. Bars represent the percentage of each strain within the population (right y axis) at the different time points. The error bars represent standard errors for three independent measurements. * p-value <0.01.</p

Immunodetection of MgtA in <i>Salmonella</i> wild-type and mutant strains.

The MgtA-Flag protein was immunodetected in Salmonella wild-type and mutant strains grown for 18 h at 37°C in LB supplemented or not with MgCl2 10 mM or EDTA 2 mM. MgtA is a 95-kDa protein. In most immunodetection experiments, two MgtA-Flag products were found (a full-length product at about 98-kDa and a smaller product of about 38-kDa), suggesting that the MgtA-flag protein was partially degraded. Membranes used to reveal the Flag-tagged proteins with the anti-Flag antibody were then incubated in the presence of antibodies directed against GroEL used as a loading control of total protein amounts. A representative experiment is shown. Similar results were found in repeat experiments (S7 Fig).</p

Details for protein-protein interaction network on Fig 6.

Details for protein-protein interaction network on Fig 6.</p