Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

Européanisation au XXe siècle : un regard historique

International audienceThe growing interest in the concept of Europeanisation reflects the transformation of European societies since the end of the Cold War. Since then it has become less and less legitimate to consider the contemporary history of European states from a purely national angle. Indeed, the concept of Europeanisation allows national frontiers to be overcome and to write a shared European history from a broader perspective than just the European integration project. The contributions in this volume show that the phenomenon of Europeanisation is not limited to the post1945 period, nor is it limited to Western Europe.L'intérêt grandissant pour le concept d'européanisation ces dernières années fait écho à la transformation des sociétés européennes depuis la fin de la guerre froide. Dès lors, il apparaît de moins en moins légitime d'enfermer l'histoire contemporaine des pays européens dans un cadre exclusivement national. En effet, le concept d'européanisation permet à la fois de dépasser le cadre national et d'écrire une histoire de l'Europe à partir d'un angle plus large que celui de l'intégration européenne. Les contributions présentées dans ce volume démontrent à l'évidence que le phénomène de l'européanisation ne se limite pas à l'après-1945, ni à la seule partie occidentale de l'Europe

One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

International audienceCandida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains

Long-term co-incubation of <i>C. glabrata</i> with RAW 264.7 macrophages yields a <i>C. glabrata</i> strain with pseudohyphae-like morphology.

<p>A. Daily passage of <i>C. glabrata</i> with RAW 264.7 macrophages led to the formation of a pseudohyphae-like growth phenotype. The left panel shows the <i>C. glabrata</i> Evo strain interacting with RAW 264.7 cells. In contrast to the parental strain (WT; middle picture) the evolved strain (Evo) formed clumps (right picture) in liquid media. B. The parental wild type (WT) formed smooth colonies on YPD agar; colonies of the evolved strain (Evo) grew with a strongly wrinkled morphology. Both strains appeared purple colored on CHROMagar plates (right picture), characteristic for <i>C. glabrata</i>. C. Transmission electron micrographs indicate a cell separation defect of the evolved (Evo) strain in comparison to the WT, and show enlarged septa between mother and daughter cells, as well as an increased thickness of the outer cell wall layer.</p

Microevolutionary adaptation results in altered host-pathogen interactions.

<p>A. The uptake by RAW 246.7 macrophages after 15 min to 6 hours of co-incubation was analyzed by differential staining (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004478#s4" target="_blank">Material and Methods</a>). Both strains were internalized to a similar extent a all time points. (n≥3). B. Following 24 h co-incubation, the evolved strain (Evo) damaged RAW 246.7 macrophages, but not epithelial TR 146 cells, to a higher extent than the parental strain (WT) as measured by lactate dehydrogenase (LDH) release. (n≥3).</p

Introduction of a single nucleotide exchange into <i>CHS2</i> results in increased macrophage damage.

<p>Following 24 h co-incubation with macrophages, the CHS2^Evo strain, containing the Evo allele of the <i>CHS2</i> gene, elicited the same increased LDH release from macrophages as the Evo strain. Reintroduction of the wild type <i>CHS2</i> gene (CHS^WT) into the WT strain did not lead to a significant change in its damage potential. (n≥3).</p

No large-scale genomic changes, but single SNP differences can be detected between WT and Evo strains.

<p>Following alignment of Solexa/Illumina reads for the genomes of strains ATCC2001 and Evo on the <i>C. glabrata</i> reference genome, an average coverage score was calculated for each 1 kb region and normalized to the coverage obtained across the whole genome. These coverage ratio are shown in log2 scale. <i>C. glabrata</i> chromosomes A to M are shown in alternating black and grey colors. The location of SNPs identified in both strains relative to the reference genome is shown with green diamonds. The location of SNPs that distinguish the two strains is shown with red diamonds, the SNP on chromosome I responsible for the phenotype of strain Evo being shown in larger size. Note that several 1 kb regions harbored more than one SNP and are nevertheless represented using a single diamond.</p

A single nucleotide exchange is sufficient to produce the evolved phenotype.

<p>A. Sanger sequencing confirmed the sequence alteration in <i>CHS2</i> of the Evo strain (identified by whole genome sequencing), which led to an Asn→Lys (WT→Evo) amino acid exchange in the protein. Following a counter-selection experiment, the gene reverted to its original sequence (Rev), concomitant with the reversal to the original yeast growth form. B. This single nucleotide exchange observed in of the Evo strain was introduced into the WT strain by PCR amplification of <i>CHS2</i> from the Evo strain, and cotransformation of this fragment with an PCR-amplified <i>HIS3</i> marker including an overlapping (U1) region. C. The resulting strain was called CHS^Evo, and correct integration was tested by sequencing. Similarly, CHS^WT was created by amplifying the WT <i>CHS2</i> gene and following the same cloning strategy. D. Morphologies of the WT, Evo, CHS^WT and CHS^Evo strain. The introduction of the Evo <i>CHS2</i> gene into CHS^Evo resulted in a growth form indistinguishable from the original Evo strain. The reintroduction of the WT gene did not change morphology (CHS^WT).</p

Increased fitness of the evolved strain in macrophages in direct competition.

<p>Macrophages were infected with WT and Evo cells at a ratio of 100∶1 at day 0 and the relative proportion between the two strains monitored daily. The ratio reversed after a few days of coincubation, demonstrating an advantage for the Evo strain during interaction with macrophages. Mean values and standard deviations of three independent experiments are shown.</p