Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.

The longstanding model is that most bloodstream infections (BSIs) are caused by a single organism. We perform whole genome sequencing of five-to-ten strains from blood culture (BC) bottles in each of ten patients with Candida glabrata BSI. We demonstrate that BCs contain mixed populations of clonal but genetically diverse strains. Genetically distinct strains from two patients exhibit phenotypes that are potentially important during BSIs, including differences in susceptibility to antifungal agents and phagocytosis. In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identify mixed fluconazole-susceptible and -resistant populations. Diversity in drug susceptibility is likely clinically relevant, as fluconazole-resistant strains were subsequently recovered by the clinical laboratory during persistent or relapsing infections. In one patient, unrecognized respiration-deficient small colony variants are fluconazole-resistant and significantly attenuated for virulence during murine candidiasis. Our data suggest a population-based model of C. glabrata genotypic and phenotypic diversity during BSIs.

Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.

The longstanding paradigm is that most bloodstream infections (BSIs) are caused by a single organism. We performed whole genome sequencing of five-to-ten strains from blood culture (BC) bottles in each of ten patients with Candida glabrata BSI. We demonstrated that BCs contained mixed populations of clonal but genetically diverse strains. Genetically distinct strains from two patients exhibited phenotypes that were potentially important during BSIs, including differences in susceptibility to antifungal agents and phagocytosis. In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identified mixed fluconazole-susceptible and â€"resistant populations. Diversity in drug susceptibility was likely clinically relevant, as fluconazole-resistant strains were subsequently recovered by the clinical laboratory during persistent or relapsing infections. In one patient, unrecognized respiration-deficient small colony variants were fluconazole-resistant and significantly attenuated for virulence during murine candidiasis. Our data suggest a new population-based paradigm of C. glabrata genotypic and phenotypic diversity during BSIs.

Candida albicans Transcriptional Profiling Within Biliary Fluid From a Patient With Cholangitis, Before and After Antifungal Treatment and Surgical Drainage.

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates the yeast cell wall integrity pathway. Candida albicans exposure to caspofungin results in the rapid redistribution of PI(4,5)P2 and septins to plasma membrane foci and subsequent fungicidal effects. We studied C. albicans PI(4,5)P2 and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P2 and septins were visualized by live imaging of C. albicans cells coexpressing green fluorescent protein (GFP)-pleckstrin homology (PH) domain and red fluorescent protein-Cdc10p, respectively. PI(4,5)P2 was also visualized in GFP-PH domain-expressing C. albicans mkc1 mutants. Mkc1p phosphorylation was measured as a marker of PKC-Mkc1 pathway activation. Fungicidal activity was assessed using 20-h time-kill assays. Caspofungin immediately induced PI(4,5)P2 and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P2 levels increased in a dose-response manner at caspofungin concentrations of ≤4× MIC and progressively decreased at concentrations of ≥8× MIC. Caspofungin exposure resulted in broad-based mother-daughter bud necks and arrested septum-like structures, in which PI(4,5)P2 and Cdc10 colocalized. PKC-Mkc1 pathway activation was maximal within 10 min, peaked in response to caspofungin at 4× MIC, and declined at higher concentrations. The caspofungin-induced PI(4,5)P2 redistribution remained apparent in mkc1 mutants. Caspofungin exerted dose-dependent killing and paradoxical effects at ≤4× and ≥8× MIC, respectively. Fluconazole, amphotericin B, calcofluor white, and H2O2 did not impact the PI(4,5)P2 or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P2-septin and PKC-Mkc1 responses that correlate with the extent of C. albicans killing, and the responses are not induced by other antifungal agents. PI(4,5)P2-septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.

A competitive infection model of hematogenously disseminated candidiasis in mice redefines the role of Candida albicans IRS4 in pathogenesis.

Candida albicans IRS4 encodes a protein that regulates phosphatidylinositol-(4,5)-bisphosphate, which was shown to contribute to hematogenously disseminated candidiasis (DC) after several days in the standard mouse model. Our objective was to more accurately define the temporal contributions of IRS4 to pathogenesis. During competition assays in vitro, an irs4-null (Δirs4) mutant exhibited wild-type fitness. In DC experiments, mice were infected intravenously with the Δirs4 mutant, strain CAI-12 (1 × 10(5) CFU), or a mixture of the strains (0.5 × 10(5) CFU each). In single-strain infections, quantitative PCR revealed reduced Δirs4 mutant burdens within kidneys at days 1, 4, and 7 but not 6 h. In competitive infections, the Δirs4 mutant was outcompeted by CAI-12 in each mouse at ≥6 h (competitive indices, P ≤ 0.0001). At 4 and 7 days, the Δirs4 mutant burdens during competitive infections were significantly lower than those during single-strain infections (P = 0.01 and P < 0.001, respectively), suggesting increased susceptibility to inflammatory responses. Phagocytic infiltration of kidneys in response to CAI-12 or competitive infections was significantly greater than that in response to Δirs4 mutant infection at days 1 and 4 (P < 0.001), and the Δirs4 mutant was more susceptible to phagocytosis and killing by human polymorphonuclear cells (P = 0.01 and P = 0.006, respectively) and mouse macrophages in vitro (P = 0.04 and P = 0.01, respectively). Therefore, IRS4 contributes to tissue invasion at early stages of DC and mediates resistance to phagocytosis as DC progresses. Microarray analysis revealed remarkably similar gene expression by the Δirs4 mutant and reference strain CAI-12 within blood, suggesting that IRS4 is not significantly involved in the hematogenous stage of disease. A competitive DC model detects attenuated virulence that is not evident with the standard model.

Rapid redistribution of phosphatidylinositol-(4,5)-bisphosphate and septins during the Candida albicans response to caspofungin.

We previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintaining Candida albicans cell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response of C. albicans to caspofungin. First, we studied caspofungin-hypersusceptible C. albicans irs4 and inp51 mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5'-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes, irs4 and inp51 mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants. irs4 and inp51 were linked most closely to the gin4 mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-type C. albicans to caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those of irs4, inp51, and gin4 mutants. Taken together, our data suggest that the C. albicans Irs4-Inp51 5'-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

The Candida albicans phosphatase Inp51p interacts with the EH domain protein Irs4p, regulates phosphatidylinositol-4,5-bisphosphate levels and influences hyphal formation, the cell integrity pathway and virulence.

We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P(2) levels and the cell integrity pathway, and that PI(4,5)P(2) homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.

Candida albicans IRS4 contributes to hyphal formation and virulence after the initial stages of disseminated candidiasis.

Candida albicans is a common cause of mucosal and bloodstream infections. As a screening strategy to identify novel candidal virulence factors, sera recovered from HIV-infected patients with active oropharyngeal candidiasis (OPC) were previously used to probe a C. albicans genomic expression library. IRS4 was identified as a gene that encodes an immunogenic protein. In the present study, the presence of IRS4 transcripts was verified within OPC pseudomembranes recovered from patients. Having confirmed that the gene is expressed during human candidiasis, gene disruption strains were created and this implicated IRS4 in diverse processes, including hyphal formation on solid media and under embedded conditions, cell wall integrity and structure, and adherence to human epithelial cells in vitro. IRS4 disruption, however, did not influence hyphal formation or virulence in a murine model of OPC. Rather, the gene was found to be necessary for normal morphogenesis and full virulence during murine intravenously disseminated candidiasis (DC). IRS4's effects on hyphal formation and virulence during DC were not evident on the first day after intravenous inoculation, even though transcripts were detected within murine kidneys. After 4 days, however, an irs4 null mutant strain was associated with attenuated mortality, diminished tissue burdens, less extensive infections, impaired C. albicans hyphal formation and decreased kidney damage. Taken together, these findings suggest that IRS4 makes distinct temporal-spatial contributions to the pathogenesis of candidiasis, which appear to vary between different tissue sites as well as within a given tissue over time.

MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system.

We have previously reported on the isolation of in vivo inducible genes of Pseudomonas aeruginosa using IVET system. One of such genes isolated from burn mouse infection model encodes a short open reading frame with unknown function. In this study, we demonstrate that this gene product specifically suppresses the expression of type III secretion genes in P. aeruginosa, thus named PtrA (Pseudomonas type III repressor A). A direct interaction between the PtrA and type III transcriptional activator ExsA was demonstrated, suggesting that its repressor function is probably realized through inhibition of the ExsA protein function. Indeed, an elevated expression of the exsA compensates the repressor effect of the PtrA. Interestingly, expression of the ptrA is highly and specifically induced by copper cation. A copper- responsive two-component regulatory system, copR-copS, has also been identified and shown to be essential for the copper resistance in P. aeruginosa as well as the activation of ptrA in response to the copper signal. Elevated expression of the ptrA during the infection of mouse burn wound suggests that P. aeruginosa has evolved tight regulatory systems to shut down energy-expensive type III secretion apparatus in response to specific environmental signals, such as copper stress.

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles.

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.

Biomedical research in developing countries: the case of Morocco in the 1990s.

Moroccan biomedical research occupies the third place among African or Arab countries, and its outputs considerably increased during the last decade. The quality of publications from developing countries should be improved as suggested by the comparison with developed countries. The gap between developed and developing countries is very large considering the number of publications and their quality, the number of edited journals, and the number of patented inventions, thus making developing countries more as consumers then producers. Accordingly, there is a large gap between developing and developed countries when considering the human and financial resources devoted to scientific research.

Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae.

Genome rearrangements, especially amplifications and deletions, have regularly been observed as responses to sustained application of the same strong selective pressure in microbial populations growing in continuous culture. We studied eight strains of budding yeast (Saccharomyces cerevisiae) isolated after 100-500 generations of growth in glucose-limited chemostats. Changes in DNA copy number were assessed at single-gene resolution by using DNA microarray-based comparative genomic hybridization. Six of these evolved strains were aneuploid as the result of gross chromosomal rearrangements. Most of the aneuploid regions were the result of translocations, including three instances of a shared breakpoint on chromosome 14 immediately adjacent to CIT1, which encodes the citrate synthase that performs a key regulated step in the tricarboxylic acid cycle. Three strains had amplifications in a region of chromosome 4 that includes the high-affinity hexose transporters; one of these also had the aforementioned chromosome 14 break. Three strains had extensive overlapping deletions of the right arm of chromosome 15. Further analysis showed that each of these genome rearrangements was bounded by transposon-related sequences at the breakpoints. The observation of repeated, independent, but nevertheless very similar, chromosomal rearrangements in response to persistent selection of growing cells parallels the genome rearrangements that characteristically accompany tumor progression.