11 research outputs found
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 NFÎşB 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 NFÎşB 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
Bacterial colonization dynamics and antibiotic resistance gene dissemination in the hospital environment after first patient occupancy: a longitudinal metagenetic study
Background!#!Humans spend the bulk of their time in indoor environments. This space is shared with an indoor ecosystem of microorganisms, which are in continuous exchange with the human inhabitants. In the particular case of hospitals, the environmental microorganisms may influence patient recovery and outcome. An understanding of the bacterial community structure in the hospital environment is pivotal for the prevention of hospital-acquired infections and the dissemination of antibiotic resistance genes. In this study, we performed a longitudinal metagenetic approach in a newly opened ward at the Charité Hospital (Berlin) to characterize the dynamics of the bacterial colonization process in the hospital environment after first patient occupancy.!##!Results!#!The sequencing data showed a site-specific taxonomic succession, which led to stable community structures after only a few weeks. This data was further supported by network analysis and beta-diversity metrics. Furthermore, the fast colonization process was characterized by a significant increase of the bacterial biomass and its alpha-diversity. The compositional dynamics could be linked to the exchange with the patient microbiota. Over a time course of 30 weeks, we did not detect a rise of pathogenic bacteria in the hospital environment, but a significant increase of antibiotic resistance determinants on the hospital floor.!##!Conclusions!#!The results presented in this study provide new insights into different aspects of the environmental microbiome in the clinical setting, and will help to adopt infection control strategies in hospitals and health care-related buildings. Video Abstract
Axenic Long-Term Cultivation of <i>Pneumocystis jirovecii</i>
Pneumocystis jirovecii, a fungus causing severe Pneumocystis pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with P. jirovecii and a small number of experiments involving animal-derived Pneumocystis species have been published to date. However, P. jirovecii culture conditions may differ significantly from those of animal-derived Pneumocystis, as there are major genotypic and phenotypic differences between them. Establishing a well-performing P. jirovecii cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for Pneumocystis jirovecii. To identify promising approaches for cultivation, a literature survey encompassing animal-derived Pneumocystis cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for P. jirovecii culture. This allowed us to develop a medium that produced a 42.6-fold increase in P. jirovecii qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing Pneumocystis clusters in the final medium, DMEM-O3. P. jirovecii doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated P. jirovecii under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable
Combination of Classifiers Identifies Fungal-Specific Activation of Lysosome Genes in Human Monocytes
Blood stream infections can be caused by several pathogens such as viruses, fungi and bacteria and can cause severe clinical complications including sepsis. Delivery of appropriate and quick treatment is mandatory. However, it requires a rapid identification of the invading pathogen. The current gold standard for pathogen identification relies on blood cultures and these methods require a long time to gain the needed diagnosis. The use of in situ experiments attempts to identify pathogen specific immune responses but these often lead to heterogeneous biomarkers due to the high variability in methods and materials used. Using gene expression profiles for machine learning is a developing approach to discriminate between types of infection, but also shows a high degree of inconsistency. To produce consistent gene signatures, capable of discriminating fungal from bacterial infection, we have employed Support Vector Machines (SVMs) based on Mixed Integer Linear Programming (MILP). Combining classifiers by joint optimization constraining them to the same set of discriminating features increased the consistency of our biomarker list independently of leukocyte-type or experimental setup. Our gene signature showed an enrichment of genes of the lysosome pathway which was not uncovered by the use of independent classifiers. Moreover, our results suggest that the lysosome genes are specifically induced in monocytes. Real time qPCR of the identified lysosome-related genes confirmed the distinct gene expression increase in monocytes during fungal infections. Concluding, our combined classifier approach presented increased consistency and was able to “unmask” signaling pathways of less-present immune cells in the used datasets
A simple sequence-based filtering method for the removal of contaminants in low-biomass 16S rRNA amplicon sequencing approaches
Effect of phagosome pH on <i>C. glabrata</i> survival.
<p>(A) Viable and heat killed <i>C. glabrata</i> containing phagosomes acquire similar levels of V-ATPase. Representative fluorescence microscopy images of viable or heat killed <i>C. glabrata</i> 180 min post-infection phagocytosed by murine J774E cells expressing a V-ATPase-GFP fusion protein (left panel). V-ATPase is shown in green while non-phagocytosed yeasts (stained with concanavalin A [ConA]) are indicated in yellow (marked with red arrows). Phagocytosed yeasts are labeled with white arrows. Co-localization with V-ATPase was quantified for phagosomes containing viable or heat killed <i>C. glabrata</i> at indicated time points (right panel). (B) Rising phagosome pH with chloroquine but not bafilomycin A1 reduces <i>C. glabrata</i> survival in MDMs. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 24 h. Co-incubation samples contained no drug (untreated), chloroquine (50 µM), chloroquine plus iron nitriloacetate (20 µM, FeNTA) or bafilomycin A1 (50 nM). (C) Chloroquine or bafilomycin A1 are not toxic to <i>C. glabrata in vitro</i>. Growth in presence of the drugs is comparable to untreated cultures. Statistical analysis was performed comparing heat killed with viable <i>C. glabrata</i> at indicated time points (A) or comparing untreated and drug-treated samples (B) (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p
Phagosome maturation arrest occurs in different macrophage differentiation or activation states and is yeast phagosome-specific.
<p>(A) Human M1-polarized and M2-polarized MDMs do not differ in central aspects of <i>C. glabrata</i>-macrophage interaction: phagocytosis, phagosome acidification and killing. Phagocytosis (MOI of 5) was quantified microscopically by determining the percentage of internalized (Concanavalin A stain-negative) yeasts out of total yeasts after 90 min. Phagosome acidification was quantified microscopically by determining the percentage of LysoTracker-positive phagosomes after 90 min. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 3 h of co-incubation and comparing to yeasts incubated without macrophages. (B) Treatment with vitamin D<sub>3</sub> (calcitriol) has no influence on the number of LysoTracker-positive viable <i>C. glabrata</i> containing phagosomes of human MDMs. (C) MDMs co-infected with <i>C. glabrata</i> and latex beads show a acidification defect specific to <i>C. glabrata</i> containing phagosomes (LysoTracker-negative staining; white arrow) but acidify latex bead containing phagosomes (LysoTracker-positive staining; white asterisk). Representative image 90 min post infection. GFP-expressing <i>C. glabrata</i> is indicated in green and non-phagocytosed yeasts stained with Concanavalin A (ConA) are in yellow (marked with red arrows). Statistical analysis was performed comparing M1-type with M2-type macrophages (A) or drug treated with untreated viable <i>C. glabrata</i> (B) (n≥3; *p<0.05, **p<0.01 by unpaired Student’s t test).</p
<i>C. glabrata</i> does not induce MAP-kinase or NFÎşB signaling cascades upon phagocytosis but activates Syk.
<p>RAW264.7 macrophages were stimulated with LPS (1 µg/ml) or infected with viable or heat killed (Hk) <i>C. glabrata</i> (MOI of 5) for indicated time points. (A) Cell lysates were subjected to Western Blot analyses by using antibodies detecting either the phosphorylated or unphosphorylated form (as a loading control) of p38, p44/42 (Erk1/2), SAPK/JNK, IKKαβ, IκBα and p65. Only LPS treatment induced changes in phosphorylation patterns of analyzed proteins. Data shown are representatives of three independent experiments. (B) Cell lysates were resolved on SDS-PAGE and membranes blotted for phosphorylated Syk (P-Syk) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone.0096015-Mansour1" target="_blank">[25]</a> (C) Localization of the NFκB subunit p65 was analyzed by immunofluorescence microscopy. Representative pictures of macrophages treated with LPS or viable <i>C. glabrata</i> for 10 min are shown on the left site, a quantification of indicated time points on the right site. Percentage of NFκB nuclear localization was quantified for all macrophages (LPS) or for yeast-bound macrophages (viable, heat killed). While LPS induced the translocation of p65 to the nucleus, <i>C. glabrata</i> independent of its viability, did not. Statistical analysis was performed for <i>C. glabrata-</i>infected versus LPS-treated macrophages at the indicated time points (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p
Alkalinization-defective <i>C. glabrata</i> mutants.
<p>Listed are mutants that showed reduced <i>in vitro</i> alkalinization of phenol red containing YNB medium with 1% casamino acids as sole carbon and nitrogen source in a screen of 647 mutants. Alkalinization defects were verified in independent assays and with two independent clones.</p>A<p>+reduced alkalinization (same phenotype as <i>bcy1</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>), ++ strongly reduced alkalinization (same phenotype as <i>put3</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>) as compared to the wild type.</p>B, C<p>Growth was monitored in parallel in YPD and in YNB medium with 1% casamino acids without phenol red by measuring absorption at 600 nm. ++ strong growth defect, + weak growth defect, - unaltered growth as compared to the wild type.</p>D<p>Mutants were co-incubated with MDMs for 90 min and phagosome acidification was monitored by LysoTracker staining. At least three independent microscopic fields were scored per mutant. ++ strong increase in LysoTracker signal, + medium increase in LysoTracker signal, - no change in LysoTracker signal as compared to the wild type.</p
The influence of mannosyltransferases of <i>C. glabrata</i> on environmental alkalinization and acidification of phagosomes.
<p>(A) Representative fluorescence microscopy images of wild type (wt <i>hlt</i>Δ) <i>C. glabrata</i> and <i>mnn10</i>Δ mutant 90 min post infection, phagocytosed by human MDMs (left panels). LysoTracker staining is shown in red, while non-phagocytosed yeasts, stained with Concanavalin A (ConA), are shown in yellow. Phagocytosed yeasts are labeled with a white arrow while non-phagocytosed yeasts are marked with red arrows. (B) Co-localization with LysoTracker was quantified for phagosomes containing wild type (wt <i>hlt</i>Δ or wt <i>t</i>Δ) or mutant (<i>mnn10</i>Δ, <i>mnn11</i>Δ, <i>anp1</i>Δ) <i>C. glabrata</i> at 90 min post infection. Statistical analysis was performed comparing mutant with wild type <i>C. glabrata</i> (n≥3; **p<0.01, ***p<0.005 by unpaired Student’s t test). (C) <i>mnn10</i>Δ and <i>mnn11</i>Δ mutants showed severe defects in environmental alkalinization <i>in vitro</i>, while alkalinization by the <i>anp1</i>Δ mutant was comparable to isogenic wild type levels. 1×10<sup>6 </sup><i>C. glabrata</i> cells/ml were inoculated in a 24 well plate with liquid YNB medium with 1% casamino acids and 20 mg/l phenol red and incubated for 24 h.</p