Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.post-print10768 K

The Continuing Emergence of <i>Candida blankii</i> as a Pathogenic Fungus: A New Case of Fungemia in a Patient Infected with SARS-CoV-2

Candida blankii is a recently recognized human pathogen, with most cases of the infection being reported in the immunocompromised. We here describe the case of a critically ill elderly woman with COVID-19 who developed a C. blankii bloodstream infection from a femoral central venous catheter. Aspergillus niger was also isolated from her respiratory secretions. The patient was started on voriconazole for empiric coverage of both A. niger, and at that time, unidentified yeast was found in the blood. Fevers persisted, and the patient expired six days after the yeast was first isolated. Almost one month after her death, C. blankii was identified as the cause of fungemia by sequencing of the internal transcribed spacer (ITS) region of the ribosomal gene and BLAST searching against two databases (performed by a reference laboratory). The isolate demonstrated high minimum inhibitory concentrations (MICs) to azoles and low MICs to amphotericin B, similar to previously described isolates. Timely identification of C. blankii would have prompted different empiric antifungal choices and possibly changed the final outcome. Clinicians should be aware of the pathological potential of C. blankii, the challenges of correctly identifying the organism, and its susceptibility patterns to common antifungals. There is an urgent need to improve assays for C. blankii identification, which will aid in accurate and timely pathogen identification, and appropriate therapeutic management

Identification of Candidate Adherent-Invasive E. coli Signature Transcripts by Genomic/Transcriptomic Analysis.

Adherent-invasive Escherichia coli (AIEC) strains are detected more frequently within mucosal lesions of patients with Crohn's disease (CD). The AIEC phenotype consists of adherence and invasion of intestinal epithelial cells and survival within macrophages of these bacteria in vitro. Our aim was to identify candidate transcripts that distinguish AIEC from non-invasive E. coli (NIEC) strains and might be useful for rapid and accurate identification of AIEC by culture-independent technology. We performed comparative RNA-Sequence (RNASeq) analysis using AIEC strain LF82 and NIEC strain HS during exponential and stationary growth. Differential expression analysis of coding sequences (CDS) homologous to both strains demonstrated 224 and 241 genes with increased and decreased expression, respectively, in LF82 relative to HS. Transition metal transport and siderophore metabolism related pathway genes were up-regulated, while glycogen metabolic and oxidation-reduction related pathway genes were down-regulated, in LF82. Chemotaxis related transcripts were up-regulated in LF82 during the exponential phase, but flagellum-dependent motility pathway genes were down-regulated in LF82 during the stationary phase. CDS that mapped only to the LF82 genome accounted for 747 genes. We applied an in silico subtractive genomics approach to identify CDS specific to AIEC by incorporating the genomes of 10 other previously phenotyped NIEC. From this analysis, 166 CDS mapped to the LF82 genome and lacked homology to any of the 11 human NIEC strains. We compared these CDS across 13 AIEC, but none were homologous in each. Four LF82 gene loci belonging to clustered regularly interspaced short palindromic repeats region (CRISPR)--CRISPR-associated (Cas) genes were identified in 4 to 6 AIEC and absent from all non-pathogenic bacteria. As previously reported, AIEC strains were enriched for pdu operon genes. One CDS, encoding an excisionase, was shared by 9 AIEC strains. Reverse transcription quantitative polymerase chain reaction assays for 6 genes were conducted on fecal and ileal RNA samples from 22 inflammatory bowel disease (IBD), and 32 patients without IBD (non-IBD). The expression of Cas loci was detected in a higher proportion of CD than non-IBD fecal and ileal RNA samples (p <0.05). These results support a comparative genomic/transcriptomic approach towards identifying candidate AIEC signature transcripts

Cryptococcus neoformans glucuronoxylomannan and sterylglucoside are required for host protection in an animal vaccination model

© 2019 Colombo et al. Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis. IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipidcontaining structures in a vaccination model to prevent cryptococcosis

List of genes upregulated in old <i>C</i>. <i>glabrata</i> cells known to be involved in antifungal resistance.

<p>List of genes upregulated in old <i>C</i>. <i>glabrata</i> cells known to be involved in antifungal resistance.</p

Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.

<p>Gene ontology enrichment analysis for genes upregulated in old <i>C</i>. <i>glabrata</i> cells.</p

Summary of the phenotypic characteristics of 14-generation-old <i>C</i>. <i>glabrata</i> cells compared to young cells (strain BG2).

<p>Summary of the phenotypic characteristics of 14-generation-old <i>C</i>. <i>glabrata</i> cells compared to young cells (strain BG2).</p

Switch variants have altered lifespan.

<p>(A) Phenotypic switching in BG2 from S to DB colony morphology (inset) increased consistently with aging to 17 fold (experiments were run in duplicates). (B) RLS of DB was shortened over 50% relative to S and reconstituted in the revertant colony (n = 20 cells over two experiments; DB v. S RLS compared by Wilcoxon Rank Sum Test). ***<i>P <</i> 0.001.</p