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Candida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections. Phagocytic cells are crucial components of the innate immune system preventing Candida albicans mucosal infections. Streptococcus gordonii and Pseudomonas aeruginosa often colonize mucosal sites, along with C. albicans , and yet interkingdom interactions that might alter the survival and escape of fungi from macrophages are not understood. Murine macrophages were coinfected with S. gordonii or P. aeruginosa , along with C. albicans to evaluate changes in fungal survival. S. gordonii increased C. albicans survival and filamentation within macrophage phagosomes, while P. aeruginosa reduced fungal survival and filamentation. Coinfection with S. gordonii resulted in greater escape of C. albicans from macrophages and increased size of fungal microcolonies formed on macrophage monolayers, while coinfection with P. aeruginosa reduced macrophage escape and produced smaller microcolonies. Microcolonies formed in the presence of P. aeruginosa cells outside macrophages also had significantly reduced size that was not found with P. aeruginosa phenazine deletion mutants. S. gordonii cells, as well as S. gordonii heat-fixed culture supernatants, increased C. albicans microcolony biomass but also resulted in microcolony detachment. A heat-resistant, trypsin-sensitive pheromone processed by S. gordonii Eep was needed for these effects. The majority of fungal microcolonies formed on human epithelial monolayers with S. gordonii supernatants developed as large floating structures with no detectable invasion of epithelium, along with reduced gene expression of C. albicans HYR1 , EAP1 , and HWP2 adhesins. However, a subset of C. albicans microcolonies was smaller and had greater epithelial invasiveness compared to microcolonies grown without S. gordonii . Thus, bacteria can alter the killing and escape of C. albicans from macrophages and contribute to changes in C. albicans pathogenicity. IMPORTANCE Candida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease. In this Resource article, the authors integrate genomic, bioinformatic and flow cytometric data from human bone marrow to provide an atlas of hematopoietic progenitor cell states in health and disease.

Candida albicans is an opportunistic fungal pathogen colonizing the mucosa of healthy hosts and is the major causative agent of oropharyngeal candidiasis. Its behavior can be modified by the innate immunity and the presence of microbial flora. As part of the host innate immune system, phagocytic cells are crucial players in initial defense against Candida albicans infection. In turn, C. albicans utilizes MAPKinases and Ras1-cAMP stress response signaling pathways to protect itself from killing. We began this study by testing the importance of MAPKinase pathways on C. albicans phagocytosis by neutrophils and subsequent phagosomal survival (Chapter 1). Phagocytosis was influenced by C. albicans morphology, so that hyphal length > 10 μm reduced the phagocytic index in human neutrophils. Primary human neutrophils were significantly more efficient in killing phagocytosed C. albicans, compared to their mouse counterpart. We found that both C. albicans Cek1 and Hog1 pathways were required for survival of phagocytosed yeast, whereas deletion of C. albicans RAS1 resulted in a significantly increase in survival within neutrophils. We hypothesized that this resistant phenotype was as result of an increase resistance to oxidative and non-oxidative killing mechanisms of neutrophils. The absence of Ras1 reduced almost completely ROS release by neutrophils. Moreover, C. albicans Δ/Δras1 cells had increased resistance to hydrogen peroxide as a result of high levels of catalase activity. This phenotype was specific to Ras1, since these effects were not observed in the absence of its partner Cyr1 or with its downstream target Efg1. In addition, the absence of Ras1 in C. albicans had a significantly increased resistance to non-oxidative killing by human neutrophil peptide 1 (alpha-defensin) that was reversed by restoring cellular cAMP levels. These data show that C. albicans Ras1 inactivation and subsequent dampening of cAMP leads to fungal resistance to both oxidative and non-oxidative mechanisms of neutrophil phagosomal killing.In addition to C. albicans signaling pathways, we hypothesized that the presence of oral microbiota would modify C. albicans response to phagosomal killing (Chapter 3). By studying the response of macrophages after the phagocytosis of C. albicans and Streptoccocus gordonii (synergistic) or Pseudomonas aeruginosa (antagonistic specie), we demonstrated that bacteria modify C. albicans survival during phagosomal killing by macrophages. The presence of S. gordonii SK12 strain did not modify uptake of C. albicans however, it increased fungal survival in the phagosome. This was a result of a delayed phagosomal maturation and consequent increase in C. albicans germination, crucial step to escape the phagosome of macrophages. In turn, a clinical strain of P. aeruginosa decreased C. albicans survival by reducing filamentation without affecting phagosomal maturation or uptake. Bacteria species also modified microcolony formation, a pathogenic phenotype found during oropharyngeal candidiasis. While, P. aeruginosa secreted phenazines decrease microcolony formation, S. gordonii increase these structures by releasing a heat stable peptide processed by the lipoprotein signal peptidase II (LspA). These microcolonies also display defective adherence properties driven by a reduction in adhesins expression, while their virulence remains unaltered. This data demonstrated that bacterial interactions modify C. albicans pathogenic phenotypes, which result in an altered C. albicans –macrophage interactions.

ABSTRACT Phagocytic cells are crucial components of the innate immune system preventing Candida albicans mucosal infections. Streptococcus gordonii and Pseudomonas aeruginosa often colonize mucosal sites, along with C. albicans, and yet interkingdom interactions that might alter the survival and escape of fungi from macrophages are not understood. Murine macrophages were coinfected with S. gordonii or P. aeruginosa, along with C. albicans to evaluate changes in fungal survival. S. gordonii increased C. albicans survival and filamentation within macrophage phagosomes, while P. aeruginosa reduced fungal survival and filamentation. Coinfection with S. gordonii resulted in greater escape of C. albicans from macrophages and increased size of fungal microcolonies formed on macrophage monolayers, while coinfection with P. aeruginosa reduced macrophage escape and produced smaller microcolonies. Microcolonies formed in the presence of P. aeruginosa cells outside macrophages also had significantly reduced size that was not found with P. aeruginosa phenazine deletion mutants. S. gordonii cells, as well as S. gordonii heat-fixed culture supernatants, increased C. albicans microcolony biomass but also resulted in microcolony detachment. A heat-resistant, trypsin-sensitive pheromone processed by S. gordonii Eep was needed for these effects. The majority of fungal microcolonies formed on human epithelial monolayers with S. gordonii supernatants developed as large floating structures with no detectable invasion of epithelium, along with reduced gene expression of C. albicans HYR1, EAP1, and HWP2 adhesins. However, a subset of C. albicans microcolonies was smaller and had greater epithelial invasiveness compared to microcolonies grown without S. gordonii. Thus, bacteria can alter the killing and escape of C. albicans from macrophages and contribute to changes in C. albicans pathogenicity. IMPORTANCE Candida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections.

Background To evaluate the analgesic effect and tolerability of paracetamol syrup compared to placebo and ketoprofen lysine salt in children with pharyngotonsillitis cared by family pediatricians. Methods A double-blind, randomized, placebo-controlled trial of a 12 mg/kg single dose of paracetamol paralleled by open-label ketoprofren lysine salt sachet 40 mg. Six to 12 years old children with diagnosis of pharyngo-tonsillitis and a Children's Sore Throat Pain (CSTP) Thermometer score > 120 mm were enrolled. Primary endpoint was the Sum of Pain Intensity Differences (SPID) of the CSTP Intensity scale by the child. Results 97 children were equally randomized to paracetamol, placebo or ketoprofen. Paracetamol was significantly more effective than placebo in the SPID of children and parents ( P < 0.05) but not in the SPID reported by investigators, 1 hour after drug administration. Global evaluation of efficacy showed a statistically significant advantage of paracetamol over placebo after 1 hour either for children, parents or investigators. Patients treated in open fashion with ketoprofen lysine salt, showed similar improvement in pain over time. All treatments were well-tolerated. Conclusions A single oral dose of paracetamol or ketoprofen lysine salt are safe and effective analgesic treatments for children with sore throat in daily pediatric ambulatory care.