Cell-Type–Specific Transcriptional Profiles of the Dimorphic Pathogen Penicillium marneffei Reflect Distinct Reproductive, Morphological, and Environmental Demands

Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify “phase or cell-state–specific” gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase–encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity

Structural and functional characterization of Legionella pneumophila effector MavL

Abstract: Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitinconjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila

Infrared based saliva screening test for COVID-19

Abstract: Severe acute respiratory syndrome coronavirus 2 has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 patients testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Here, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells.

The genus Legionella comprises 65 species, among which Legionella pneumophila is a human pathogen causing severe pneumonia. To understand the evolution of an environmental to an accidental human pathogen, we have functionally analyzed 80 Legionella genomes spanning 58 species. Uniquely, an immense repository of 18,000 secreted proteins encoding 137 different eukaryotic-like domains and over 200 eukaryotic-like proteins is paired with a highly conserved type IV secretion system (T4SS). Specifically, we show that eukaryotic Rho- and Rab-GTPase domains are found nearly exclusively in eukaryotes and Legionella Translocation assays for selected Rab-GTPase proteins revealed that they are indeed T4SS secreted substrates. Furthermore, F-box, U-box, and SET domains were present in >70% of all species, suggesting that manipulation of host signal transduction, protein turnover, and chromatin modification pathways are fundamental intracellular replication strategies for legionellae. In contrast, the Sec-7 domain was restricted to L. pneumophila and seven other species, indicating effector repertoire tailoring within different amoebae. Functional screening of 47 species revealed 60% were competent for intracellular replication in THP-1 cells, but interestingly, this phenotype was associated with diverse effector assemblages. These data, combined with evolutionary analysis, indicate that the capacity to infect eukaryotic cells has been acquired independently many times within the genus and that a highly conserved yet versatile T4SS secretes an exceptional number of different proteins shaped by interdomain gene transfer. Furthermore, we revealed the surprising extent to which legionellae have coopted genes and thus cellular functions from their eukaryotic hosts, providing an understanding of how dynamic reshuffling and gene acquisition have led to the emergence of major human pathogens

Comparative analysis of dimorphic growth in the pathogenic fungus Penicillium marneffei

© 2013 Dr. Shivani PasrichaFungal infection is a major cause of mortality in immunocompromised individuals and an escalating health threat in the 21st century. With very little known about the mechanisms behind its virulence, Penicillium marneffei is a dimorphic opportunistic fungal pathogen of humans. At 25°C P. marneffei grows as multicellular nonpathogenic hyphae that through differentiation produces asexual conidia, the infectious agent. Infection begins with the inhalation of conidia, followed by a dimorphic switch of these conidia to pathogenic yeast cells upon entering the 37°C environment of the host. In the host P. marneffei resides within immune phagocytic cells, where it then grows as a fission yeast. The objective of this study was to identify differences between P. marneffei hyphal and yeast cells. For this purpose, firstly extensive microarray-based expression profiling data was analysed, identifying a variety of differentially expressed genes that were organized into clusters based on function and spatiotemporal expression patterns. One key activity highlighted in the data related to the acquisition of the essential trace metal iron. Genes involved in high-affinity iron assimilation systems, their regulation and the production of key iron acquisition secondary metabolites (siderophores) were characterised in both hyphal and yeast cells. It was found that compared to hyphal cells, yeast cells rely more heavily on iron acquisition through high-affinity reductive iron assimilation. Also, at least under rich iron conditions there is a yeast cell-specific upregulation of genes involved in the biosynthesis of extracellular siderophores, via the de-repression of transcriptional regulation by SreA. Secondly, metabolite profiling and ‘footprint’ analyses of hyphal and yeast cells were performed. This snapshot of P. marneffei metabolism revealed the metabolite differences between the two cell types and pointed to the utilisation of specific pathways in each growth form. Further, 13C-labeled glucose experiments revealed an increase in specific carbon metabolic pathways in yeast cells, particularly the TCA cycle and the pentose phosphate cycle, indicative of responses to stressful environments. By combining data derived from multiple molecular approaches, key metabolic networks were reconstructed and results highlight that P. marneffei hyphal and yeast cells have definitively different developmental and nutritional profiles, reminiscent of their mode of reproduction and likely adaptations to the environmental niche they inhabit

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei

Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. Talaromyces marneffei grows as a non-pathogenic hyphal form at 25°C but undergoes a dimorphic transition to a pathogenic yeast form at 37°C in vitro and following inhalation of asexual conidia by a host. Here we show that this transition is associated with major changes in central carbon metabolism, and that these changes are correlated with increased virulence of the yeast form. Comprehensive metabolite profiling and 13C-labeling studies showed that hyphal cells exhibited very active glycolytic metabolism and contain low levels of internal carbohydrate reserves. In contrast, yeast cells fully catabolized glucose in the mitochondrial TCA cycle, and store excess glucose in large intracellular pools of trehalose and mannitol. Inhibition of the yeast TCA cycle inhibited replication in culture and in host cells. Yeast, but not hyphae, were also able to use myo-inositol and amino acids as secondary carbon sources, which may support their survival in host macrophages. These analyses suggest that T. marneffei yeast cells exhibit a more efficient oxidative metabolism and are capable of utilizing a diverse range of carbon sources, which contributes to their virulence in animal tissues, highlighting the importance of dimorphic switching in pathogenic yeast

Talaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages

ABSTRACT Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth

LMS og samarbeidslæring

I denne oppgåva har eg arbeidd med temaet kollaborativ samarbeidslæring i den vidaregåande skule og utgangspunktet har vore samarbeidslæring i elevgrupper på den tradisjonelle læringsarenaen (som regel klasserommet). Elevane er i dette samarbeidet store ressursar for kvarandre og dei samarbeider med stort utbyte, først og fremst ved at dei gjev kvarandre innsyn i kvarandre sitt arbeid. Ulempa med slikt samarbeid i klasseromet er at det for det meste berre skjer med sidemannen, dersom dei sit parvis i klasserommet. Fokuset i denne masteroppgåva har vore retta mot i kva grad LMS (Learning Management System) er eigna til å fremja kollaborativt samarbeid. Tanken er at det kan oppstå eit kollaborativt samarbeid av større omfang dersom den tradisjonelle , fysiske læringsarenaen vert utvida med ein digital arena. Bruk av LMS kan i beste fall gje alle innsyn i alle sitt digitale arbeid, dersom elevane er viljuge til å dela digitalt, dvs å leggja det dei arbeider med ut på LMS, slik at dei kan få innsyn i arbeidet til kvarandre. Innleiingsvis gjer eg greie for eigen bakgrunn som utgangspunkt for val av fagområde og problemstilling. Vidare har eg prøvd å sjå problemstillinga i lys av den auka satsinga på IKT i skulen og i samfunnet elles. I teoridelen er læring, samarbeidslæring, Computer Supported Collaborative Learning (CSCL) og LMS sentrale tema. Metodekapitlet er for ein stor del via den kvalitative metode for datainnsamling der instrumentet er intervjumetoden. Den sentrale delen er presentasjon av innsamla data og analysen av desse

Infrared based saliva screening test for COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening