Reverse genetics through random mutagenesis in Histoplasma capsulatum

<p>Abstract</p> <p>Background</p> <p>The dimorphic fungal pathogen <it>Histoplasma capsulatum </it>causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms underlying the biology and the pathogenesis of <it>Histoplasma </it>has been hindered by a shortage of methodologies for mutating a gene of interest.</p> <p>Results</p> <p>We describe a reverse genetics process that combines the random mutagenesis of <it>Agrobacterium</it>-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a PCR-addressing method. Application of this procedure facilitated the isolation of a <it>cbp1 </it>mutant in a North American type 2 strain, a <it>Histoplasma </it>strain recalcitrant to gene knock-outs through homologous recombination. Optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants.</p> <p>Conclusion</p> <p>This methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of <it>Histoplasma </it>biology. The procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.</p

TRIM5alpha Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation

Tripartite motif-containing protein 5alpha (TRIM5alpha) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5alpha is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5alpha suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5alpha-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5alpha suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5alpha contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5alpha possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern

Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival

<i>Histoplasma</i> Sod3 encodes an extracellular Cu⁺⁺-dependent superoxide dismutase.

<p>(<b>A</b>) PCR validation of deletion of the <i>SOD3</i> gene. Genomic DNA from the <i>SOD3(+)</i> parental strain (WU8) and the <i>sod3Δ</i> strain (OSU13) were tested by PCR for the ribosomal subunit gene <i>RPS15</i>, the wild-type <i>SOD3</i> gene, and the mutant allele marked with the hygromycin resistance gene (<i>hph</i>). (<b>B</b>) Superoxide dismutase activity in culture filtrates harvested from <i>SOD3(+)</i> (OSU45), <i>sod3Δ</i> (OSU15), and the <i>sod3Δ/SOD3</i> complemented (OSU49) strains. Detection of superoxide was determined through superoxide-dependent reduction of the WST-1 tetrazolium dye after generation of superoxide using hypoxanthine and xanthine oxidase. Reduction of WST-1 was monitored by absorbance at 438 nm. Buffer or culture filtrates contained 5 µg ovalbumin or total culture filtrate protein, respectively. Asterisks represent significant difference (*** p<0.001) in the inhibition of WST-1 reduction between <i>SOD3(+)</i> and <i>sod3Δ</i> culture filtrates. Data shown is representative of three independent experiments, each performed with triplicate samples. (<b>C</b>) Sod3 activity following Cu⁺⁺ depletion. Culture filtrates containing 5 µg total protein from <i>SOD3(+)</i> (OSU45) and <i>sod3Δ</i> (OSU15) strains were tested for their ability to inhibit WST-1 reduction by superoxide before (no chelator), after Cu⁺⁺ depletion (+DDC), and after subsequent repletion with 50 mM Cu⁺⁺ (+CuSO₄). Values represent relative inhibition of WST-1 reduction by culture filtrate samples (n = 3) compared to buffer controls treated in parallel. Asterisks represent significant differences from <i>SOD3(+)</i> culture filtrates (* p<0.05, ** p<0.01).</p

<i>Histoplasma capsulatum</i> strains.

a<p>all strains were constructed in the G186A (ATCC# 26027) background.</p>b<p>uracil auxotroph of G186A (Marion CM, et al., 2006 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002713#ppat.1002713-Marion1" target="_blank">[78]</a>).</p>c<p>GFP sentinel RNAi background (Edwards, et al. 2011 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002713#ppat.1002713-Edwards1" target="_blank">[26]</a>).</p

Sod3 facilitates infection through detoxification of host reactive oxygen.

<p>Kinetics of sublethal lung infection by <i>Histoplasma</i> in animals competent for ROS production (<b>A</b>) or animals lacking the NADPH-oxidase function (<b>B</b>). Mice were intranasally infected with approximately 1×10⁴<i>SOD3(+)</i> (OSU45) or <i>sod3Δ</i> (OSU15) <i>Histoplasma</i> yeasts. At 2, 4, 8, and 15 days post-infection, the fungal burden in lungs was determined by quantitative platings for <i>Histoplasma</i> cfu. (<b>A</b>) Respiratory infection of Phox(+/+) mice isogenic to the p47^phox knock-outs. (<b>B</b>) Respiratory infection of p47^phox knock-out (Phox(−/−)) mice. Each data point represents cfu counts per lung from an individual animal (n = 3 per time point) and horizontal bars represent the mean fungal burden. Non-significant (ns) or significant differences (* p<0.05, ** p<0.01) from animals infected with <i>SOD3(+)</i> organisms is indicated above the respective columns. The actual inoculum dose is shown in graphs at day 0. The limit of detection is 100 cfu.</p

Definition of the Extracellular Proteome of Pathogenic-Phase <i>Histoplasma capsulatum</i>

The dimorphic fungal pathogen <i>Histoplasma capsulatum</i> causes respiratory and systemic disease. Within the mammalian host, pathogenic <i>Histoplasma</i> yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to <i>Histoplasma</i> virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of <i>Histoplasma</i>. In addition to secreted Cbp1, the extracellular proteome of pathogenic <i>Histoplasma</i> yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic <i>Histoplasma</i> mycelia, suggesting that these proteins are linked to <i>Histoplasma</i> pathogenesis. In addition, <i>Histoplasma</i> yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how <i>Histoplasma</i> alters the host-pathogen interaction to its advantage

Definition of the Extracellular Proteome of Pathogenic-Phase <i>Histoplasma capsulatum</i>

The dimorphic fungal pathogen <i>Histoplasma capsulatum</i> causes respiratory and systemic disease. Within the mammalian host, pathogenic <i>Histoplasma</i> yeast infect, replicate within, and ultimately kill host phagocytes. Surprisingly, few factors have been identified that contribute to <i>Histoplasma</i> virulence. To address this deficiency, we have defined the constituents of the extracellular proteome using LC−MS/MS analysis of the proteins in pathogenic-phase culture filtrates of <i>Histoplasma</i>. In addition to secreted Cbp1, the extracellular proteome of pathogenic <i>Histoplasma</i> yeast consists of 33 deduced proteins. The proteins include glycanases, extracellular enzymes related to oxidative stress defense, dehydrogenase enzymes, chaperone-like factors, and five novel culture filtrate proteins (Cfp’s). For independent verification of proteomics-derived identities, we employed RNA interference (RNAi)-based depletion of candidate factors and showed loss of specific proteins from the cell-free culture filtrate. Quantitative RT-PCR revealed the expression of 10 of the extracellular factors was particularly enriched in pathogenic yeast cells as compared to nonpathogenic <i>Histoplasma</i> mycelia, suggesting that these proteins are linked to <i>Histoplasma</i> pathogenesis. In addition, <i>Histoplasma</i> yeast express these factors within macrophages and during infection of murine lungs. As extracellular proteins are positioned at the interface between host and pathogen, the definition of the pathogenic-phase extracellular proteome provides a foundation for the molecular dissection of how <i>Histoplasma</i> alters the host-pathogen interaction to its advantage

N-terminal and C-terminal signals direct extracellular localization of Sod3.

<p>(<b>A</b>) Schematic of the Sod3 protein highlighting the predicted signal peptide (SP) and the glycophosphatidyl inositol anchor (GPI) signal motifs. Numbers represent amino acid residues in the Sod3 protein. Shading beneath the Sod3 protein indicates amino acid sequence similarity between G186A, G217B and NAm1 Sod3 proteins ranging from dark (>90% sequence identity) to light (<50% identity). (<b>B</b>) Relative Sod3 activity associated with the yeast cell and soluble extracellular fraction. Superoxide dismutase activities were determined by inhibition of superoxide-dependent WST-1 reduction in the presence of 1×10⁸ yeasts (cell-associated) or the corresponding culture filtrate (soluble) of <i>SOD3(+)</i> (OSU45) and <i>sod3Δ</i> (OSU15) strains (n = 3, each). Inhibition of WST-1 reduction was normalized to reactions in the absence of yeasts or culture filtrate. Asterisks represent significant differences (p<0.001) from <i>SOD3(+)</i> samples. (<b>C</b>) Determination of the localization of GFP when fused to the N-terminus of Sod3. Extracellular or intracellular GFP localization was determined by α-FLAG immunoblot of culture filtrates or cellular lysates from <i>Histoplasma</i> yeast strains expressing FLAG epitope-tagged GFP (GFP:FLAG; OSU88) or GFP with the first 26 amino acids of Sod3 (Sod3_1–26:GFP:FLAG; OSU102). Cellular lysates were also tested for α-tubulin to demonstrate equal loadings. (<b>D</b>) Localization of Sod3 activity after removal of the C-terminal 26 amino acids. Cell-associated and soluble superoxide dismutase activities of <i>Histoplasma</i> yeasts were determined using 1×10⁸ intact yeasts or their corresponding culture filtrates, respectively. Samples were collected from <i>SOD3(+)</i> (OSU45), <i>sod3Δ</i> (OSU15), and yeasts expressing full length Sod3 (<i>sod3Δ/FLAG:SOD3</i>; OSU116) or Sod3 lacking the putative GPI signal (sod3Δ/FLAG:<i>SOD3</i>_ΔGPI; OSU117). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002713#s2" target="_blank">Results</a> were normalized to uninhibited reactions and plotted as the proportion of total inhibitory activity. Asterisks represent significant difference from full length Sod3 (** p<0.01, *** p<0.001). Relative quantitation of Sod3 in culture filtrates was determined by α-FLAG immunoblot and is indicated numbers below.</p