Eric D. Chow

Systematic Genetic Analysis of Virulence in the Human Fungal Pathogen Cryptococcus neoformans

The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes-polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3‐one to cholest‐4‐en‐3‐one‐27‐oic acid. We determined the X‐ray structure of cholest‐4‐en‐3‐one‐bound CYP125A1 at a resolution of 1.58 Å. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest‐4‐en‐3‐one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest‐4‐en‐3‐one.

Exploration of whole-genome responses of the human AIDS-associated yeast pathogen Cryptococcus neoformans var grubii : nitric oxide stress and body temperature

Cryptococcus neoformans var grubii is an opportunistic basidiomycete yeast pathogen that is a significant cause of HIV/AIDS-related deaths worldwide. We describe a whole-genome oligonucleotide microarray for this pathogen. These arrays have been used to elucidate the transcriptional responses of the genome to heat shock as well as to two conditions relevant to human infections: body temperature and nitric oxide (NO) stress produced by the NO donor DPTA-NONOate. This analysis revealed an NO-inducible C. neoformans-specific four-gene family that showed a highly similar transcriptional profile to that of FHB1, a previously described NO dioxygenase/flavohemoglobin required for virulence. NO treatment also induced genes involved in the synthesis of the antioxidant mannitol, a polyol that accumulates in the cerebrospinal fluid of infected patients. Exposure to NO also caused increased expression of the sole C. neoformans var grubii protein with HHE/hemerythrin cation binding motifs. Notably, a similar gene in E. coli, ytfE, has been shown to be NO-inducible and protects bacterial cells from killing by NO. Genes induced by NO were highly enriched for those repressed at 37°C, indicating an unexpected interplay between temperature and NO regulation in this basidiomycete. Resources described here should facilitate future investigations of this lethal human yeast pathogen.

Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications.

Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are ‘DASHed’ with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost.

Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Childrens Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006).

A cysteine protease inhibitor rescues mice from a lethal Cryptosporidium parvum infection

ABSTRACT Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

ABSTRACT The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an α-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel β-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections

Detection of intraocular infections relies heavily on molecular diagnostics. A fundamental challenge is that only 100e300 ml of intraocular fluid can be safely obtained at any given time for diagnostic testing. The most widely available molecular diagnostic panel for infections in ophthalmology includes 4 separate pathogendirected polymerase chain reactions (PCRs): cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii. Not surprisingly, more than 50% of all presumed intraocular infections fail to have a pathogen identified. Metagenomic deep sequencing has the potential to improve diagnostic yield as it is unbiased and hypothesis-free; it can theoretically detect all pathogens in a clinical sample. Previously, we demonstrated that unbiased RNA sequencing (RNA-seq) of intraocular fluid detects fungi, parasites, DNA and RNA viruses in uveitis patients. One obvious drawback regarding RNA-seq is that optimal RNA sequencing requires proper specimen handling, including either flash-freezing or immediate placement of the specimen on dry ice. Although commercial room-temperature RNA-preservatives may address this issue, practicing ophthalmologists may find these collection techniques impractical in an outpatient setting. For pathogens with DNA genomes, metagenomic DNA sequencing (DNA-seq) can circumvent this challenge, as DNA is more tolerant of ambient temperature. This study compares the performance of DNA-seq with conventional pathogen-directed PCRs to diagnose intraocular infections. De-identified archived vitreous samples received by the Proctor Foundation for pathogen-directed PCR testing from 2010e2015 were included. Samples were previously boiled and stored at 80 C. A total of 31 pathogen-positive reference samples and 36 pathogen-negative reference samples by Proctor PCRs were randomized and subjected to DNA-seq. The sensitivity and specificity of the Proctor’s pathogen-directed PCRs range from 85%e 100% and 98%e100%, respectively. The DNA-seq workflow included the following steps: 50 ml of each vitreous sample was used to isolate DNA using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) per the manufacturer’s recommendations, 5 ml of the extracted DNA were used to prepare libraries using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and amplified with 12 PCR cycles, and library size and concentration were determined as described. Samples were then sequenced to an average depth of 15 10 reads/sample on an Illumina HiSeq 4000 instrument using 125 nucleotide (nt) paired-end sequencing. Sequencing data were analyzed using a rapid computational pipeline developed in-house to classify sequencing reads and identify potential pathogens against the entire National Center for Biotechnology Information (NCBI) nt reference database. Any infectious agent that had 2 nonoverlapping reads to the reference pathogen genome was considered positive if it met both of the following criteria: (1) It is a pathogen known to be associated with infectious uveitis, and (2) reads from this pathogen were not present in the no-template (water only) control on the same run and library preparation. Discrepant samples were evaluated at the Clinical Laboratory Improvement Amendments (CLIA)-certified Stanford Clinical Microbiology and Clinical Virology laboratories, except for human T-cell leukemia virus type 1 (HTLV-1), which was confirmed at the University of California San Francisco using primers targeting the tax gene and subsequent amplicon sequencing (Elim Biopharmaceuticals, Inc, Hayward, CA). Laboratory personnel involved in sample preparation, analysis, and confirmatory testing were masked to the reference PCR results until all analyses were completed. Of the 31 positive-reference samples tested, 27 samples were identified correctly with DNA-seq (Fig 1A, Table S1, available at www.aaojournal.org). Three samples positive for T. gondii and 1 sample positive for VZV by directed-PCR were not detected by DNA-seq. The positivity of these 4 samples was confirmed by directed real-time PCRs. The cycle threshold for VZVwas 28.2, while the cycle thresholds for the 3 T. gondii samples ranged from 27e36.5. The positive agreement between DNA-seq and directed-PCR was 87%. Thirty-six archived vitreous samples that tested negative by all Proctor pathogen-directed PCRs (CMV, VZV, HSV, and T. gondii) were randomly selected for DNA-seq. Twenty eight of 36 samples yielded no additional pathogen (Fig 1B, Table S1, available at www.aaojournal.org), while 8 samples (22%) resulted in 6 additional pathogens either not detected or not tested with pathogen-directed PCRs. Those organisms included CMV, human herpesvirus 6 (HHV-6), HSV-2, HTLV-1, Klebsiella pneumoniae, and Candida dubliniensis. All of these organisms are known to be associated with infectious uveitis (Fig 1B). The results for HHV-6, HTLV-1, Klebsiella pneumoniae, and Candida dubliniensis were confirmed. Two samples tested positive for HSV-2 and CMV by DNA-seq were not confirmed by directed-PCR. Here, it was unclear if DNA-seq can achieve higher sensitivity for CMV and HSV-2 compared with PCR, or whether these were false-positive results by DNA-seq, as these samples yielded only 2 reads on DNA-seq. It should be noted that DNA-seq detected 100% of all samples that tested positive for CMV and HSV by PCR. An advantage of metagenomic deep sequencing is the ability to apply sequence information to infer the phenotypic behavior of the identified pathogen. We compared samples in which CMV sequences were adequately recovered for the UL54 and UL97 genes, coding for the DNA polymerase and phosphotransferase respectively, and compared with a CMV antiviral drug resistance database developed at Stanford University. Of the 7 samples analyzed, 3 had mutations in UL97 (phosphotransferase) that confer ganciclovir and valganciclovir resistance. Two samples were found to have C592G mutations, and 1 sample had both C592G and L595S mutations (Table S2, available at www.aaojournal.org). In summary, we showed that metagenomic DNA sequencing was highly concordant with pathogen-directed PCRs, despite nonideal sample handling conditions (boiled, long-term frozen). The unbiased nature of metagenomic DNA sequencing allowed an expanded scope of pathogen detection, including bacteria, fungal

Chronic Meningitis Investigated via Metagenomic Next-Generation Sequencing

Importance Identifying infectious causes of subacute or chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed. Objective To present a case series of patients with diagnostically challenging subacute or chronic meningitis using metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) supported by a statistical framework generated from mNGS of control samples from the environment and from patients who were noninfectious. Design, Setting, and Participants In this case series, mNGS data obtained from the CSF of 94 patients with noninfectious neuroinflammatory disorders and from 24 water and reagent control samples were used to develop and implement a weighted scoring metric based on z scores at the species and genus levels for both nucleotide and protein alignments to prioritize and rank the mNGS results. Total RNA was extracted for mNGS from the CSF of 7 participants with subacute or chronic meningitis who were recruited between September 2013 and March 2017 as part of a multicenter study of mNGS pathogen discovery among patients with suspected neuroinflammatory conditions. The neurologic infections identified by mNGS in these 7 participants represented a diverse array of pathogens. The patients were referred from the University of California, San Francisco Medical Center (n = 2), Zuckerberg San Francisco General Hospital and Trauma Center (n = 2), Cleveland Clinic (n = 1), University of Washington (n = 1), and Kaiser Permanente (n = 1). A weighted z score was used to filter out environmental contaminants and facilitate efficient data triage and analysis. Main Outcomes and Measures Pathogens identified by mNGS and the ability of a statistical model to prioritize, rank, and simplify mNGS results. Results The 7 participants ranged in age from 10 to 55 years, and 3 (43%) were female. A parasitic worm (Taenia solium, in 2 participants), a virus (HIV-1), and 4 fungi (Cryptococcus neoformans, Aspergillus oryzae, Histoplasma capsulatum, and Candida dubliniensis) were identified among the 7 participants by using mNGS. Evaluating mNGS data with a weighted z score–based scoring algorithm reduced the reported microbial taxa by a mean of 87% (range, 41%-99%) when taxa with a combined score of 0 or less were removed, effectively separating bona fide pathogen sequences from spurious environmental sequences so that, in each case, the causative pathogen was found within the top 2 scoring microbes identified using the algorithm. Conclusions and Relevance Diverse microbial pathogens were identified by mNGS in the CSF of patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for 1 year, the first reported case of CNS vasculitis caused by Aspergillus oryzae, and the fourth reported case of C dubliniensis meningitis. Prioritizing metagenomic data with a scoring algorithm greatly clarified data interpretation and highlighted the problem of attributing biological significance to organisms present in control samples used for metagenomic sequencing studies.