g-Ming Qin

Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.

RNAi Screen of Endoplasmic Reticulum–Associated Host Factors Reveals a Role for IRE1α in Supporting Brucella Replication

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Phylogenetic Analyses of Phytopathogenic Isolates of Verticillium spp.

ABSTRACT To better understand the genetic relationships between Verticillium dahliae isolates from lettuce and other phytopathogenic Verticillium spp. isolates from various hosts and geographic locations, the complete intergenic spacer (IGS) region of the nuclear ribosomal RNA gene (rDNA) and the beta-tubulin gene were amplified and sequenced. The sequences of the complete IGS region and the beta-tubulin gene were used alone and in combination to infer genetic relationships among different isolates of Verticillium with the maximum-likelihood distance method. Phylogenetic analyses set sequences into four distinct groups comprising isolates of V. albo-atrum, V. tricorpus, and V. dahliae from cruciferous and noncruciferous hosts. Within the four Verticillium groups, isolates of V. dahliae from cruciferous hosts displayed the closest affinity to V. dahliae from noncruciferous hosts. Isolates of V. dahliae from noncruciferous hosts could be further divided into four subgroups based on sequence similarities within the IGS region. Cross-pathogenicity tests demonstrated that most Verticillium isolates were as virulent on other hosts as on their hosts of origin. A phenogram based on the cross pathogenicity of individual isolates resembled those derived from the IGS and beta-tubulin sequence comparisons. On the basis of the data presented, the potential origin of some isolates of V. dahliae pathogenic on lettuce is proposed.

Characterization of Race-Specific Interactions Among Isolates of Verticillium dahliae Pathogenic on Lettuce

ABSTRACT Verticillium wilt, caused by Verticillium dahliae, poses a major threat to lettuce (Lactuca sativa) production in California. Incorporation of resistance into commercial lettuce cultivars offers the least expensive technique of sustaining production in infested areas. To test the breadth of the resistance identified in field experiments, a pair of susceptible (Salinas and Sniper) and resistant (La Brillante and Little Gem) lettuce cultivars were used as differentials and individually inoculated with 29 isolates of V. dahliae and two isolates of V. albo-atrum from several hosts, including lettuce, in replicated greenhouse experiments. The reactions of the four cultivars were determined based on the disease severity at maturity. None of the V. albo-atrum isolates or V. dahliae isolates from cruciferous hosts caused significant disease on lettuce. Both Salinas and Sniper were susceptible to many isolates of V. dahliae (21 of 23) from noncruciferous hosts, and the isolates varied in their overall virulence. However, of these, only three isolates caused significant disease on the resistant cvs. La Brillante and Little Gem. These three isolates also were distinct from the other V. dahliae isolates based on sequence data from the intergenic spacer (IGS) region of the nuclear ribosomal RNA gene, suggesting that they form a phylogenetically distinct subgroup that differs in virulence toward specific lettuce genotypes. Accordingly, isolates of V. dahliae virulent on all tested cultivars, including the resistant La Brillante and Little Gem, were designated as race 2, whereas those virulent only on the susceptible Salinas and Sniper were designated as race 1. Although a range of virulence among isolates has been described in other hosts, this is the first description of distinct virulence phenotypes in V. dahliae since a similar race structure was described in tomato in the 1960s.

Variation for Resistance to Verticillium Wilt in Lettuce (Lactuca sativa L.)

Host resistance offers the most cost-effective method of Verticillium wilt control in lettuce (Lactuca sativa). In 2004 and 2005, 107 and 22 lettuce cultivars, respectively, were screened for resistance in a field infested with Verticillium dahliae, and disease progress on resistant and susceptible cultivars was determined. Greenhouse experiments were conducted to evaluate 16 cultivars for resistance to a race 1 and a race 2 isolate. Significant differences for resistance were observed within cultivated lettuce. In susceptible cultivars, disease levels increased through the season, whereas disease in resistant cultivars remained constant. Resistance in greenhouse tests was dependant upon the race used. Seven cultivars were resistant to race 1, whereas all were susceptible to race 2. Cultivar reactions to race 1 in greenhouse and field experiments were correlated, indicating the utility of greenhouse evaluations. The identification of resistance in diverse lettuce types is beneficial to the breeding process. However, because of the existence of resistance-breaking race 2 isolates, this resistance may not be durable. Alternatively, targeted releases of race-1-resistant cultivars to fields with only race 1 pathogen genotypes may extend the life of these cultivars.

Characterization of Verticillium dahliae and V. tricorpus Isolates from Lettuce and Artichoke

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of <1 (0-to-5 scale) on lettuce and artichoke. In contrast, isolates of V. dahliae consistently caused severe wilt with a median DS of >3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.

DNA watermarking of infectious agents: progress and prospects.

Following the 2001 anthrax attacks, infectious disease research laboratories and personnel were subjected to increased scrutiny amid concerns that the released agent originated from within such facilities. Since then, enhanced regulatory controls have been implemented to thwart the possibility of future releases. However, improved microbial forensics technologies have not been employed to facilitate fault attribution or to control and track agent inventories. n nWe believe that novel systems employing enhanced identity protection will instill new public confidence in scientists and avoid erroneous assignment of liability in the case of a release. We propose a DNA watermarking system that includes institution-, laboratory-, and/or investigator-specific watermarks in the genomes of organisms, especially Select Agents. The system will achieve five key goals critical to any watermarking system, phrased in general information theoretic terms: message fidelity, error tolerance, ease of interpretation, availability of signatures, and resistance to attack (Table 1). n n n nTable 1 n nGoals and features of watermarking system. n n n nA DNA watermark is a unique synthetic DNA sequence embedded into the genome of a genetically tractable organism. The watermark provides a means for agent, isolate, or strain identification and tracking by PCR amplification and sequencing of the embedded tag. The power of watermarking for agent control emerges when the technology is linked to the activities of a trusted authorizing entity (Figure 1). This entity could, for example, be charged with distributing organisms containing unique watermark sequences to individual laboratories and/or investigators. These watermarks would distinguish their organisms from those of others in the research community. Laboratories would be encouraged, permitted, or required to use only strains that contain their approved, and confidential, watermark. In the event of release, the offending pathogen would be interrogated for the presence of an approved watermark. If such a watermark were present, then information about the possible source would become immediately available. Of course, pathogen-specific standard operating procedures (SOPs) that ensure the integrity of the watermarking system (to prevent cross-contamination, manage the sharing of strains, and prevent accidental or intentional misuse) would be a necessary component of any watermarking strategy. n n n nFigure 1 n nProposed watermark implementation strategies. n n n nPreviously developed watermarking technologies include approaches for embedding watermarks in microbial genomes [1]–[4] and strategies for encryption [2], [5], [6]. Each method seeks to develop a genetic cipher that is 1) robust to mutation, 2) easy for intended users to decipher, and 3) difficult for third parties to decipher or alter. While these strategies for manipulating watermarks have been successful at watermark encoding, placement in a genome, retrieval from a genome, and decoding, none of the techniques achieves all of the five goals (outlined in Table 1) that are necessary for a watermarking system for Select Agent tracking. n nIn our opinion, however, these techniques are worthy of further investigation, as regards their utility for the research and biosecurity communities. We propose investigation proceed on three discrete but interconnected fronts. First, the theoretical mathematical and information aspects of watermarking systems must be examined and rigorously tested in silico. Second, insertion and removal of watermarks from microbial genomes must be assessed, and the phenotypic invisibility of the watermarks tested. Finally, pathogen-specific SOPs must be developed, keeping in mind the need for transparency and collaboration in research, and tested in a “role playing” scenario. Our initial work indicates that the model is mathematically plausible. Previous work in the use of watermarks suggests that appropriately placed watermarks can be phenotypically neutral [1], [3]. The technologies to introduce watermarks into several of the highest risk Select Agent genomes are currently available, using site-specific insertion tools such as Targetron (intron-based homing) and Lambda red mutagenesis. Adaptation of these or comparable genetic tools for less tractable Select Agents would require technological advances that would also broadly benefit research of each agent. n nAdoption of a watermarking strategy by research groups would need to be justified by a cost-benefit analysis, from an institutional liability perspective, and from the perspective of the research community. Several salient concerns can be readily identified. Our proposed system does not protect against covert usage of naturally occurring wild-type strains or remediate existing stocks, but instead provides a forward-looking strategy. To address cost concerns, funding agencies that require enhanced inventory control could be encouraged or required to support the cost of implementing watermarking systems. Similarly, these agencies could support or collaborate with private or public authorizing entities to develop SOPs for strain management. Finally, convincingly establishing phenotypic neutrality of genomic modifications will be non-trivial, and thus, will constitute an important area for future research. Despite these potential impediments, watermarking would nearly eliminate the potential for mistaken assignment of source for a suspected agent release. Moreover, the development and implementation costs may prove to be much less than other proposed measures for enhancing laboratory security, including around-the-clock security patrols. n nWe considered two variations on the operational infrastructure required (Figure 1). An authorizing entity could (Figure 1A) design, insert, and distribute, or (Figure 1B) simply distribute, the secured watermark to requesting laboratory. In the latter scenario, the requesting laboratory would be responsible for adapting genetic technology to deliver the watermark. We do not propose that previously generated modified strains (mutant collections, etc.) would be modified and restocked. The transition to marked strains would be incremental but stable. We speculate that an efficient approach to this scenario would be to provide funding opportunities to establish and validate agent-specific systems. While there are several potential impediments to implementing the proposed watermarking systems, the combination of positive impact on lay perception of responsible scientific activity and an increased confidence in control of liability by investigators and institutions provides a rationale to investigate the development of watermarking tools for Select Agent research.

Glutathione S-transferase of brown planthoppers (Nilaparvata lugens) is essential for their adaptation to gramine-containing host plants.

Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms.

Nonlinear colony extension of Sclerotinia minor and S. sclerotiorum

Fungal colonies initially extend exponentially and reach a constant linear extension rate determined solely by their growth in the peripheral zone. However the radial extension rates of Sclerotinia sclerotiorum and S. minor accelerate over time on PDA. Experiments were conducted to analyze the variable extension rates of the two Sclerotinia species and compare them with those of Verticillium dahliae and Cladosporium sp. In addition, the effects of starter disk size, disk position in the parent colony, the age of the parent colony, the concentration of potato dextrose broth and of incubation temperature also were determined. While the growth of Cladosporium sp. and V. dahliae followed established linear trends, the radial extension of S. sclerotiorum and S. minor colonies continuously accelerated over time until they reached the edge of the (150 mm diam) Petri dish. A polynomial model fitted the radial extension of colonies of Sclerotinia spp. Furthermore the accelerating colony extension rate was partly due to increasing colony radius. The rates of extension from mycelial disks transferred from the parental colony were positively correlated with the radius of the mycelial disks transferred. The rates of extension also were dependent on where the transferred disks were taken from parent colonies and the age and radius of the parent colony. On potato dextrose agar medium the extension rates of colonies of S. sclerotiorum and S. minor also were affected by broth concentration and temperature. With increasing nutrient concentration colony extension rates increased and were highest at 25 C. This study revealed a novel pattern of radial growth for Sclerotinia spp. that diverged from the established growth patterns of fungal colonies. Knowledge of the differences in growth behavior may be exploited in the laboratory studies on fungal competition and hyperparasitism and potentially in disease control strategies.

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection

Cryptococcus neoformans (Cn) is a deadly fungal pathogen whose intracellular lifestyle is important for virulence. Host mechanisms controlling fungal phagocytosis and replication remain obscure. Here, we perform a global phosphoproteomic analysis of the host response to Cryptococcus infection. Our analysis reveals numerous and diverse host proteins that are differentially phosphorylated following fungal ingestion by macrophages, thereby indicating global reprogramming of host kinase signaling. Notably, phagocytosis of the pathogen activates the host autophagy initiation complex (AIC) and the upstream regulatory components LKB1 and AMPKα, which regulate autophagy induction through their kinase activities. Deletion of Prkaa1, the gene encoding AMPKα1, in monocytes results in resistance to fungal colonization of mice.xa0Finally, the recruitment of AIC components to nascent Cryptococcus-containing vacuoles (CnCVs) regulates the intracellular trafficking and replication of the pathogen. These findings demonstrate that host AIC regulatory networks confer susceptibility to infection and establish a proteomic resource for elucidating host mechanisms that regulate fungal intracellular parasitism.