Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize due to cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a new twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wildtype, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1-2, 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wildtype chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form "biosynthetic gene clusters" (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%-60% of the crude organic extracts.

Completeness of HIV-1 Envelope Glycan Shield at Transmission Determines Neutralization Breadth.

Densely arranged N-linked glycans shield the HIV-1 envelope (Env) trimer from antibody recognition. Strain-specific breaches in this shield (glycan holes) can be targets of vaccine-induced neutralizing antibodies that lack breadth. To understand the interplay between glycan holes and neutralization breadth in HIV-1 infection, we developed a sequence- and structure-based approach to identify glycan holes for individual Env sequences that are shielded in most M-group viruses. Applying this approach to 12 longitudinally followed individuals, we found that transmitted viruses with more intact glycan shields correlated with development of greater neutralization breadth. Within 2 years, glycan acquisition filled most glycan holes present at transmission, indicating escape from hole-targeting neutralizing antibodies. Glycan hole filling generally preceded the time to first detectable breadth, although time intervals varied across hosts. Thus, completely glycan-shielded viruses were associated with accelerated neutralization breadth development, suggesting that Env immunogens with intact glycan shields may be preferred components of AIDS vaccines.

Effective treatment of SIVcpz-induced immunodeficiency in a captive western chimpanzee.

BACKGROUND: Simian immunodeficiency virus of chimpanzees (SIVcpz), the progenitor of human immunodeficiency virus type 1 (HIV-1), is associated with increased mortality and AIDS-like immunopathology in wild-living chimpanzees (Pan troglodytes). Surprisingly, however, similar findings have not been reported for chimpanzees experimentally infected with SIVcpz in captivity, raising questions about the intrinsic pathogenicity of this lentivirus. FINDINGS: Here, we report progressive immunodeficiency and clinical disease in a captive western chimpanzee (P. t. verus) infected twenty years ago by intrarectal inoculation with an SIVcpz strain (ANT) from a wild-caught eastern chimpanzee (P. t. schweinfurthii). With sustained plasma viral loads of 105 to 106 RNA copies/ml for the past 15 years, this chimpanzee developed CD4+ T cell depletion (220 cells/µl), thrombocytopenia (90,000 platelets/µl), and persistent soft tissue infections refractory to antibacterial therapy. Combination antiretroviral therapy consisting of emtricitabine (FTC), tenofovir disoproxil fumarate (TDF), and dolutegravir (DTG) decreased plasma viremia to undetectable levels (<200 copies/ml), improved CD4+ T cell counts (509 cell/µl), and resulted in the rapid resolution of all soft tissue infections. However, initial lack of adherence and/or differences in pharmacokinetics led to low plasma drug concentrations, which resulted in transient rebound viremia and the emergence of FTC resistance mutations (M184V/I) identical to those observed in HIV-1 infected humans. CONCLUSIONS: These data demonstrate that SIVcpz can cause immunodeficiency and other hallmarks of AIDS in captive chimpanzees, including P. t. verus apes that are not naturally infected with this virus. Moreover, SIVcpz-associated immunodeficiency can be effectively treated with antiretroviral therapy, although sufficiently high plasma concentrations must be maintained to prevent the emergence of drug resistance. These findings extend a growing body of evidence documenting the immunopathogenicity of SIVcpz and suggest that experimentally infected chimpanzees may benefit from clinical monitoring and therapeutic intervention.

Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness.

Sexual transmission of HIV-1 is an inefficient process, with only one or few variants of the donor quasispecies establishing the new infection. A critical, and as yet unresolved, question is whether the mucosal bottleneck selects for viruses with increased transmission fitness. Here, we characterized 300 limiting dilution-derived virus isolates from the plasma, and in some instances genital secretions, of eight HIV-1 donor and recipient pairs. Although there were no differences in the amount of virion-associated envelope glycoprotein, recipient isolates were on average threefold more infectious (P = 0.0001), replicated to 1.4-fold higher titers (P = 0.004), were released from infected cells 4.2-fold more efficiently (P < 0.00001), and were significantly more resistant to type I IFNs than the corresponding donor isolates. Remarkably, transmitted viruses exhibited 7.8-fold higher IFNα2 (P < 0.00001) and 39-fold higher IFNß (P < 0.00001) half-maximal inhibitory concentrations (IC50) than did donor isolates, and their odds of replicating in CD4+ T cells at the highest IFNα2 and IFNß doses were 35-fold (P < 0.00001) and 250-fold (P < 0.00001) greater, respectively. Interestingly, pretreatment of CD4+ T cells with IFNß, but not IFNα2, selected donor plasma isolates that exhibited a transmitted virus-like phenotype, and such viruses were also detected in the donor genital tract. These data indicate that transmitted viruses are phenotypically distinct, and that increased IFN resistance represents their most distinguishing property. Thus, the mucosal bottleneck selects for viruses that are able to replicate and spread efficiently in the face of a potent innate immune response.

A study of the impact of the 21-gene breast cancer assay on the use of adjuvant chemotherapy in women with breast cancer in a Mexican public hospital.

BACKGROUND: The majority of breast cancer patients in Mexico are treated through the public health system and >80% receive adjuvant chemotherapy. The aim of this prospective study was to characterize the impact of the Oncotype DX assay on adjuvant therapy decision making and the confidence in those decisions amongst public sector physicians in Mexico. METHODS: Ninety-eight consecutive patients with ER+, HER2-, stage I-IIIa, N0/N1-3 node-positive breast cancer from the Instituto Nacional de Cancerología were eligible for the study. The primary endpoint was the overall change in treatment recommendations after receiving the assay results. RESULTS: Of 96 patients, 48% received a chemohormonal therapy recommendation prior to testing. Following receipt of results, treatment decisions changed for 31/96 (32%) patients, including 17/62 (27%) node-negative patients and 14/34 (41%) node-positive patients. The proportion of patients with a chemotherapy-based recommendation decreased from 48% pre- to 34% post-assay (P=0.024). 92% of physicians agreed that they were more confident in their treatment recommendation after ordering the assay. CONCLUSIONS: These results suggest that use of the 21-gene assay in the Mexican public health system has a meaningful impact on adjuvant treatment recommendations that may reduce the overall use of chemotherapy.