Added Diagnostic Utility of Clinical Metagenomics for the Diagnosis of Pneumonia in Immunocompromised Adults.

BACKGROUND: In the evaluation of community-acquired pneumonia, 30% to 60% of cases remain undiagnosed, despite extensive conventional microbiologic testing (CMT). Clinical metagenomics (CM) is an unbiased pathogen detection method that can increase diagnostic yield. RESEARCH QUESTION: Does adding clinical metagenomics to conventional microbiologic testing improve the diagnostic yield for pneumonia in immunocompromised adults? STUDY DESIGN AND METHODS: We performed a noninterventional prospective study of immunocompromised adults with pneumonia who underwent bronchoscopy and BAL over 2 years. CMT was performed per standard of care. A commercial CM test was performed on residual BAL fluid. Final microbiologic diagnoses were based on CMT vs CMT + CM. Final clinical diagnoses for CMT and CMT + CM were made based on laboratory results in conjunction with clinical and radiologic findings. Hypothetical impact of CMT + CM on management and antimicrobial stewardship was also assessed. RESULTS: A total of 30 immunocompromised adult patients (31 episodes of pneumonia) were included. Final microbiologic diagnoses were made in 11 cases (35%) with the use of CMT and in 18 cases (58%) with the use of CMT + CM. Bacterial pneumonia was diagnosed in five cases (16%) by CMT and in 13 cases (42%) by CMT + CM; fungal pneumonia was diagnosed in six cases (19%) by CMT and in seven cases (23%) by CMT + CM, and viral pneumonia was diagnosed in two cases (6%) by CMT and in five cases (16%) by CMT + CM. The hypothetical impact of CMT + CM on management was deemed probable in one case, possible in eight cases, and unlikely in two cases, whereas the impact on antimicrobial stewardship was possible in 13 cases and unlikely in seven cases. Final clinical diagnoses were made in 20 of 31 cases (65%) based on CMT and in 23 of 31 cases (74%) based on CMT + CM. INTERPRETATION: CMT + CM increased diagnostic yield in immunocompromised adults with pneumonia from 35% to 58%, mostly by the detection of additional bacterial causes but was less useful for fungal pneumonia.

miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2.

Human cytomegalovirus (HCMV) impacts more than one-half of the human population owing to its capacity to manipulate the cell and create latent reservoirs in the host. Despite an extensive understanding of HCMV biology during acute infection in fibroblasts, the molecular basis for latency in myeloid cells remains incomplete. This knowledge gap is due largely to the fact that the existing genetic systems require virus rescue in fibroblasts, precluding the study of genes that are essential during acute infection, yet likely play unique roles in myeloid cells or the establishment of latency. Here we present a solution to address this restriction. Through the exploitation of a hematopoietic-specific microRNA, we demonstrate a one-step recombineering approach that enables gene silencing only in cells associated with latency. As a proof of concept, here we describe a TB40/E variant that undergoes hematopoietic targeting of the Immediate Early-2 (IE2) gene to explore its function during infection of myeloid cells. While virus replication of the hematopoietic-targeted IE2 variant was unimpaired in fibroblasts, we observed a >100-fold increase in virus titers in myeloid cells. Virus replication in myeloid cells demonstrated that IE2 has a significant transcriptional footprint on both viral and host genes. These data implicate IE2 as an essential mediator of virus biology in myeloid cells and illustrate the utility of cell-specific microRNA-based targeting.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

C-TAK1 interacts with microphthalmia-associated transcription factor, Mitf, but not the related family member Tfe3.

Microphthalmia-associated transcription factor, Mitf, has been shown to be necessary for regulating genes involved in osteoclast differentiation. Previously it was shown by others that Mitf translocates from the cytoplasm to the nucleus upon M-CSF/RANKL signaling in osteoclasts. Mitf's movement is regulated by its interaction with 14-3-3 and the kinase C-TAK1. Here we demonstrate that the related family member, Tfe3, does not shuttle from the cytoplasm to the nucleus and does not interact with C-TAK1. We also demonstrate that overexpression of C-TAK1 inhibits the expression of Acp5 while a kinase dead C-TAK1 or a Mitf mutant that cannot interact with C-TAK1 increased expression of Acp5. Finally, we show that the catalytic subunit of protein phosphatase 2A is up-regulated in osteoclasts with M-CSF/RANKL signaling, indicating a possible mechanism for dephosphorylating Mitf on its 14-3-3 binding site and allowing Mitf to be translocated to the nucleus of osteoclasts.

The 19S proteasomal lid subunit POH1 enhances the transcriptional activation by Mitf in osteoclasts.

The microphthalmia-associated transcription factor (Mitf) regulates gene expression required for osteoclast differentiation. Genes regulated by Mitf have been previously identified. However, proteins that interact and regulate Mitf's activity in osteoclasts are not well known. Here, we report that POH1, a subunit of the 19S proteasome lid is a regulator of Mitf. We show that POH1 and Mitf interact in osteoclasts and that this interaction is dependent on RANKL signaling. Overexpression of POH1 increased Mitf's activation of 5XGal4-TK and Acp5 promoters. The amino terminus of POH1 mediates the binding to Mitf and is sufficient to increase Mitf's transcriptional activity. Finally, we show that mutations in the JAMM motif of POH1 reduced Mitf activation of promoters. In summary, our results identify a novel mechanism of Mitf regulation in osteoclasts by POH1.

Enhanced osteoclastogenesis causes osteopenia in twisted gastrulation-deficient mice through increased BMP signaling.

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1-deficient (Twsg1(-/-)) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1(-/-) mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1(-/-) mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL-mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1(-/-) mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1(-/-) mice was reversed in vitro in a dose-dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.