BACKGROUND. Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) — a resistant subpopulation within a susceptible strain — is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS. Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.
Neil R.H. Stone, Johanna Rhodes, Matthew C. Fisher, Sayoki Mfinanga, Sokoine Kivuyo, Joan Rugemalila, Ella Shtifman Segal, Leor Needleman, Síle F. Molloy, June Kwon-Chung, Thomas S. Harrison, William Hope, Judith Berman, Tihana Bicanic
Chromosomal integration of genome-intact HIV-1 sequences into the host genome creates a reservoir of virally infected cells that persists throughout life, necessitating indefinite antiretroviral suppression therapy. During effective antiviral treatment, the majority of these proviruses remain transcriptionally silent, but mechanisms responsible for viral latency are insufficiently clear. Here, we used matched integration site and proviral sequencing (MIP-Seq), an experimental approach involving multiple displacement amplification of individual proviral species, followed by near-full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact and defective proviruses in 3 HIV-1–infected individuals undergoing long-term antiretroviral therapy. Simultaneously, chromatin accessibility and gene expression in autologous CD4+ T cells were analyzed by assays for transposase-accessible chromatin using sequencing (ATAC-Seq) and RNA-Seq. We observed that in comparison to proviruses with defective sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently showed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated in either relative proximity to or increased distance from active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be informative for targeting the genome-intact viral reservoir.
Kevin B. Einkauf, Guinevere Q. Lee, Ce Gao, Radwa Sharaf, Xiaoming Sun, Stephane Hua, Samantha M.Y. Chen, Chenyang Jiang, Xiaodong Lian, Fatema Z. Chowdhury, Eric S. Rosenberg, Tae-Wook Chun, Jonathan Z. Li, Xu G. Yu, Mathias Lichterfeld
Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications.
Luchang Zhu, Randall J. Olsen, Stephen B. Beres, Jesus M. Eraso, Matthew Ojeda Saavedra, Samantha L. Kubiak, Concepcion C. Cantu, Leslie Jenkins, Amelia R. L. Charbonneau, Andrew S. Waller, James M. Musser
Mast cells (MCs) are immune sentinels but whether they also function as antigen-presenting cells (APCs) remains elusive. Using mouse models of MC-deficiency, we report MC-dependent recruitment and activation of multiple T cell subsets to the skin and draining lymph nodes (LNs) during dengue virus (DENV) infection. Newly-recruited and locally-proliferating γδT cells were the first responding T cell subset to MC-driven inflammation and their production of IFN-γ was MC-dependent. MC-γδ T cell conjugates were observed consistently in infected peripheral tissues, suggesting a new role for MCs as non-conventional APCs for γδT cells. MC-dependent γδT cell activation and proliferation during DENV infection required TCR signaling and the non-conventional antigen presentation molecule EPCR on MCs. γδT cells, not previously implicated in DENV host defense, killed infected target dendritic cells and contributed to clearance of DENV in vivo. We believe immune synapse formation between MCs and γδT cells is a novel mechanism to induce specific and protective immunity at sites of viral infection.
Chinmay Kumar Mantri, Ashley L. St. John
X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic NEMO mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose three boys with EDA-ID died of immunodeficiency. We identify the same private variant in an intron of IKBKG/NEMO, IVS4+866 C>T, which was inherited from and occurred de novo in the European and Japanese mothers, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudo-exon generating a frameshift. Its leakiness accounts for NF-κB activation being impaired, but not abolished in the boys’ cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in iPSC-derived macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the pseudo-exon, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG/NEMO underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.
Bertrand Boisson, Yoshitaka Honda, Masahiko Ajiro, Jacinta Bustamante, Matthieu Bendavid, Andrew R. Gennery, Yuri Kawasaki, Jose Ichishima, Mitsujiro Osawa, Hiroshi Nihira, Takeshi Shiba, Takayuki Tanaka, Maya Chrabieh, Benedetta Bigio, Hong Hur, Yuval Itan, Yupu Liang, Satoshi Okada, Kazushi Izawa, Ryuta Nishikomori, Osamu Ohara, Toshio Heike, Laurent Abel, Anne Puel, Megumu K. Saito, Jean-Laurent Casanova, Masatoshi Hagiwara, Takahiro Yasumi
Long-lived HIV-1 reservoirs that persist despite antiretroviral therapy (ART) are a major impediment to a cure for HIV-1. We examined whether human liver macrophages (LMs), the largest tissue macrophage population, comprise an HIV-1 reservoir. We purified LMs from liver explants and included treatment with a T cell immunotoxin to reduce T cells to 1% or less. LMs were purified from 9 HIV-1–infected persons, 8 of whom were on ART (range 8–140 months). Purified LMs were stimulated ex vivo and supernatants from 6 of 8 LMs from persons on ART transmitted infection. However, HIV-1 propagation from LMs was not sustained except in LMs from 1 person taking ART for less than 1 year. Bulk liver sequences matched LM-derived HIV-1 in 5 individuals. Additional in vitro experiments undertaken to quantify the decay of HIV-1–infected LMs from 3 healthy controls showed evidence of infection and viral release for prolonged durations (>170 days). Released HIV-1 propagated robustly in target cells, demonstrating that viral outgrowth was observable using our methods. The t1/2 of HIV-1–infected LMs ranged from 3.8–55 days. These findings suggest that while HIV-1 persists in LMs during ART, it does so in forms that are inert, suggesting that they are defective or restricted with regard to propagation.
Abraham J. Kandathil, Sho Sugawara, Ashish Goyal, Christine M. Durand, Jeffrey Quinn, Jaiprasath Sachithanandham, Andrew M. Cameron, Justin R. Bailey, Alan S. Perelson, Ashwin Balagopal
Ferroptosis is a death program executed via selective oxidation of arachidonic acid–phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa–associated diseases such as CF and persistent lower respiratory tract infections.
Haider H. Dar, Yulia Y. Tyurina, Karolina Mikulska-Ruminska, Indira Shrivastava, Hsiu-Chi Ting, Vladimir A. Tyurin, James Krieger, Claudette M. St. Croix, Simon Watkins, Erkan Bayir, Gaowei Mao, Catherine R. Armbruster, Alexandr Kapralov, Hong Wang, Matthew R. Parsek, Tamil S. Anthonymuthu, Abiola F. Ogunsola, Becca A. Flitter, Cody J. Freedman, Jordan R. Gaston, Theodore R. Holman, Joseph M. Pilewski, Joel S. Greenberger, Rama K. Mallampalli, Yohei Doi, Janet S. Lee, Ivet Bahar, Jennifer M. Bomberger, Hülya Bayır, Valerian E. Kagan
Enterovirus 71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand-foot-and-mouth disease and life-threatening encephalitis. We identified an interferon-gamma (IFNγ)-inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pull-down, antibody/antigen blocking, and CRISPR/Cas9. Upon IFNγ treatment, induced hyperexpression and plasma membrane translocation of hWARS were observed, which sensitized semi-permissive (human neuronal NT2)/non-permissive (mouse fibroblast L929) cells to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to patients with severe EV-A71 infection. The expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important CV-A16 and EV-D68. This is the first report on the discovery of an entry factor, hWARS, which can be induced by IFNγ for EV-A71. Given that a high level of IFNγ was observed in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to the expression of entry factor upon IFNγ stimulation and the therapeutic options for treating severe EV-A71-associated complications.
Man Lung Yeung, Lilong Jia, Cyril C.Y. Yip, Jasper F.W. Chan, Jade L.L. Teng, Kwok-Hung Chan, Jian-Piao Cai, Chaoyu Zhang, Anna J. Zhang, Wan-Man Wong, Kin-Hang Kok, Susanna K.P. Lau, Patrick C.Y. Woo, Janice Y.C. Lo, Dong-Yan Jin, Shin-Ru Shih, Kwok-Yung Yuen
B cells are increasingly recognised to play an important role in the ongoing control of hepatitis B virus (HBV). The development of antibodies against the viral surface antigen (HBsAg) constitutes the hallmark of resolution of acute infection and is a therapeutic goal for functional cure of chronic HBV (CHB). We characterised B cells directly ex vivo from the blood and liver of patients with CHB to investigate constraints on their antiviral potential. Unexpectedly, we found that HBsAg-specific B cells persisted in the blood and liver of many patients with CHB and were enriched for T-bet, a signature of antiviral potential in B cells. However purified, differentiated HBsAg-specific B cells from patients with CHB had defective antibody production, consistent with undetectable anti-HBs antibodies in vivo. HBsAg-specific and global B cells had an accumulation of CD21–CD27– atypical memory B cells (atMBC) with high expression of inhibitory receptors including PD-1. These atMBC demonstrated altered signalling, homing, differentiation into antibody-producing cells, survival and antiviral/pro-inflammatory cytokine production, that could be partially rescued by PD-1 blockade. Analysis of B cells within healthy and HBV-infected livers implicated the combination of this tolerogenic niche and HBV infection in driving PD-1hiatMBC and impairing B cell immunity.
Alice R. Burton, Laura J. Pallett, Laura E. McCoy, Kornelija Suveizdyte, Oliver E. Amin, Leo Swadling, Elena Alberts, Brian R. Davidson, Patrick T.F. Kennedy, Upkar S. Gill, Claudia Mauri, Paul A. Blair, Nadege Pelletier, Mala K. Maini
Chronic HBV (CHB) infection suppresses virus-specific T cells, but its impact on humoral immunity has been poorly analyzed. Here, we developed a dual staining method, which utilizes HBsAg labelled with fluorochromes as “baits”, for specific ex vivo detection of HBsAg-specific B cells and analysis of their quantity, function and phenotype. We studied healthy vaccinated subjects (n=18) and patients with resolved (n=21), acute (n=11) or chronic (n=96) HBV infection and observed that frequencies of circulating HBsAg-specific B cells are independent of the HBV infection status. In contrast, serum HBsAg presence affects function and phenotype of HBsAg-specific B cells that were unable to mature in vitro into antibody-secreting cells and displayed an increased expression of markers linked to hyperactivation (CD21low) and exhaustion (PD-1). Importantly, B cell alterations were not limited to HBsAg-specific B cells but affected the global B cell population. HBsAg-specific B cell maturation could be partially restored by a method involving the combination of IL-2, IL-21 and CD40L-expressing feeder cells, and further boosted by addition of anti-PD-1 antibodies.In conclusion, HBV infection has a marked impact on global and HBV-specific humoral immunity, yet HBsAg-specific B cells are amenable to a partial rescue by B cell maturing cytokines and PD-1 blockade.
Loghman Salimzadeh, Nina Le Bert, Charles-A. Dutertre, Upkar S. Gill, Evan W. Newell, Christian Frey, Magdeleine Hung, Nikolai Novikov, Simon Fletcher, Patrick T.F. Kennedy, Antonio Bertoletti
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