Cell Host Microbe Research Articles

MSphere of Influence: Coordinating effective clearance of bacterial bloodstream infections.

Caitlyn Holmes works in the field of bacterial pathogenesis and host-pathogen interactions. In this mSphere of Influence article, she reflects on how the papers "Innate Lymphocyte/Ly6Chi Monocyte Crosstalk Promotes Klebsiella pneumoniae Clearance" by Xiong et al. (H. Xiong, J. W. Keith, D. W. Samilo, R. A. Carter, et al., Cell 165:679-89, 2016, https://doi.org/10.1016/j.cell.2016.03.017) and "Dual-Track Clearance of Circulating Bacteria Balances Rapid Restoration of Blood Sterility with Induction of Adaptive Immunity" by Broadley et al. (S. P. Broadley, A. Plaumann, R. Coletti, C. Lehmann, et al., Cell Host Microbe 20:36-48, 2016, https://doi.org/10.1016/j.chom.2016.05.023) impacted her research by highlighting the tangled web of immune responses that influence bacterial bloodstream infections.

Iron Deficiency Modulates Metabolic Landscape of Bacteroidetes Promoting Its Resilience during Inflammation.

Bacteria have to persist under low iron conditions in order to adapt to the nutritional immunity of a host. Since the knowledge of iron stimulon of Bacteroidetes is sparse, we examined oral (Porphyromonas gingivalis and Prevotella intermedia) and gut (Bacteroides thataiotaomicron) representatives for their ability to adapt to iron deplete and iron replete conditions. Our transcriptomics and comparative genomics analysis show that many iron-regulated mechanisms are conserved within the phylum. They include genes upregulated in low iron, as follows: fldA (flavodoxin), hmu (hemin uptake operon), and loci encoding ABC transporters. Downregulated genes were frd (ferredoxin), rbr (rubrerythrin), sdh (succinate dehydrogenase/fumarate reductase), vor (oxoglutarate oxidoreductase/dehydrogenase), and pfor (pyruvate:ferredoxin/flavodoxin oxidoreductase). Some genus-specific mechanisms, such as the sus of B. thetaiotaomicron coding for carbohydrate metabolism and the xusABC coding for xenosiderophore utilization were also identified. While all bacteria tested in our study had the nrfAH operon coding for nitrite reduction and were able to reduce nitrite levels present in culture media, the expression of the operon was iron dependent only in B. thetaiotaomicron. It is noteworthy that we identified a significant overlap between regulated genes found in our study and the B. thetaiotaomicron colitis study (W. Zhu, M. G. Winter, L. Spiga, E. R. Hughes et al., Cell Host Microbe 27:376-388, 2020, http://dx.doi.org/10.1016/j.chom.2020.01.010). Many of those commonly regulated genes were also iron regulated in the oral bacterial genera. Overall, this work points to iron being the master regulator enabling bacterial persistence in the host and paves the way for a more generalized investigation of the molecular mechanisms of iron homeostasis in Bacteroidetes. IMPORTANCE Bacteroidetes are an important group of anaerobic bacteria abundant both in the oral and gut microbiomes. Although iron is a required nutrient for most living organisms, the molecular mechanisms of adaptation to the changing levels of iron are not well known in this group of bacteria. We defined the iron stimulon of Bacteroidetes by examination of the transcriptomic response of Porphyromonas gingivalis and Prevotella intermedia (both belong to the oral microbiome) and Bacteroidetes thetaiotaomicron (belongs to the gut microbiome). Our results indicate that many of the iron-regulated operons are shared among the three genera. Furthermore, using bioinformatics analysis, we identified a significant overlap between our in vitro studies and transcriptomic data derived from a colitis study, thus underscoring the biological significance of our work. Defining the iron-dependent stimulon of Bacteroidetes can help to identify the molecular mechanisms of iron-dependent regulation as well as better understand the persistence of the anaerobes in the human host.

Always a bridesmaid, never a bride: Committing to flu neuraminidase as a vaccine target

Influenza A viruses pose an ongoing threat due to their role in seasonal epidemics and the potential for the emergence of novel viruses from the animal reservoir. 1 Kerstetter L.J. Buckley S. Bliss C.M. Coughlan L. Adenoviral vectors as vaccines for emerging avian influenza viruses. Front. Immunol. 2020; 11607333https://doi.org/10.3389/fimmu.2020.607333 Crossref PubMed Scopus (12) Google Scholar Extensive efforts are being made to develop vaccines that elicit protection against multiple influenza virus antigens, as well as universal vaccines that confer broad protection against antigenically distinct seasonal and/or pandemic viruses. In order to achieve long-lived and cross-protective immunity, it is considered that combining distinct vaccine platforms in heterologous prime:boost regimens may be required. 2 Coughlan L. Palese P. Overcoming barriers in the path to a universal influenza virus vaccine. Cell Host Microbe. 2018; 24: 18-24https://doi.org/10.1016/j.chom.2018.06.016 Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

Development of highly effective LCB1-based lipopeptides targeting the spike receptor-binding motif of SARS-CoV-2

LCB1 is a computationally designed 56-mer miniprotein targeting the spike (S) receptor-binding motif of SARS-CoV- 2 with high potent activity (Science, 2020; Cell host microbe, 2021); however, recent studies have demonstrated that emerging SARS-CoV-2 variants are highly resistant to LCB1's inhibition. In this study, we first identified a truncated peptide termed LCB1v8, which maintained the high antiviral potency. Then, a group of lipopeptides were generated by modifying LCB1v8 with diverse lipids, and of two lipopeptides, the C-terminally stearicacid-conjugtaed LCB1v17 and cholesterol-conjugated LCB1v18, were highly effective in inhibiting both S protein-pseudovirus and authentic SARS-CoV-2 infections. We further showed that LCB1-based inhibitors had similar α-helicity and thermostability in structure and bound to the target-mimic RBD protein with high affinity, and the lipopeptides exhibited greatly enhanced binding with the viral and cellular membranes, improved inhibitory activities against emerging SARS-CoV-2 variants. Moreover, LCB1v18 was validated with high preventive and therapeutic efficacies in K18-hACE2 transgenic mice against lethal SARS-CoV-2 challenge. In conclusion, our studies have provided important information for understanding the structure and activity relationship (SAR) of LCB1 inhibitor and would guide the future development of novel antivirals.

The benefit of vaccination after previous SARS-CoV-2 infection in the omicron era

The benefit of vaccination after previous SARS-CoV-2 infection in the omicron era

INFLAMMATION OVERDRIVE: A CONFIRMED DIAGNOSIS OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS RESULTING FROM COVID-19

INFLAMMATION OVERDRIVE: A CONFIRMED DIAGNOSIS OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS RESULTING FROM COVID-19

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Beyond neutralization for BNT162b2 mRNA vaccination

Mounting a robust immune response against SARS-CoV-2 requires neutralization as well as effector T cell functions. In this issue of Cell Host Microbe, Tauzin et al. characterize the humoral and T cell responses after a single dose of BNT162b2 mRNA vaccine in individuals with or without previous exposure to SARS-CoV-2.

Nutritional Support from the Intestinal Microbiota Improves Hematopoietic Reconstitution after Bone-Marrow Transplantation in Mice

Bone-marrow transplantation (BMT) offers curative potential for patients with high-risk hematologic malignancies, but the post-transplantation period is characterized by profound immunodeficiency. Delayed immune reconstitution is an important contributor to transplant-related morbidity and mortality (Maury et al., Br J Haematol, 2001; Small et al., Blood, 1999). Thus, understanding the mechanisms by which the immune system reconstitutes and how environmental exposures might influence this process is an important objective in improving outcomes after BMT.The intestinal microbiota shapes both mucosal and systemic immunity and contributes to steady-state hematopoiesis (Belkaid and Hand, Cell, 2014). To determine the role of the intestinal flora in hematopoietic reconstitution following BMT, we performed syngeneic BMT after lethal irradiation in mice with an intact intestinal flora (control, C/No abx) and in mice treated with two different antibiotic (abx) regimens administered in the drinking water: ampicillin + enrofloxacin (AmpEnro/AE) or vancomycin + amikacin (VancAmik/VA). While both regimens depleted the intestinal flora 1000-fold, ampicillin and enrofloxacin are both relatively well absorbed in the intestine while vancomycin and amikacin both have poor oral bioavailability with negligible systemic effects. BMT recipients treated with either of the abx regimens had lower white blood cell (WBC) counts and, in particular, lower lymphocyte counts compared to untreated BMT recipients (Fig 1A). Fecal microbiota transfer (FMT) with an abx-resistant intestinal flora (Caballero et al., Cell Host Microbe, 2017) rescued the impaired hematopoietic reconstitution in AE-treated mice (Fig 1B). These data rule out a direct inhibitory effect of abx on hematopoietic recovery. When we examined multiple compartments of the hematopoietic cascade we observed that flora depletion significantly impaired hematopoiesis specifically in the lymphoid and myeloid compartments of the bone marrow, thymus, and peripheral blood, while the hematopoietic stem and progenitor compartment and erythroid lineage were largely unaffected. These data are summarized in a radial plot (Fig 1C) in which each compartment of the hematopoietic cascade is a spoke and the distance from the center is proportional the cell count.Treatment of mice with AmpEnro or VancAmik also reduced dietary energy uptake (Fig 1D), resulting in reduction of visceral adipose tissue (Fig 1E). The mice are normally fed a standard chow diet based on complex plant carbohydrates. To test if reduced uptake of calories in flora-depleted animals contributed to impaired hematopoiesis post-BMT, we supplemented the drinking water with 5% sucrose, a carbohydrate source that can be directly utilized by the host in the absence of microbiota metabolism. Sucrose supplementation increased peripheral WBC counts and lymphocyte counts in AE-treated mice (Fig 1F), indicating that a critical function of the intestinal microbiota in supporting hematopoiesis post-BMT is to improve dietary energy uptake.We conclude that apart from specific and direct effects of the microbiota on immuno-hematopoiesis as previously described (Balmer et al., J Immunol, 2014; Clarke et al., Nat Med, 2010; Mazmanian et al., Cell, 2005), the intestinal flora can contribute to post-transplant hematopoietic reconstitution through improved dietary energy uptake. [Display omitted] DisclosuresPeled:Seres: Research Funding. Jenq:Seres: Research Funding. van den Brink:Therakos Institute: Other: Speaking engagement; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Seres: Research Funding.

MSphere of Influence: Forgotten Questions.

ABSTRACTLaura-Isobel McCall studies the relationship between location and disease pathogenesis, with a focus on infectious diseases and neglected diseases of poverty. In this mSphere of Influence article, she reflects on how three papers, “Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation” (A. Wang, S. C. Huen, H. H. Luan, S. Yu, et al., Cell 166:1512−1525.e12, 2016, https://doi.org/10.1016/j.cell.2016.07.026), “Three-dimensional microbiome and metabolome cartography of a diseased human lung” (N. Garg, M. Wang, E. Hyde, R. R. da Silva, et al., Cell Host Microbe 22:705−716.e4, 2017, https://doi.org/10.1016/j.chom.2017.10.001), and “‘It’s like a phantom disease’: patient perspectives on access to treatment for Chagas disease in the United States” (C. J. Forsyth, S. Hernandez, C. A. Flores, M. F. Roman, et al., Am J Trop Med Hyg 98:735−741, 2018, https://doi.org/10.4269/ajtmh.17-0691), shaped her spatial approach to infectious disease pathogenesis and helped her broaden her perspective from a pathogen-centric focus to a holistic view that include diseases tolerance mechanisms and barriers to health care access.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Isolation and characterization of cross-neutralizing coronavirus antibodies from COVID-19+ subjects.

SummarySARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

Dynamics of B cell repertoires and emergence of cross-reactive responses in patients with different severities of COVID-19.

SummaryIndividuals with the 2019 coronavirus disease (COVID-19) show varying severity of the disease, ranging from asymptomatic to requiring intensive care. Although monoclonal antibodies specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified, we still lack an understanding of the overall landscape of B cell receptor (BCR) repertoires in individuals with COVID-19. We use high-throughput sequencing of bulk and plasma B cells collected at multiple time points during infection to characterize signatures of the B cell response to SARS-CoV-2 in 19 individuals. Using principled statistical approaches, we associate differential features of BCRs with different disease severity. We identify 38 significantly expanded clonal lineages shared among individuals as candidates for responses specific to SARS-CoV-2. Using single-cell sequencing, we verify the reactivity of BCRs shared among individuals to SARS-CoV-2 epitopes. Moreover, we identify the natural emergence of a BCR with cross-reactivity to SARS-CoV-1 and SARS-CoV-2 in some individuals. Our results provide insights important for development of rational therapies and vaccines against COVID-19.

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

Risk of SARS-CoV-2 reinfection after natural infection

Risk of SARS-CoV-2 reinfection after natural infection

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.

Harnessing Tissue Engineering Tools to Interrogate Host-Microbiota Crosstalk in Cancer.

SummaryRecent studies have begun to highlight the diverse and tumor-specific microbiomes across multiple cancer types. We believe this work raises the important question of whether the classical “Hallmarks of Cancer” should be expanded to include tumor microbiomes. To answer this question, the causal relationships and co-evolution of these microbiotic tumor ecosystems must be better understood. Because host-microbe interactions should be studied in a physiologically relevant context, animal models have been preferred. Yet these models are often poor mimics of human tumors and are difficult to interrogate at high spatiotemporal resolution. We believe that in vitro tissue engineered platforms could provide a powerful alternative approach that combines the high-resolution of in vitro studies with a high degree of physiological relevance. This review will focus on tissue engineered approaches to study host-microbe interactions and to establish their role as an emerging hallmark of cancer with potential as a therapeutic target.