Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199–3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250–258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

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.

mSphere of Influence: Experimental Evolution of RNA Viruses

ABSTRACT Gonzalo Moratorio works in the field of experimental evolution of viruses. In this mSphere of Influence article, he reflects on how the papers “Virus attenuation by genome-scale changes in codon pair bias” by Coleman et al. (Science 320:1784–1787, 2008, https://doi.org/10.1126/science.1155761) and “Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus” by Lauring et al. (Cell Host Microbe 12:623–632, 2012, https://doi.org/10.1016/j.chom.2012.10.008) made an impact on his thinking about how to employ synthetic biology to study experimental evolution of viruses.

mSphere of Influence: Decoding Transcriptional Regulatory Networks To Illuminate the Mechanisms of Microbial Pathogenicity

ABSTRACT Sadri Znaidi works in the field of molecular mycology with a focus on functional genomics in Candida albicans. In this mSphere of Influence article, he reflects on how the paper “An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis” by Chen et al. (C. Chen, K. Pande, S. D. French, B. B. Tuch, and S. M. Noble, Cell Host Microbe 10:118–135, 2011, https://doi.org/10.1016/j.chom.2011.07.005) made an impact on his research on how transcriptional regulatory networks function to control C. albicans’ ability to efficiently interact with the host environment.

FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells

ABSTRACT The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368–380, 2016, https://doi.org/10.1016/j.chom.2016.07.015 ) and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lütgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018–9028, 2016, https://doi.org/10.1128/JVI.01448-16 ) reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio, Grau-Expósito et al. (J. Grau-Expósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaña, E. Ribera, A. Torrella, B. Planas, R. Badía, J. Castellví, V. Falcó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17 !) reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication.

Metagenomic shotgun sequencing to identify specific human gut microbes associated with immune checkpoint therapy efficacy in melanoma patients.

9516 Background: Immune checkpoint inhibitor therapy, ICT, achieves durable remissions in 30-50% of patients (pts) with metastatic melanoma (Larkin et al. NEJM 2015). It is still unclear what host factors modulate response to ICT. Preclinical mouse studies with B16 melanoma demonstrated that ICT response was dependent on the presence of specific commensal gut bacteria (Vetizou et al. Science 2015; Sivan et al. Science2015). These specific gut bacteria induced the maturation of dendritic cells (DCs) and T-cells needed for effective ICT. We sought to determine whether specific gut microbiota are associated with improved response to ICT in melanoma patients. Methods: 37 melanoma pts treated with ICT (nivolumab plus ipilimumab or pembrolizumab alone) at UTSW Medical Center were enrolled. Fecal samples were collected prior to ICT. Genomic DNA was extracted, and metagenomic shotgun sequencing (MSS) performed on an Illumina HiSeq 2500 PE-100. Taxonomic (MetaPhlAn) and functional (HUMAnN) analysis was performed on MSS data. Disease status was assessed by CT scans and physical exams every two months. Results: Among the 23 evaluable pts, 8 were classified as RECIST responders, 5 with stable disease and 10 with progression. RECIST responder microbiomes were significantly enriched with Methanobrevibacter smithii(p = 0.03; LDA coupled with effect size measurements, LEfSe; Kruskal-Wallis test) , Bacteroides thetaiotamicron(p = 0.03) , Lactobacillus plantarum(p = 0.04), and Eubacterium limosum(p = 0.01) compared to those with progressive disease. Conclusions: MSS identified 4 specific gut microbiota associated with improved response to ICT therapy in melanoma pts. All of these bacteria have been shown to modulate host immune response (Bang PLoS One 2014; Hickey Cell Host Microbe 2016; Rigaux Allergy 2009; Kaunachi World J Gastroentertol 2006). To gain mechanistic insight and confirm causality, shotgun metabolomics on the same fecal specimens used for MSS, in vitroimmune cell assays using the gut microbiota identified, and preclinical modeling in a mouse melanoma model with ICT are underway. These studies may lay the foundation for optimizing the host response to ICT.

Targeting Iron/Heme in Infectious Disease

Inflammation and immunity can be associated with varying degrees of hemolysis and as such with heme release from cell-free hemoglobin (Hb), i.e. free heme. Accumulation of free heme in plasma can lead, eventually, to tissue iron (Fe) overload, oxidative stress, and tissue damage. Presumably, these deleterious effects contribute critically to the pathogenesis of systemic infections, as illustrated for severe sepsis (1) as well as for severe forms of malaria (2). Free heme sensitizes parenchyma cells to undergo programmed cell death in response to pro-inflammatory agonists (1). This cytotoxic effect is driven by the intracellular accumulation of free radicals, which sustain the activation of the c-Jun N-terminal Kinase (JNK) signaling transduction pathway. While heme catabolism by heme oxygenase1 (HO-1) prevents heme driven programmed cell death, this cytoprotective effect requires the co-expression of ferritin H chain (FTH), which controls the pro-oxidant labile Fe released from the protoporphyrin IX ring of heme (3). The antioxidant effect of FTH restrains JNK activation, while JNK activation inhibits FTH expression, a crosstalk that controls metabolic adaptation to cellular Fe overload during systemic infections (4). 1. Larsen, R. et al., Sci Transl Med 2 (51), 51ra71 (2010). 2. Pamplona, A. et al., Nat Med 13 (6), 703 (2007); Ferreira, A. et al., J Mol Med 86 (10), 1097 (2008); Ferreira, A. et al., Cell 145 (April 29), 398 (2011). 3. Gozzelino, R. et al., Cell Host Microbe 12 (5), 693 (2012) 4.Medzhitov, R, Schneider, DS, and Soares. Science 335, 936 (2012). Disclosures: No relevant conflicts of interest to declare.

RNA Structures Required for Production of Subgenomic Flavivirus RNA

ABSTRACT Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNVKUN) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.