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Burkholderia ubonensis Meropenem Resistance: Insights into Distinct Properties of Class A {beta}-Lactamases in Burkholderia cepacia Complex and Burkholderia pseudomallei Complex Bacteria

ABSTRACT

Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB β-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A β-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a β-lactamase. Regulated expression of a transcriptional penB'-lacZ (β-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible β-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from β-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.

IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis β-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A β-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.




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A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.




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Targeting Hidden Pathogens: Cell-Penetrating Enzybiotics Eradicate Intracellular Drug-Resistant Staphylococcus aureus

ABSTRACT

Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

IMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.




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Erratum for Teymournejad et al., "Isolation and Molecular Analysis of a Novel Neorickettsia Species That Causes Potomac Horse Fever"




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Human Serum Albumin Facilitates Heme-Iron Utilization by Fungi

ABSTRACT

A large portion of biological iron is found in the form of an iron-protoporphyrin IX complex, or heme. In the human host environment, which is exceptionally poor in free iron, heme iron, particularly from hemoglobin, constitutes a major source of iron for invading microbial pathogens. Several fungi were shown to utilize free heme, and Candida albicans, a major opportunistic pathogen, is able both to capture free heme and to extract heme from hemoglobin using a network of extracellular hemophores. Human serum albumin (HSA) is the most abundant host heme-scavenging protein. Tight binding of heme by HSA restricts its toxic chemical reactivity and could diminish its availability as an iron source for pathogenic microbes. We found, however, that rather than inhibiting heme utilization, HSA greatly increases availability of heme as an iron source for C. albicans and other fungi. In contrast, hemopexin, a low-abundance but high-affinity heme-scavenging serum protein, does inhibit heme utilization by C. albicans. However, inhibition by hemopexin is mitigated in the presence of HSA. Utilization of albumin-bound heme requires the same hemophore cascade as that which mediates hemoglobin-iron utilization. Accordingly, we found that the C. albicans hemophores are able to extract heme bound to HSA in vitro. Since many common drugs are known to bind to HSA, we tested whether they could interfere with heme-iron utilization. We show that utilization of albumin-bound heme by C. albicans can be inhibited by the anti-inflammatory drugs naproxen and salicylic acid.

IMPORTANCE Heme constitutes a major iron source for microorganisms and particularly for pathogenic microbes; to overcome the iron scarcity in the animal host, many pathogenic bacteria and fungi have developed systems to extract and take up heme from host proteins such as hemoglobin. Microbial heme uptake mechanisms are usually studied using growth media containing free heme or hemoglobin as a sole iron source. However, the animal host contains heme-scavenging proteins that could prevent this uptake. In the human host in particular, the most abundant serum heme-binding protein is albumin. Surprisingly, however, we found that in the case of fungi of the Candida species family, albumin promoted rather than prevented heme utilization. Albumin thus constitutes a human-specific factor that can affect heme-iron utilization and could serve as target for preventing heme-iron utilization by fungal pathogens. As a proof of principle, we identify two drugs that can inhibit albumin-stimulated heme utilization.




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Simian Immunodeficiency Virus-Infected Memory CD4+ T Cells Infiltrate to the Site of Infected Macrophages in the Neuroparenchyma of a Chronic Macaque Model of Neurological Complications of AIDS

ABSTRACT

Simian immunodeficiency virus (SIV)-infected nonhuman primates can serve as a relevant model for AIDS neuropathogenesis. Current SIV-induced encephalitis (SIVE)/neurological complications of AIDS (neuroAIDS) models are generally associated with rapid progression to neuroAIDS, which does not reflect the tempo of neuroAIDS progression in humans. Recently, we isolated a neuropathogenic clone, SIVsm804E-CL757 (CL757), obtained from an SIV-infected rhesus macaque (RM). CL757 causes a more protracted progression to disease, inducing SIVE in 50% of inoculated animals, with high cerebral spinal fluid viral loads, multinucleated giant cells (MNGCs), and perivascular lymphocytic cuffing in the central nervous system (CNS). This latter finding is reminiscent of human immunodeficiency virus (HIV) encephalitis in humans but not generally observed in rapid progressor animals with neuroAIDS. Here, we studied which subsets of cells within the CNS were targeted by CL757 in animals with neurological symptoms of SIVE. Immunohistochemistry of brain sections demonstrated infiltration of CD4+ T cells (CD4) and macrophages (Ms) to the site of MNGCs. Moreover, an increase in mononuclear cells isolated from the brain tissues of RMs with SIVE correlated with increased cerebrospinal fluid (CSF) viral load. Subset analysis showed a specific increase in brain CD4+ memory T cells (Br-mCD4), brain-Ms (Br-Ms), and brain B cells (Br-B cells). Both Br-mCD4s and Br-Ms harbored replication-competent viral DNA, as demonstrated by virus isolation by coculture. However, only in animals exhibiting SIVE/neuroAIDS was virus isolated from Br-Ms. These findings support the use of CL757 to study the pathogenesis of AIDS viruses in the central nervous system and indicate a previously unanticipated role of CD4s cells as a potential reservoir in the brain.

IMPORTANCE While the use of combination antiretroviral therapy effectively suppresses systemic viral replication in the body, neurocognitive disorders as a result of HIV infection of the central nervous system (CNS) remain a clinical problem. Therefore, the use of nonhuman primate models is necessary to study mechanisms of neuropathogenesis. The neurotropic, molecular clone SIVsm804E-CL757 (CL757) results in neuroAIDS in 50% of infected rhesus macaques approximately 1 year postinfection. Using CL757-infected macaques, we investigate disease progression by examining subsets of cells within the CNS that were targeted by CL757 and could potentially serve as viral reservoirs. By isolating mononuclear cells from the brains of SIV-infected rhesus macaques with and without encephalitis, we show that immune cells invade the neuroparenchyma and increase in number in the CNS in animals with SIV-induced encephalitis (SIVE). Of these cells, both brain macrophages and brain memory CD4+ T cells harbor replication-competent SIV DNA; however, only brain CD4+ T cells harbored SIV DNA in animals without SIVE. These findings support use of CL757 as an important model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.




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Temporal Dynamics of the Adult Female Lower Urinary Tract Microbiota

ABSTRACT

Temporal dynamics of certain human microbiotas have been described in longitudinal studies; variability often relates to modifiable factors or behaviors. Early studies of the urinary microbiota preferentially used samples obtained by transurethral catheterization to minimize vulvovaginal microbial contributions. Whereas voided specimens are preferred for longitudinal studies, the few studies that reported longitudinal data were limited to women with lower urinary tract (LUT) symptoms, due to ease of accessing a clinical population for sampling and the impracticality and risk of collecting repeated catheterized urine specimens in a nonclinical population. Here, we studied the microbiota of the LUT of nonsymptomatic, premenopausal women using midstream voided urine (MSU) specimens to investigate relationships between microbial dynamics and personal factors. Using 16S rRNA gene sequencing and a metaculturomics method called expanded quantitative urine culture (EQUC), we characterized the microbiotas of MSU and periurethral swab specimens collected daily for approximately 3 months from a small cohort of adult women. Participants were screened for eligibility, including the ability to self-collect paired urogenital specimens prior to enrollment. In this population, we found that measures of microbial dynamics related to specific participant-reported factors, particularly menstruation and vaginal intercourse. Further investigation of the trends revealed differences in the composition and diversity of LUT microbiotas within and across participants. These data, in combination with previous studies showing relationships between the LUT microbiota and LUT symptoms, suggest that personal factors relating to the genitourinary system may be an important consideration in the etiology, prevention, and/or treatment of LUT disorders.

IMPORTANCE Following the discovery of the collective human urinary microbiota, important knowledge gaps remain, including the stability and variability of this microbial niche over time. Initial urinary studies preferentially utilized samples obtained by transurethral catheterization to minimize contributions from vulvovaginal microbes. However, catheterization has the potential to alter the urinary microbiota; therefore, voided specimens are preferred for longitudinal studies. In this report, we describe microbial findings obtained by daily assessment over 3 months in a small cohort of adult women. We found that, similarly to vaginal microbiotas, lower urinary tract (LUT) microbiotas are dynamic, with changes relating to several factors, particularly menstruation and vaginal intercourse. Our study results show that LUT microbiotas are both dynamic and resilient. They also offer novel opportunities to target LUT microbiotas by preventative or therapeutic means, through risk and/or protective factor modification.




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Ehrlichia chaffeensis Uses an Invasin To Suppress Reactive Oxygen Species Generation by Macrophages via CD147-Dependent Inhibition of Vav1 To Block Rac1 Activation

ABSTRACT

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C–DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.

IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.




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Tracing the Evolutionary History and Global Expansion of Candida auris Using Population Genomic Analyses

ABSTRACT

Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

IMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.




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APOBEC3C Tandem Domain Proteins Create Super Restriction Factors against HIV-1

ABSTRACT

Humans encode proteins, called restriction factors, that inhibit replication of viruses such as HIV-1. The members of one family of antiviral proteins, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; shortened here to A3), act by deaminating cytidines to uridines during the reverse transcription reaction of HIV-1. The A3 locus encodes seven genes, named A3A to A3H. These genes have either one or two cytidine deaminase domains, and several of these A3s potently restrict HIV-1. A3C, which has only a single cytidine deaminase domain, however, inhibits HIV-1 only very weakly. We tested novel double domain protein combinations by genetically linking two A3C genes to make a synthetic tandem domain protein. This protein created a "super restriction factor" that had more potent antiviral activity than the native A3C protein, which correlated with increased packaging into virions. Furthermore, disabling one of the active sites of the synthetic tandem domain protein resulted in an even greater increase in the antiviral activity—recapitulating a similar evolution seen in A3F and A3G (double domain A3s that use only a single catalytically active deaminase domain). These A3C tandem domain proteins do not have an increase in mutational activity but instead inhibit formation of reverse transcription products, which correlates with their ability to form large higher-order complexes in cells. Finally, the A3C-A3C super restriction factor largely escaped antagonism by the HIV-1 viral protein Vif.

IMPORTANCE As a part of the innate immune system, humans encode proteins that inhibit viruses such as HIV-1. These broadly acting antiviral proteins do not protect humans from viral infections because viruses encode proteins that antagonize the host antiviral proteins to evade the innate immune system. One such example of a host antiviral protein is APOBEC3C (A3C), which weakly inhibits HIV-1. Here, we show that we can improve the antiviral activity of A3C by duplicating the DNA sequence to create a synthetic tandem domain and, furthermore, that the proteins thus generated are relatively resistant to the viral antagonist Vif. Together, these data give insights about how nature has evolved a defense against viral pathogens such as HIV.




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Optimization of an Experimental Vaccine To Prevent Escherichia coli Urinary Tract Infection

ABSTRACT

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship.

IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.




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Novel Divisome-Associated Protein Spatially Coupling the Z-Ring with the Chromosomal Replication Terminus in Caulobacter crescentus

ABSTRACT

Cell division requires proper spatial coordination with the chromosome, which undergoes dynamic changes during chromosome replication and segregation. FtsZ is a bacterial cytoskeletal protein that assembles into the Z-ring, providing a platform to build the cell division apparatus. In the model bacterium Caulobacter crescentus, the cellular localization of the Z-ring is controlled during the cell cycle in a chromosome replication-coupled manner. Although dynamic localization of the Z-ring at midcell is driven primarily by the replication origin-associated FtsZ inhibitor MipZ, the mechanism ensuring accurate positioning of the Z-ring remains unclear. In this study, we showed that the Z-ring colocalizes with the replication terminus region, located opposite the origin, throughout most of the C. crescentus cell cycle. Spatial organization of the two is mediated by ZapT, a previously uncharacterized protein that interacts with the terminus region and associates with ZapA and ZauP, both of which are part of the incipient division apparatus. While the Z-ring and the terminus region coincided with the presence of ZapT, colocalization of the two was perturbed in cells lacking zapT, which is accompanied by delayed midcellular positioning of the Z-ring. Moreover, cells overexpressing ZapT showed compromised positioning of the Z-ring and MipZ. These findings underscore the important role of ZapT in controlling cell division processes. We propose that ZapT acts as a molecular bridge that physically links the terminus region to the Z-ring, thereby ensuring accurate site selection for the Z-ring. Because ZapT is conserved in proteobacteria, these findings may define a general mechanism coordinating cell division with chromosome organization.

IMPORTANCE Growing bacteria require careful tuning of cell division processes with dynamic organization of replicating chromosomes. In enteric bacteria, ZapA associates with the cytoskeletal Z-ring and establishes a physical linkage to the chromosomal replication terminus through its interaction with ZapB-MatP-DNA complexes. However, because ZapB and MatP are found only in enteric bacteria, it remains unclear how the Z-ring and the terminus are coordinated in the vast majority of bacteria. Here, we provide evidence that a novel conserved protein, termed ZapT, mediates colocalization of the Z-ring with the terminus in Caulobacter crescentus, a model organism that is phylogenetically distant from enteric bacteria. Given that ZapT facilitates cell division processes in C. crescentus, this study highlights the universal importance of the physical linkage between the Z-ring and the terminus in maintaining cell integrity.




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Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone

ABSTRACT

All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the "decorations" of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called "EPA decorations" consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor.

IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called "EPA decorations" essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.




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Advances in Understanding the Human Urinary Microbiome and Its Potential Role in Urinary Tract Infection

ABSTRACT

Recent advances in the analysis of microbial communities colonizing the human body have identified a resident microbial community in the human urinary tract (UT). Compared to many other microbial niches, the human UT harbors a relatively low biomass. Studies have identified many genera and species that may constitute a core urinary microbiome. However, the contribution of the UT microbiome to urinary tract infection (UTI) and recurrent UTI (rUTI) pathobiology is not yet clearly understood. Evidence suggests that commensal species within the UT and urogenital tract (UGT) microbiomes, such as Lactobacillus crispatus, may act to protect against colonization with uropathogens. However, the mechanisms and fundamental biology of the urinary microbiome-host relationship are not understood. The ability to measure and characterize the urinary microbiome has been enabled through the development of next-generation sequencing and bioinformatic platforms that allow for the unbiased detection of resident microbial DNA. Translating technological advances into clinical insight will require further study of the microbial and genomic ecology of the urinary microbiome in both health and disease. Future diagnostic, prognostic, and therapeutic options for the management of UTI may soon incorporate efforts to measure, restore, and/or preserve the native, healthy ecology of the urinary microbiomes.




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Minnesota association acknowledges states ancestral lands, residents

In a nod to the people who came before them — and those who still live among them — the Minnesota Public Health Association is acknowledging ancestral lands.




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Building local connections could help reduce violent encounters between police, black men

Finding common ground and building trust between local stakeholders could help prevent violent encounters between police and young black men, new research finds.




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Many people face high risk of PTSD after being injured, research finds

A significant number of injury survivors experience post-traumatic stress disorder, and better screening practices could help connect them to mental health services.




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We need equity-oriented solutions to COVID-19: Asians facing stigma, discrimination, fear during pandemic

APHA member Elena Ong, PHN, MS, past president and founding CEO of the Asian & Pacific Islander Caucus for Public Health, a recent APHA Executive Board member, and a past vice president of the Southern California Public Health Association, discusses discrimination against Asians in the U.S. and beyond.




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CDC: Vaccine for recent flu season cut visits to doctors by nearly half

This season’s flu vaccine was 45% effective overall and 55% effective among children and teens, the Centers for Disease Control and Prevention reported in February.




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Long-acting, Injectable Buprenorphine: Great Promise, but Significant Barriers to Use

To the Editor—A 30-day injectable form of buprenorphine branded as SublocadeTM (Buprenorphine XR SQ) was approved by the FDA in 2017. This medication is administered by a health care professional subcutaneously in the abdomen to treat opioid use disorder. This long-acting delivery system holds great promise for many patients who have barriers to taking daily transmucosal buprenorphine-containing medications such as those with poor adherence to a daily medication. It is beneficial for those who have difficulty safely storing their medications, including patients who have children in the home, unstable housing, or live with others who have a use disorder. This product is also an option for patients who prefer mono-product buprenorphine. As Buprenorphine XR SQ is administered directly by a health care professional, it does not contain the abuse-deterrent naloxone that some patients feel causes side effects.

There are two ways to acquire Buprenorphine XR SQ: 1) order product from the distributor (buy and bill); or 2) dispensed from a specialty pharmacy for a specific patient (specialty pharmacy) [1]. For the buy and bill option, the health care setting must be certified through the Risk Evaluation and Mitigation Strategy (REMS) program and adhere to dispensing regulations [2]. We found this challenging to implement in the outpatient setting, thus we pursued the specialty pharmacy option. It ultimately took us nearly one year to complete the process.

The following are the barriers we faced with our first attempt. As a controlled substance, the medication must be stored in a refrigerated lockbox. Before...




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A Call to Action for Philanthropy in North Carolina Health Care

The conversation about how we create and maintain health has evolved. We have now clearly expanded our thinking beyond an exclusive focus on traditional medical care, and philanthropy can play an important role




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A Call to Action for North Carolina Legislators on Improving Access to Health

To better the health of all North Carolinians, policymakers must come together to improve access to care, expand broadband, and close the coverage gap.




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Historical Geography and Health Equity: An Exploratory View of North Carolina Slavery and Sociohealth Factors

Current health inequities are rooted in more than simple systems failures and inefficiencies. Historical legacy has corrupted health outcomes, and resolution requires both acknowledgment and intention.




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Moving Upstream to Impact Health: Building a Physician Workforce that Understands Social Determinants

Decades of rallying cries from professional societies, medical education and training programs, and government stakeholders have distilled the conversation of social determinants of health (SDOH) from theoretical proposals into practical solutions [1-3]. No longer standing on the precipice of change, we are now in the trenches. The nation's health care system recognizes SDOH as important drivers of health and is taking steps to address them in the practice environment.

More widespread action and attention by the health care system drives the need to train the next generation of physicians in the concepts and actions related to SDOH. This includes SDOH as a core part of the medical curriculum, offering clinical and research experiences and service in the community [4-5]. Unfortunately, to date only a handful of programs have brought this vision to fruition. Across the country, most programs offer educational content that is largely didactic and provided in short or one-time sessions [6]. Though a start, such approaches are insufficient to prepare the next generation of physicians for their important work ahead.

In New Orleans, the NOLA Hotspotters are an interdisciplinary group of medical, public health, nursing, and pharmacy students inspired by the work out of Camden, New Jersey, to "hot spot" patients with high utilization, which is often related to social needs [7]. While the results of the Camden program have been widely discussed following publication of their work, we argue the benefit of such a program exists beyond reduced emergency department visits or health care spending [8]. The...




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Place Matters: From Health and Health Care Disparities to Equity and Liberation

Place—a confluence of the social, economic, political, physical, and built environments—is fundamental to our understanding of health and health inequities among marginalized racial groups in the United States. Moreover, racism, defined as a system of structuring opportunity and assigning value based on the social interpretation of how one looks (i.e., race), has shaped the places people live in North Carolina. This problem is deeply imbedded in all of our systems, from housing to health care, affecting the ability of every resident of the state to flourish and thrive.




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Gathering Trauma Narratives: A Qualitative Study on the Impact of Self-Identified Traumas on People Living with HIV (PLWH)

BACKGROUND Trauma—emotional, physical, and psychological—is common and associated with increased risk behaviors, low rates of care engagement and viral suppression, and overall poor health outcomes for people living with HIV (PLWH). This article presents the results of 15 in-depth, semi-structured interviews with PLWH in the Southeastern United States in which participants identified a trauma and described its long-lasting impact on their lives. Participants' trauma narratives described a wide range of traumas, including childhood sexual abuse, the loss of a loved one, and their HIV diagnosis.

METHODS Systematic qualitative analysis was used to delineate beliefs about causes, symptoms, treatments, quality of life, and health implications of trauma.

RESULTS: Fifteen participants completed semi-structured interviews that lasted on average 32 minutes. Participants described a wide spectrum of personal trauma that occurred both prior and subsequent to their HIV diagnosis. The types of trauma identified included physical, sexual, and psychological abuse inflicted by intimate partners, family members, and/or strangers.

LIMITATIONS A chief limitation of this study is selection bias. Additionally, the participant selection and content of the trauma narratives might have been affected by the surrounding context of the parent study centered on HIV, aging, and psychosocial stress. It is also difficult to interpret the distinction between discrete trauma experiences and the diagnosis of HIV, leading to potential information bias.

CONCLUSION This study highlights the importance of social support in coping with trauma and the effect of trauma on health-related behaviors. It also illustrates the need for additional research on the topic of trauma and trauma-informed care for PLWH. Understanding how different types of trauma affect individuals' lives is necessary to inform recommendations to provide better care for PLWH.




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Acute encephalopathy after head trauma in a patient with a RHOBTB2 mutation

Objective

De novo missense mutations in the RHOBTB2 gene have been described as causative for developmental and epileptic encephalopathy.

Methods

The clinical phenotype of this disorder includes early-onset epilepsy, severe intellectual disability, postnatal microcephaly, and movement disorder. Three RHOBTB2 patients have been described with acute encephalopathy and febrile epileptic status. All showed severe EEG abnormalities during this episode and abnormal MRI with hemisphere swelling or reduced diffusion in various brain regions.

Results

We describe the episode of acute encephalopathy after head trauma in a 5-year-old RHOBTB2 patient. At admission, Glasgow coma scale score was E4M4V1. EEG was severely abnormal showing a noncontinuous pattern with slow activity without epileptic activity indicating severe encephalopathy. A second EEG on day 8 was still severely slowed and showed focal delta activity frontotemporal in both hemispheres. Gradually, he recovered, and on day 11, he had regained his normal reactivity, behavior, and mood. Two months after discharge, EEG showed further decrease in slow activity and increase in normal electroencephalographic activity. After discharge, parents noted that he showed more hyperkinetic movements compared to before this period of encephalopathy. Follow-up MRI showed an increment of hippocampal atrophy. In addition, we summarize the clinical characteristics of a second RHOBTB2 patient with increase of focal periventricular atrophy and development of hemiparesis after epileptic status.

Conclusions

Acute encephalopathy in RHOBTB2 patients can also be triggered by head trauma.




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Biallelic LINE insertion mutation in HACD1 causing congenital myopathy

Congenital myopathies are clinically and genetically heterogeneous, resulting from mutations in at least 30 different genes.1 The classical presentation is neonatal hypotonia and nonprogressive weakness with normal creatine phosphokinase, although there is a broad range in terms of age at onset and clinical presentation. Historically, congenital myopathies have been defined and diagnosed based on muscle biopsy. However, with advances in genomics, genetics have taken primacy in the diagnostic pathway.2




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Phenotypic variability in chorea-acanthocytosis associated with novel VPS13A mutations

Objective

To perform a comprehensive characterization of a cohort of patients with chorea-acanthocytosis (ChAc) in Sweden.

Methods

Clinical assessments, targeted genetic studies, neuroimaging with MRI, [18F]-fluorodeoxyglucose (FDG) PET, and dopamine transporter with 123I FP-CIT (DaTscan) SPECT. One patient underwent magnetic resonance spectroscopy (MRS).

Results

Four patients living in Sweden but with different ethnical backgrounds were included. Their clinical features were variable. Biallelic VPS13A mutations were confirmed in all patients, including 3 novel mutations. All tested patients had either low or absent chorein levels. One patient had progressive caudate atrophy. Investigation using FDG-PET revealed severe bilateral striatal hypometabolism, and DaTscan SPECT displayed presynaptic dopaminergic deficiency in 3 patients. MRS demonstrated reduced N-acetylaspartate/creatine (Cr) ratio and mild elevation of both choline/Cr and combined glutamate and glutamine/Cr in the striatum in 1 case. One patient died during sleep, and another was treated with deep brain stimulation, which transiently attenuated feeding dystonia but not his gait disorder or chorea.

Conclusions

Larger longitudinal neuroimaging studies with different modalities, particularly MRS, are needed to determine their potential role as biomarkers for ChAc.




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A new cheiracanthid acanthodian from the Middle Devonian (Givetian) Orcadian Basin of Scotland and its biostratigraphic and biogeographical significance

A number of partial articulated specimens of Cheiracanthus peachi nov. sp. have been collected from the Mey Flagstone Formation and Rousay Flagstone Formation within the Orcadian Basin of northern Scotland. The new, robust-bodied species is mainly distinguished by the scale ornament of radiating grooves rather than ridges. Compared to other Cheiracanthus species in the Orcadian Basin, C. peachi nov. sp. has quite a short range making it a useful zone fossil. As well as describing the general morphology of the specimens, we have also described and figured SEM images of scales and histological sections of all elements, enabling identification of other, isolated remains. Of particular biological interest is the identification of relatively robust, tooth-like gill rakers. Finally, the species has also been identified from isolated scales in Belarus, where it appears earlier and has a longer stratigraphical range, implying the species evolved in the marine deposits of the east and migrated west into the Orcadian Basin via the river systems.




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Very large convergent multi-fluted glacigenic deposits in the NW Highlands, Scotland

We describe two large convergent multi-fluted glacigenic deposits in the NW Highlands, Scotland, and point out their resemblance to a number of landforms emerging from presently deglaciating areas of Greenland and Antarctica. We suggest that they all result from locally sourced sediment being deposited by local ice-flow, which was laterally confined by the margins of much larger adjacent glaciers or ice-streams. The NW Highlands features thus seem likely to be the result of processes active during the latter part of the Devensian Glaciation. One of these deposits, on the peninsula between Loch Broom and Little Loch Broom, is evidently sourced from the west-facing Coire Dearg of Beinn Ghobhlach, but was emplaced in a WNW direction rather than along the WSW fall-line. This suggests that the ice that emplaced it was confined by the margins of large glaciers then occupying the adjacent valleys of Loch Broom and Little Loch Broom. The second much larger and more prominent deposit, in Applecross, is composed of bouldery Torridonian sandstone till emplaced on to glacially scoured bedrock; the only feasible source location for this material is about 12 km distant, which requires that the deposit was carried by ice across the trough of Strath Maol Chalum and emplaced while active ice-streams confined it laterally to its present-day location. This, in turn, requires that ice lay in the Inner Sound between Applecross and Skye to an elevation 400–500 m above present-day sea-level. The Wester Ross Re-advance of 15–14 ka left a fragment of lateral moraine against the most easterly flute and buried the distal end of the flutes with hummocky moraine. We hypothesize that the fluted deposits reflect the locations of the ice-stream margins that constrained deposition of locally derived ice-transported sediment, rather than the flow-lines of the ice-stream itself.




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Coupled hydraulic and mechanical model of surface uplift due to mine water rebound: implications for mine water heating and cooling schemes

In order to establish sustainable heat loading (heat removal and storage) in abandoned flooded mine workings it is important to understand the geomechanical impact of the cyclical heat loading caused by fluid injection and extraction. This is particularly important where significantly more thermal loading is planned than naturally occurs. A simple calculation shows that the sustainable geothermal heat flux from abandoned coal mines can provide less than a tenth of Scotland's annual domestic heating demand. Any heat removal greater than the natural heat flux will lead to heat mining unless heat storage options are also considered.

As a first step, a steady-state, fully saturated, 2D coupled hydromechanical model of a generalized section of pillar-and-stall workings has been created. Mine water rebound was modelled by increasing the hydrostatic pressure sequentially, in line with monitored mine water-level data from Midlothian, Scotland. The modelled uplift to water-level rise ratio of 1.4 mm m–1 is of the same order of magnitude (1 mm m–1) as that observed through interferometric synthetic aperture radar (InSAR) data in the coalfield due to mine water rebound. The modelled magnitude of shear stress at the pillar corners, as a result of horizontal and vertical displacement, is shown to increase linearly with water level. Mine heat systems are expected to cause smaller changes in pressure than those modelled but the results provide initial implications on the potential geomechanical impacts of mine water heat schemes which abstract or inject water and heat into pillar-and-stall coal mine workings.

Thematic collection: This article is part of the SJG Collection on Early-Career Research available at: https://www.lyellcollection.org/cc/SJG-early-career-research




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Digging deeper: The influence of historical mining on Glasgow's subsurface thermal state to inform geothermal research

Studies of the former NE England coalfield in Tyneside demonstrated that heat flow perturbations in boreholes were due to the entrainment and lateral dispersion of heat from deeper in the subsurface through flooded mine workings. This work assesses the influence of historical mining on geothermal observations across Greater Glasgow. The regional heat flow for Glasgow is 60 mW m–2 and, after correction for palaeoclimate, is estimated as c. 80 mW m–2. An example of reduced heat flow above mine workings is observed at Hallside (c. 10 km SE of Glasgow), where the heat flow through a 352 m deep borehole is c. 14 mW m–2. Similarly, the heat flow across the 199 m deep GGC01 borehole in the Glasgow Geothermal Energy Research Field Site is c. 44 mW m–2. The differences between these values and the expected regional heat flow suggest a significant component of horizontal heat flow into surrounding flooded mine workings. This deduction also influences the quantification of deeper geothermal resources, as extrapolation of the temperature gradient above mine workings would underestimate the temperature at depth. Future projects should consider the influence of historical mining on heat flow when temperature datasets such as these are used in the design of geothermal developments.

Supplementary material: Background information on the chronology of historical mining at each borehole location and a summary of groundwater flow in mine workings beneath Glasgow are available at https://doi.org/10.6084/m9.figshare.c.4681100

Thematic collection: This article is part of the ‘Early Career Research’ available at: https://www.lyellcollection.org/cc/SJG-early-career-research




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Rupture geometries in anisotropic amphibolite recorded by pseudotachylytes in the Gairloch Shear Zone, NW Scotland

Recent earthquakes involving complex multi-fault rupture have increased our appreciation of the variety of rupture geometries and fault interactions that occur within the short duration of coseismic slip. Geometrical complexities are intrinsically linked with spatially heterogeneous slip and stress drop distributions, and hence need incorporating into seismic hazard analysis. Studies of exhumed ancient fault zones facilitate investigation of rupture processes in the context of lithology and structure at seismogenic depths. In the Gairloch Shear Zone, NW Scotland, foliated amphibolites host pseudotachylytes that record rupture geometries of ancient low-magnitude (≤MW 3) seismicity. Pseudotachylyte faults are commonly foliation parallel, indicating exploitation of foliation planes as weak interfaces for seismic rupture. Discordance and complexity are introduced by fault segmentation, stepovers, branching and brecciated dilational volumes. Pseudotachylyte geometries indicate that slip nucleation initiated simultaneously across several parallel foliation planes with millimetre and centimetre separations, leading to progressive interaction and ultimately linkage of adjacent segments and branches within a single earthquake. Interacting with this structural control, a lithological influence of abundant low disequilibrium melting-point amphibole facilitated coseismic melting, with relatively high coseismic melt pressure encouraging transient dilational sites. These faults elucidate controls and processes that may upscale to large active fault zones hosting major earthquake activity.

Supplementary material: Supplementary Figures 1 and 2, unannotated versions of field photographs displayed in Figures 4a and 5 respectively, are available at https://doi.org/10.6084/m9.figshare.c.4573256

Thematic collection: This article is part of the SJG Collection on Early-Career Research available at: https://www.lyellcollection.org/cc/SJG-early-career-research




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Mode of Action of the Catalytic Site in the N-Terminal Ribosome-Inactivating Domain of JIP60

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163–185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.




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N-Terminal Acetylation Stabilizes SIGMA FACTOR BINDING PROTEIN1 Involved in Salicylic Acid-Primed Cell Death

N-terminal (Nt) acetylation (NTA) is an ample and irreversible cotranslational protein modification catalyzed by ribosome-associated Nt-acetyltransferases. NTA on specific proteins can act as a degradation signal (called an Ac/N-degron) for proteolysis in yeast and mammals. However, in plants, the biological relevance of NTA remains largely unexplored. In this study, we reveal that Arabidopsis (Arabidopsis thaliana) SIGMA FACTOR-BINDING PROTEIN1 (SIB1), a transcription coregulator and a positive regulator of salicylic acid-primed cell death, undergoes an absolute NTA on the initiator Met; Nt-acetyltransferase B (NatB) partly contributes to this modification. While NTA results in destabilization of certain target proteins, our genetic and biochemical analyses revealed that plant NatB-involved NTA instead renders SIB1 more stable. Given that the ubiquitin/proteasome system stimulates SIB1 degradation, it seems that the NTA-conferred stability ensures the timely expression of SIB1-dependent genes, mostly related to immune responses. Taking our findings together, here we report a noncanonical NTA-driven protein stabilization in land plants.




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Phototropin2 Contributes to the Chloroplast Avoidance Response at the Chloroplast-Plasma Membrane Interface

Blue-light-induced chloroplast movements play an important role in maximizing light utilization for photosynthesis in plants. Under a weak light condition, chloroplasts accumulate to the cell surface to capture light efficiently (chloroplast accumulation response). Conversely, chloroplasts escape from strong light and move to the side wall to reduce photodamage (chloroplast avoidance response). The blue light receptor phototropin (phot) regulates these chloroplast movements and optimizes leaf photosynthesis by controlling other responses in addition to chloroplast movements. Seed plants such as Arabidopsis (Arabidopsis thaliana) have phot1 and phot2. They redundantly mediate phototropism, stomatal opening, leaf flattening, and the chloroplast accumulation response. However, the chloroplast avoidance response is induced by strong blue light and regulated primarily by phot2. Phots are localized mainly on the plasma membrane. However, a substantial amount of phot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might have different functions. To determine the functions of plasma membrane- and chloroplast envelope-localized phot2, we tethered it to these structures with their respective targeting signals. Plasma membrane-localized phot2 regulated phototropism, leaf flattening, stomatal opening, and chloroplast movements. Chloroplast envelope-localized phot2 failed to mediate phototropism, leaf flattening, and the chloroplast accumulation response but partially regulated the chloroplast avoidance response and stomatal opening. Based on the present and previous findings, we propose that phot2 localized at the interface between the plasma membrane and the chloroplasts is required for the chloroplast avoidance response and possibly for stomatal opening as well.




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OsNAR2.1 Interaction with OsNIT1 and OsNIT2 Functions in Root-growth Responses to Nitrate and Ammonium

The nitrate transport accessory protein OsNAR2 plays a critical role in root-growth responses to nitrate and nitrate acquisition in rice (Oryza sativa). In this study, a pull-down assay combined with yeast two-hybrid and coimmunoprecipitation analyses revealed that OsNAR2.1 interacts with OsNIT1 and OsNIT2. Moreover, an in vitro nitrilase activity assay indicated that indole-3-acetonitrile (IAN) is hydrolyzed to indole-3-acetic acid (IAA) by OsNIT1, the activity of which was enhanced 3- to 4-fold by OsNIT2 and in excess of 5- to 8-fold by OsNAR2.1. Knockout (KO) of OsNAR2.1 was accompanied by repressed expression of both OsNIT1 and OsNIT2, whereas KO of OsNIT1 and OsNIT2 in the osnit1 and osnit2 mutant lines did not affect expression of OsNAR2.1 or the root nitrate acquisition rate. osnit1 and osnit2 displayed decreased primary root length and lateral root density. Double KO of OsNAR2.1 and OsNIT2 caused further decreases in lateral root density under nitrate supply. Ammonium supply repressed OsNAR2.1 expression whereas it upregulated OsNIT1 and OsNIT2 expression. Both osnit1 and osnit2 showed root growth hypersensitivity to external ammonium; however, less root growth sensitivity to external IAN, higher expression of three IAA-amido synthetase genes, and a lower rate of 3H-IAA movement toward the roots were observed. Taken together, we conclude that the interaction of OsNIT1 and OsNIT2 activated by OsNAR2.1 and nitrogen supply is essential for maintaining root growth possibly via altering the IAA ratio of free to conjugate forms and facilitating its transportation.




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The Calcium Sensor CBL2 and Its Interacting Kinase CIPK6 Are Involved in Plant Sugar Homeostasis via Interacting with Tonoplast Sugar Transporter TST2

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK)-mediated calcium signaling has been widely reported to function in plant development and various stress responses, particularly in ion homeostasis. Sugars are the most important primary metabolites, and thus sugar homeostasis requires precise regulation. Here, we describe a CBL2-CIPK6-Tonoplast-Localized Sugar Transporter2 (TST2) molecular module in cotton (Gossypium hirsutum) that regulates plant sugar homeostasis, in particular Glc homeostasis. GhCIPK6 is recruited to the tonoplast by GhCBL2 and interacts with the tonoplast-localized sugar transporter GhTST2. Overexpression of either GhCBL2, GhCIPK6, or GhTST2 was sufficient to promote sugar accumulation in transgenic cotton, whereas RNAi-mediated knockdown of GhCIPK6 expression or CRISPR-Cas9-mediated knockout of GhTST2 resulted in significantly decreased Glc content. Moreover, mutation of GhCBL2 or GhTST2 in GhCIPK6-overexpressing cotton reinstated sugar contents comparable to wild-type plants. Heterologous expression of GhCIPK6 in Arabidopsis (Arabidopsis thaliana) also promoted Glc accumulation, whereas mutation of AtTST1/2 in GhCIPK6-overexpressing Arabidopsis similarly reinstated wild-type sugar contents, thus indicating conservation of CBL2-CIPK6-TST2-mediated sugar homeostasis among different plant species. Our characterization of the molecular players behind plant sugar homeostasis may be exploited to improve sugar contents and abiotic stress resistance in plants.




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Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air

Photorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activity.




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Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula x alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1. Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.




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Dehydroascorbate Reductases and Glutathione Set a Threshold for High-Light-Induced Ascorbate Accumulation

Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated dhar Arabidopsis plants as well as a quadruple mutant line combining dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ~30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with dhar, there was near-complete inhibition of high-light–dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light–induced ascorbate accumulation.




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Transcription Factors BLH2 and BLH4 Regulate Demethylesterification of Homogalacturonan in Seed Mucilage

The polysaccharide pectin is a major component of the plant cell wall. The pectic glycan homogalacturonan (HG) is a proportionally small but important component of a specialized seed cell wall called mucilage. HG is synthesized in a highly methylesterified form, and, following secretion, is de-methylesterified by pectin methylesterases (PMEs). The degree of methylesterification of HG determines the structural and functional properties of pectin, but how methylesterification is regulated remains largely unknown. Here, we identified two BEL1-Like homeodomain (BLH) transcription factors, BLH2 and BLH4, as positive regulators of HG de-methylesterification in Arabidopsis (Arabidopsis thaliana) seed coat mucilage. BLH2 and BLH4 were significantly expressed in mucilage secretory cells during seed mucilage production. BLH2 and BLH4 single mutants exhibited no obvious mucilage phenotype, but the blh2 blh4 double mutant displayed significantly reduced mucilage adherence to the seed. Reduced mucilage adherence in blh2 blh4 was caused by decreased PME activity in the seed coat, which increased the degree of methylesterification of HG in mucilage. The expression of several PME metabolism-related genes, including PME58, PECTIN METHYLESTERASE INHIBITOR6, SEEDSTICK, and MYB52 was significantly altered in blh2 blh4 seeds. BLH2 and BLH4 directly activated PME58 expression by binding to its TGACAGGT cis-element. Moreover, pme58 mutants exhibited reduced mucilage adherence similar to that of blh2 blh4, and the blh2 blh4 pme58 triple mutant exhibited no additional mucilage adherence defects. Furthermore, overexpression of PME58 in blh2 blh4 rescued the mucilage adherence defect. Together, these results demonstrate that BLH2 and BLH4 redundantly regulate de-methylesterification of HG in seed mucilage by directly activating PME58.




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Sensory-Directed Genetic and Biochemical Characterization of Volatile Terpene Production in Kiwifruit

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A’ kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a–AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A. chinensis. AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia. Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.




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SUMOylation Stabilizes the Transcription Factor DREB2A to Improve Plant Thermotolerance

Heat stress (HS) has serious effects on plant development, resulting in heavy agricultural losses. A critical transcription factor network is involved in plant adaptation to high temperature. DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) is a key transcription factor that functions in plant thermotolerance. The DREB2A protein is unstable under normal temperature and is degraded by the 26S proteasome; however, the mechanism by which DREB2A protein stability dramatically increases in response to HS remains poorly understood. In this study, we found that the DREB2A protein of Arabidopsis (Arabidopsis thaliana) is stabilized under high temperature by the posttranslational modification SUMOylation. Biochemical data indicated that DREB2A is SUMOylated at K163, a conserved residue adjacent to the negative regulatory domain during HS. SUMOylation of DREB2A suppresses its interaction with BPM2, a ubiquitin ligase component, consequently increasing DREB2A protein stability under high temperature. In addition, analysis of plant heat tolerance and marker gene expression indicated that DREB2A SUMOylation is essential for its function in the HS response. Collectively, our data reveal a role for SUMOylation in the maintenance of DREB2A stability under high temperature, thus improving our understanding of the regulatory mechanisms underlying HS response in plant cells.




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Starting Off Right: N-Terminal Acetylation Stabilizes an Immune-Activating Protein




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Location, Location, Location: Phototropin 2 Action at the Chloroplast Membrane




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What Are the Roles for Dehydroascorbate Reductases and Glutathione in Sustaining Ascorbate Accumulation?




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Severe Pulmonary Hypertension Management Across Europe (PHAROS): an ERS Clinical Research Collaboration

The past 20 years have seen major advances in the understanding and treatment of pulmonary arterial hypertension (PAH; group 1 of the pulmonary hypertension (PH) clinical classification) [1]. A strong basis of knowledge has been acquired in: 1) large randomised clinical trials for drug development; 2) national registries for epidemiology and outcome; and 3) smaller studies on the pathophysiological mechanisms of the disease. This knowledge has been reviewed at World Symposia on Pulmonary Hypertension (the most recent in 2018 [2]) and summarised in European Respiratory Society (ERS)/European Society of Cardiology (ESC) clinical guidelines (the most recent in 2015 [3, 4]). We are, however, much less knowledgeable on specific aspects such as 1) the implementation of guidelines and access to therapies in different European countries; 2) the management of PH crises and progressive (acute on chronic) heart failure; and 3) other groups of PH, such as PH due to lung diseases. Therapeutic strategies also need to be optimised, in particular regarding the combination of drugs, the use of anticoagulants, the place for new medications targeting different pathophysiological pathways, etc.




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Looking back to go forward: adherence to inhaled therapy before biologic therapy in severe asthma

For decades inhaled corticosteroids have been central to the management of asthma and are proven to be effective in maintaining symptom control, reducing exacerbations and preserving quality of life through mediation of airway inflammation. However, a small minority of patients have disease which is refractory to high dose inhaled corticosteroid (ICS) therapy and require additional oral corticosteroids to achieve acceptable control of symptoms and exacerbations. Severe asthma represents less than 10% of the total asthma population [1] but is the most serious, life-affecting and costly form of the condition [2].