Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

First PCR Confirmed anthrax outbreaks in Ethiopia—Amhara region, 2018–2019

BackgroundAnthrax is a disease that affects humans and animals. In Ethiopia, anthrax is a reportable disease and assumed to be endemic, although laboratory confirmation has not been routinely performed until recently. We describe the findings from the investigation of two outbreaks in Amhara region.MethodsFollowing reports of suspected outbreaks in Wag Hamra zone (Outbreak 1) and South Gondar zone (Outbreak 2), multi-sectoral teams involving both animal and public health officials were deployed to investigate and establish control programs. A suspect case was defined as: sudden death with rapid bloating or bleeding from orifice(s) with unclotted blood (animals); and signs compatible with cutaneous, ingestion, or inhalation anthrax ≤7 days after exposure to a suspect animal (humans). Suspect human cases were interviewed using a standard questionnaire. Samples were collected from humans with suspected anthrax (Outbreak 1 and Outbreak 2) as well as dried meat of suspect animal cases (Outbreak 2). A case was confirmed if a positive test was returned using real-time polymerase chain reaction (qPCR).ResultsIn Outbreak 1, a total of 49 cows died due to suspected anthrax and 22 humans developed symptoms consistent with cutaneous anthrax (40% attack rate), two of whom died due to suspected ingestion anthrax. Three people were confirmed to have anthrax by qPCR. In Outbreak 2, anthrax was suspected to have caused the deaths of two livestock animals and one human. Subsequent investigation revealed 18 suspected cases of cutaneous anthrax in humans (27% attack rate). None of the 12 human samples collected tested positive, however, a swab taken from the dried meat of one animal case (goat) was positive by qPCR.ConclusionWe report the first qPCR-confirmed outbreaks of anthrax in Ethiopia. Both outbreaks were controlled through active case finding, carcass management, ring vaccination of livestock, training of health professionals and outreach with livestock owners. Human and animal health authorities should work together using a One Health approach to improve case reporting and vaccine coverage.     

Structure and biochemical function of a prototypical Arabidopsis U-box domain

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the betabetaalphabeta fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal alpha-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal alpha-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal alpha-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitin-dependent protein degradation is a prevalent regulatory mechanism in plants.     

Findings from Integrated Behavioral and Biologic Survey among Males Who Inject Drugs (MWID) — Vietnam, 2009–2010: Evidence of the Need for an Integrated Response to HIV,Hepatitis B Virus,and Hepatitis C Virus

IntroductionGiven the overlapping modes of transmission of HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV), understanding the burden and relationship of these infections is critical for an effective response. Representative data on these infections among males who inject drugs (MWID), the key high-risk population for HIV in Vietnam, are currently lacking.MethodsData and stored specimens from Vietnam’s 2009-2010 Integrated Biologic and Behavioral Survey, a cross-sectional study among high-risk populations, were used for this analysis. Plasma samples were tested for HIV, HBV, and HCV using commercial assays. A questionnaire was administered to provide demographic, behavior, and service-uptake information. Provincial-level analyses were conducted to profile MWID enrollees and to provide estimates on the prevalence of HIV, HBV, and HCV infection.ResultsAmong 3010 MWID sampled across 10 provinces, the median (range) HIV prevalence was 28.1% (1.0%-55.5%). Median prevalence for current HBV infection (HBsAg+) was 14.1% (11.7%-28.0%), for previous exposure to HBV (total anti-HBc+) was 71.4% (49.9%-83.1%), and for current or past HCV infection (HCV Ag/Ab+) was 53.8% (10.9%-80.8%). In adjusted analysis, HBsAg+ (aOR: 2.09, 1.01-4.34) and HCV Ag/Ab+ (aOR: 19.58, 13.07-29.33) status were significantly associated with HIV infection; the association with total anti-HBc+ approached significance (aOR: 1.29, 0.99-1.68).ConclusionThe prevalence and association between HIV, HBV, and HCV are high among MWID in Vietnam. These findings indicate the need for integrated policies and practice that for the surveillance, prevention, screening, and treatment of both HIV and viral hepatitis among MWID in Vietnam.     

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.     

Plasmodium malariae Infection Associated with a High Burden of Anemia: A Hospital-Based Surveillance Study

Background

Plasmodium malariae is a slow-growing parasite with a wide geographic distribution. Although generally regarded as a benign cause of malaria, it has been associated with nephrotic syndrome, particularly in young children, and can persist in the host for years. Morbidity associated with P. malariae infection has received relatively little attention, and the risk of P. malariae-associated nephrotic syndrome is unknown.

Methodology/Principal Findings

We used data from a very large hospital-based surveillance system incorporating information on clinical diagnoses, blood cell parameters and treatment to describe the demographic distribution, morbidity and mortality associated with P. malariae infection in southern Papua, Indonesia. Between April 2004 and December 2013 there were 1,054,674 patient presentations to Mitra Masyarakat Hospital of which 196,380 (18.6%) were associated with malaria and 5,097 were with P. malariae infection (constituting 2.6% of all malaria cases). The proportion of malaria cases attributable to P. malariae increased with age from 0.9% for patients under one year old to 3.1% for patients older than 15 years. Overall, 8.5% of patients with P. malariae infection required admission to hospital and the median length of stay for these patients was 2.5 days (Interquartile Range: 2.0–4.0 days). Patients with P. malariae infection had a lower mean hemoglobin concentration (9.0g/dL) than patients with P. falciparum (9.5g/dL), P. vivax (9.6g/dL) and mixed species infections (9.3g/dL). There were four cases of nephrotic syndrome recorded in patients with P. malariae infection, three of which were in children younger than 5 years old, giving a risk in this age group of 0.47% (95% Confidence Interval; 0.10% to 1.4%). Overall, 2.4% (n = 16) of patients hospitalized with P. malariae infection subsequently died in hospital, similar to the proportions for the other endemic Plasmodium species (range: 0% for P. ovale to 1.6% for P. falciparum).

Conclusions/Significance

Plasmodium malariae infection is relatively uncommon in Papua, Indonesia but is associated with significant morbidity from anemia and a similar risk of mortality to patients hospitalized with P. falciparum and P. vivax infection. In our large hospital database, one in 200 children under the age of 5 years with P. malariae infection were recorded as having nephrotic syndrome.     

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.     

Identification of Novel Loci Regulating Interspecific Variation in Root Morphology and Cellular Development in Tomato

While the Arabidopsis (Arabidopsis thaliana) root has been elegantly characterized with respect to specification of cell identity, its development is missing a number of cellular features present in other species. We have characterized the root development of a wild and a domesticated tomato species, Solanum pennellii and Solanum lycopersicum ‘M82.’ We found extensive differences between these species for root morphology and cellular development including root length, a novel gravity set point angle, differences in cortical cell layer patterning, stem cell niche structure, and radial cell division. Using an introgression line population between these two species, we identified numerous loci that regulate these distinct aspects of development. Specifically we comprehensively identified loci that regulate (1) root length by distinct mechanisms including regulation of cell production within the meristem and the balance between cell division and expansion, (2) the gravity set point angle, and (3) radial cell division or expansion either in specific cell types or generally across multiple cell types. Our findings provide a novel perspective on the regulation of root growth and development between species. These loci have exciting implications with respect to regulation of drought resistance or salinity tolerance and regulation of root development in a family that has undergone domestication.The root system is of vital importance to plants because it anchors the plant and its cells absorb and transport water, nutrients, and solutes to the shoot. The root system has a complex branching architecture with numerous cell types whose development must be dynamic, plastic, and highly responsive to the environment to maximize plant fitness and yield. To optimize root system architecture for the specific environment in which the plant is growing, developmental programs associated with distinct developmental stages, and cell types are specifically and precisely regulated by both local and global signals. For instance, local nitrogen sources induce root hair tip growth and can regulate lateral root initiation (Malamy and Ryan, 2001; Bloch et al., 2011) while the search for water regulates primary root growth (Saucedo et al., 2012). This complex architecture and plasticity complicate the ability to enumerate and study root architecture concomitantly at all cellular and tissue levels. Because of this limitation, population-level studies are typically limited to measuring architecture-level variables such as root length, number, and branching, without focusing on the development of specific cell types that give rise to this architecture.Root cell type specification and development has been extensively studied using classical genetic methods in the model plant Arabidopsis (Arabidopsis thaliana). These studies revealed the elegant simplicity of the Arabidopsis root at the cellular level (Dolan et al., 1993). In Arabidopsis there is an invariant number of cells within the single cortical and endodermal layers of the primary root but variable cell numbers within lateral roots. The core of the root stem cell niche is formed by a set of four quiescent center (QC) cells, with a set of initial cells that give rise to all cell types in the root surrounding the QC. Developmental genetic studies in Arabidopsis have identified a variety of genes that regulate root length, lateral root number, and radial patterning (Benfey and Scheres, 2000; Mähönen et al., 2000; Schiefelbein et al., 2009). This includes the identification of genes that regulate vascular cell proliferation, endodermis and epidermis cell identity, and the asymmetric division of the cortex-endodermis initial (CEI).Arabidopsis has provided an excellent base model for root cellular development, yet as with any species there are unique cellular aspects that are present and/or missing within Arabidopsis that necessitate the study of other species. For instance, Arabidopsis is unusual as it contains only four QC cells, whereas most monocot and dicot species contain a greater number of QC cells (Jiang et al., 2003). To date, the regulatory mechanisms controlling this diversity in QC cell number are completely unknown. Additionally, most monocot and dicot species contain numerous cortex layers that are the product of repeated divisions of a CEI cell, whereas Arabidopsis only contains a single cortex layer (Dolan et al., 1993). The cell number in the cortex and the endodermis is invariant in the Arabidopsis primary root, but variable in many other plant species. Regulation of radial cell number variability in these cell types as well as the pericycle has never been addressed in any plant species. Furthermore, in 80% of flowering plant species, the outer layer of the root’s cortex, or exodermis, contains a suberinized cell wall to restrict passage of solutes from the outside of the root to the inside, but Arabidopsis does not contain a suberinized exodermis. The exodermis has been reported to be derived from an independent cortical initial, suggesting it is an independent specialized cell type whose genetics are not addressable within Arabidopsis (Heimsch and Seago, 2008). Genes regulating the specification of the exodermis and the production of multiple cortical layers have not been identified in monocots or dicots. Thus, classical genetic approaches have not addressed the genetic mechanisms regulating cell proliferation and patterning decisions within many cell types not present in Arabidopsis.One approach with significant potential to identify these unresolved genetic mechanisms and integrate them into the broader control of root system architecture is the use of natural variation within and between species (Shindo et al., 2007). The use of stable mapping populations such as a homozygous introgression line (IL) between two different species provides a stable genetic pool from which to repeatedly phenotype different cellular and morphological aspects of root architecture and integrate them into a common model. This quantitative genetic analysis is typically conducted using quantitative trait locus (QTL) mapping, which has identified loci or, in a small number of cases, genes that regulate root length in monocots and dicots (Bettey et al., 2000; Mouchel et al., 2004; Loudet et al., 2005; Fitz Gerald et al., 2006; Reymond et al., 2006; Fita et al., 2008; Khan et al., 2012). These studies however have typically been limited to the analysis of large-effect loci (Loudet et al., 2005; Reymond et al., 2006) and have not coordinately dissected root architecture at both the morphological and cellular levels.To determine how tomato (Solanum spp.) root morphogenesis is determined by cellular features including radial patterning, radial cell proliferation, radial cell expansion, and compensatory changes in cell expansion when cell proliferation is altered (Hemerly et al., 1995), we performed a detailed characterization of root development in two Solanum species. We used Solanum pennellii, a wild tomato species, and cv M82 of the domesticated species Solanum lycopersicum and their derived IL population. A wild species, S. pennellii is found in coastal deserts and rocky, arid soil and exhibits drought and salt tolerance and pathogen resistance in comparison with the domesticated cv M82 (Dehan and Tal, 1978; Koca et al., 2006; Easlon and Richards, 2009). In this study, we identified significant developmental differences between the two species by measuring a large range of root traits including the cell number within individual cell types, CEI spatiotemporal patterning differences, variability in cortex cell layer and QC cell number, root growth, and a novel gravity set point angle. To explore the link between the whole organ phenotype and cellular level using interspecific genetic variation we used the IL population derived from a cross between cv M82 and S. pennellii (Eshed and Zamir, 1995). This population comprises 76 segmental ILs with marker-defined genomic regions of S. pennellii substituting for homologous intervals of the cultivated variety cv M82 that partition the tomato genome into 107 bins. Measuring the above cellular and morphological phenotypes in these lines identified numerous major- and minor-effect loci for each phenotype, showing that interspecific variation in root development involves a complex suite of genetic changes, many of which display cell type-specific effects.