Localization of <i>Plasmodium berghei</i> SUFD, E, and S to the apicoplast in liver stages.

<p>(A) Co-staining of fixed, <i>sufD::tag</i>, <i>sufE::tag</i>, or <i>sufS::tag</i> parasite-infected hepatoma cells 48 h after sporozoite infection using anti-mCherry antibodies and anti-sera against acyl carrier protein (ACP). Note substantial overlap between the SUF::tag proteins and the signature apicoplast protein. (B) Drug treatment of <i>suf::tag</i>-infected hepatoma cells to corroborate apicoplast localization of the SUF::tag proteins. During liver stage development <i>suf::tag</i>-infected cells were left untreated (control) or treated with 1 µM azithromycin. Liver stages were stained with anti-mCherry antibodies and anti-sera against upregulated in infective sporozoite protein 4 (UIS4), a signature protein of the parasitophorous vacuolar membrane (PVM). Nuclei were stained with the DNA-dye Hoechst. Bars, 10 µm.</p

<i>SUFA</i> is dispensable for blood infection <i>in vivo</i>.

<p>Parasitemias of <i>sufA</i>^–-infected animals in comparison to mice infected with wild type (WT) parasites. Female C57BL/6 mice (WT, <i>n</i> = 3; <i>sufA</i>^–1 and 2, <i>n</i> = 5 each) were injected intravenously with 10,000 freshly dissected salivary gland sporozoites and infection was monitored by microscopic examination of Giemsa-stained blood films. The two isogenic <i>sufA</i>^– parasite lines (brown and orange lines) and WT parasites (gray line) showed equal pre-patent periods (three days) and similar exponential parasite growth (<i>P</i>>0.05; two-way ANOVA followed by Bonferroni posttests).</p

Experimental Genetics of <i>Plasmodium berghei</i> NFU in the Apicoplast Iron-Sulfur Cluster Biogenesis Pathway

<div><p>Eukaryotic pathogens of the phylum <i>Apicomplexa</i> contain a non-photosynthetic plastid, termed apicoplast. Within this organelle distinct iron-sulfur [Fe-S] cluster proteins are likely central to biosynthesis pathways, including generation of isoprenoids and lipoic acid. Here, we targeted a nuclear-encoded component of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria parasite <i>Plasmodium berghei</i>. We show that ablation of the gene encoding a nitrogen fixation factor U (NifU)-like domain containing protein (<i>NFUapi</i>) resulted in parasites that were able to complete the entire life cycle indicating redundant or non-essential functions. <i>nfu</i>^– parasites displayed reduced merosome formation <i>in vitro</i>, suggesting that apicoplast NFUapi plays an auxiliary role in establishing a blood stage infection. NFUapi fused to a combined fluorescent protein-epitope tag delineates the <i>Plasmodium</i> apicoplast and was tested to revisit inhibition of liver stage development by azithromycin and fosmidomycin. We show that the branched apicoplast signal is entirely abolished by azithromycin treatment, while fosmidomycin had no effect on apicoplast morphology. In conclusion, our experimental genetics analysis supports specialized and/or redundant role(s) for NFUapi in the [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite.</p></div

A large proportion of people who inject drugs are susceptible to hepatitis B: Results from a bio-behavioural study in eight German cities

Background: People who inject drugs (PWID) are at high risk of hepatitis B virus (HBV) infection by sharing needles and drug use paraphernalia. In Germany, no routine surveillance of HBV prevalence and vaccination coverage among PWID exists. Methods: Socio-demographic and behavioural data were collected between 2011 and 2014 through face-to-face interviews, during a bio-behavioural survey of PWID recruited in eight German cities. Dried blood spots (DBS) prepared with capillary blood were tested for HBV markers. Factors associated with past/current HBV infection and vaccination status were analysed by univariable and multivariable analysis using logistic regression. The validity of self-reported HBV infection and vaccination status was analysed by comparison to the laboratory results. Results: Among 2077 participants, the prevalence of current HBV infection was 1.1%, of past HBV infection was 24%, and of vaccine-induced HBV antibodies was 32%. No detectable HBV antibodies were found in 43%. HBV infection status was significantly associated with study city, age, years of injecting, use of stimulants, migration status, and homelessness; HBV vaccination status was significantly associated with study city, age, and level of education. Correct infection status was reported by 71% and correct vaccination status by 45%. Conclusions: HBV seroprevalence among PWID was about five times higher than in the general population in Germany, confirming PWID as an important risk group. Targeted information campaigns on HBV and HBV prevention for PWID and professionals in contact with PWID need to be intensified. Routinely offered HBV vaccination during imprisonment and opioid substitution therapy would likely improve vaccination rates among PWID

Normal development of <i>sufA</i>^– parasites in the mosquito vector and during liver cell infection.

<p>(A) Percentage of <i>A. stephensi</i> mosquitoes infected with WT (gray, <i>n</i> = 4) and <i>sufA</i>^– (brown, <i>n</i> = 6) parasites. Shown is the mean percentage (± S.D.) from independent mosquito feeding experiments. For <i>sufA</i>^– infections, both isogenic strains, <i>sufA</i>^–1 (<i>n</i> = 4) and <i>sufA</i>^–2 (<i>n</i> = 2), were transmitted to mosquitoes and data combined. (B) Mean sporozoite number (± S.D.) in salivary glands (days 17–21 after infection) from the same independent mosquito feedings as shown in panel A (WT, <i>n</i> = 4; pooled <i>sufA</i>^–, <i>n</i> = 6). (C) Liver stages development of WT and <i>sufA</i>^– parasites in cultured hepatoma cells. Shown are mean numbers (± S.D.) of intracellular parasites at the time points indicated from two experiments done in quadruplicate each. (D) Merosome formation at 72 h after infection. Shown are mean values (± S.D.). None of the <i>sufA</i>^– data points were significantly different from the corresponding WT values (<i>P</i>>0.05; non-parametric, two-tailed Mann-Whitney’s test).</p

Confirmed and predicted [Fe-S] cluster-containing proteins in <i>Plasmodium</i>.

a<p>Gene IDs of the <i>P. berghei</i> and <i>P. falciparum</i> orthologs and the predicted localizations of the proteins were retrieved from PlasmoDB (<a href="http://PlasmoDB.org" target="_blank">http://PlasmoDB.org</a>) or identified by similarity searches using <i>A. thaliana</i> [Fe-S] cluster proteins as query sequences <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089718#pone.0089718-Balk1" target="_blank">[41]</a>.</p>b<p>Putative targeting of the <i>P. falciparum</i> [Fe-S] cluster-containing proteins to the apicoplast or mitochondrion was predicted using four different algorithms. PlasmoAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089718#pone.0089718-Foth1" target="_blank">[51]</a> indicates the likelihood of the presence of the required signal peptide followed by the likelihood of an apicoplast localization (“−” = unlikely, “0″ = undecided, “+” = likely, “++” = very likely). ApicoAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089718#pone.0089718-Cilingir1" target="_blank">[52]</a> is a different algorithm that can identify apicoplast proteins in multiple <i>Apicomplexa</i> (“No SP” = no signal peptide, “No ATP” = signal peptide but no transit peptide, “ATP” = apicoplast targeted protein). PlasMit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089718#pone.0089718-Bender1" target="_blank">[53]</a> predicts the likelihood of a mitochondrial localization for <i>P. falciparum</i> proteins (“non-mito” (99%), “possibly” (91%), and “mito” (99%)). MitoProtII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089718#pone.0089718-Claros1" target="_blank">[54]</a> gives a probability score for the likelihood of mitochondrial localization but is not optimized for <i>Plasmodium</i> sequences.</p

<i>Plasmodium</i> [Fe-S] biosynthesis pathway proteins of the apicoplast.

a<p>Gene IDs of the <i>P. berghei</i> and <i>P. falciparum</i> orthologs (<a href="http://PlasmoDB.org" target="_blank">http://PlasmoDB.org</a>).</p>b<p>Putative targeting of the <i>P. falciparum</i> SUF pathway proteins to the apicoplast or mitochondrion was predicted using four different algorithms. ApicoAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067269#pone.0067269-Cilingir1" target="_blank">[35]</a> predicts whether a given protein lacks the required signal peptide (“No SP”), contains a signal peptide but no transit peptide (“non-ApicoTP”), or is an apicoplast targeted protein (“ApicoTP”) that uses the bipartite signaling mechanism. PlasmoAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067269#pone.0067269-Foth1" target="_blank">[36]</a> indicates the likelihood of the presence of the required signal peptide followed by the likelihood of an apicoplast localization (“-” = unlikely, “0″ = undecided, “+” = likely, “++” = very likely). PlasMit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067269#pone.0067269-Bender1" target="_blank">[37]</a> predicts the likelihood of a mitochondrial localization for <i>P. falciparum</i> proteins (“non-mito (99%)”, “mito (91%)”, and “mito (99%)”). MitoProtII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067269#pone.0067269-Claros1" target="_blank">[38]</a> gives a probability score for the likelihood of mitochondrial localization but is not optimized <i>Plasmodium</i> sequences. Note that no analysis was done for <i>SUFB</i> as the gene is encoded on the apicoplast genome and hence needs no targeting sequences.</p

Feasibility study for the use of self‐collected nasal swabs to identify pathogens among participants of a population‐based surveillance system for acute respiratory infections (GrippeWeb‐Plus)—Germany, 2016

Background Internet‐based participatory surveillance systems, such as the German GrippeWeb, monitor the frequency of acute respiratory illnesses on population level. In order to interpret syndromic information better, we devised a microbiological feasibility study (GrippeWeb‐Plus) to test whether self‐collection of anterior nasal swabs is operationally possible, acceptable for participants and can yield valid data. Methods We recruited 103 GrippeWeb participants (73 adults and 30 children) and provided them with a kit, instructions and a questionnaire for each sample. In the first half of 2016, participants took an anterior nasal swab and sent it to the Robert Koch Institute whenever an acute respiratory illness occurred. Reporting of illnesses through the GrippeWeb platform continued as usual. We analysed swabs for the presence of human c‐myc‐DNA and 22 viral and bacterial pathogens. After the study, we sent participants an evaluation questionnaire. We analysed timeliness, completeness, acceptability and validity. Results One hundred and two participants submitted 225 analysable swabs. Ninety per cent of swabs were taken within 3 days of symptom onset. Eighty‐nine per cent of swabs had a corresponding reported illness in the GrippeWeb system. Ninety‐nine per cent of adults and 96% of children would be willing to participate in a self‐swabbing scheme for a longer period. All swabs contained c‐myc‐DNA. In 119 swabs, we identified any of 14 viruses but no bacteria. The positivity rate of influenza was similar to that in the German physician sentinel. Conclusion Self‐collection of anterior nasal swabs proofed to be feasible, was well accepted by participants, gave valid results and was an informative adjunct to syndromic data.Peer Reviewe

Targeted deletion of <i>Plasmodium berghei NFUapi</i>.

<p>(A) Replacement strategy to delete <i>PbNFUapi</i>. The endogenous <i>NFUapi</i> gene (gray arrow) was targeted with a replacement plasmid (pNFU-ko) containing 5′ and 3′ regions (dark gray bars) flanking the open reading frame (light gray arrow), a high-expressing GFP cassette (green), and the <i>hDHFR-yFcu</i> drug-selectable cassette (blue). Integration- and WT-specific primer combinations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067269#pone.0067269.s001" target="_blank">Table S2 in File S1</a>) and expected fragments are indicated. (B) PCR-based genotyping of <i>nfu^–</i> parasites to verify successful deletion of <i>NFUapi</i>. Absence of WT-specific signals in the clonal <i>nfu^–</i> line confirms purity of the knockout parasites. (C) Southern blot analysis of the clonal <i>nfu</i>^– parasite line shows bands of the expected sizes (arrows) in NdeI restriction-digested gDNA of WT (gray, 2.2 kb) and recombinant parasites (black, 7.0 kb). The 3′ homologous sequence used for targeted integration of the transfection vectors (dark gray bar in [A]) was used as the probe.</p