Transmission dynamics of symbiotic protist communities in the termite gut: association with host adult eclosion and dispersal.

The fidelity of vertical transmission is a critical factor in maintaining mutualistic associations with microorganisms. The obligate mutualism between termites and intestinal protist communities has been maintained for over 130 million years, suggesting the faithful transmission of diverse protist species across host generations. Although a severe bottleneck can occur when alates disperse with gut protists, how protist communities are maintained during this process remains largely unknown. In this study, we examined the dynamics of intestinal protist communities during adult eclosion and alate dispersal in the termite Reticulitermes speratus. We found that the protist community structure in last-instar nymphs differed significantly from that in workers and persisted intact during adult eclosion, whereas all protists disappeared from the gut during moults between worker stages. The number of protists in nymphs and alates was substantially lower than in workers, whereas the proportion of protist species exhibiting low abundance in workers was higher in nymphs and alates. Using a simulation-based approach, we demonstrate that such changes in the protist community composition of nymphs and alates improve the transmission efficiency of whole protist species communities. This study thus provides novel insights into how termites have maintained mutualistic relationships with diverse gut microbiota for generations.

Fine-scale genetic diversity and putative ecotypes of oxymonad protists coinhabiting the hindgut of Reticulitermes speratus.

The hindgut of lower termites is generally coinhabited by multiple morphologically identifiable protist species. However, it is unclear how many protist species truly coexist in this miniaturized environment, and moreover, it is difficult to define the fundamental unit of protist diversity. Species delineation of termite gut protists has therefore been guided without a theory-based concept of species. Here, we focused on the hindgut of the termite Reticulitermes speratus, where 10 or 11 morphologically distinct oxymonad cell types, that is, morphospecies, coexist. We elucidated the phylogenetic structure of all co-occurring oxymonads and addressed whether their diversity can be explained by the "ecotype" hypothesis. Oxymonad-specific 18S rRNA gene amplicon sequencing analyses of whole-gut samples, combined with single-cell 18S rRNA sequencing of the oxymonad morphospecies, identified 210 one-nucleotide-level variants. The phylogenetic analysis of these variants revealed the presence of microdiverse clusters typically within 1% sequence divergence. Each known oxymonad morphospecies comprised one to several monophyletic or paraphyletic microdiverse clusters. Using these sequence data sets, we conducted computational simulation to predict the rates of ecotype formation and periodic selection, and to demarcate putative ecotypes. Our simulations suggested that the oxymonad genetic divergence is constrained primarily by strong selection, in spite of limited population size and possible bottlenecks during intergenerational transmission. A total of 33 oxymonad ecotypes were predicted, and most of the putative ecotypes were consistently detected among different colonies and host individuals. These findings provide a possible theoretical basis for species diversity and underlying mechanisms of coexistence of termite gut protists.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Genome analyses of uncultured TG2/ZB3 bacteria in 'Margulisbacteria' specifically attached to ectosymbiotic spirochetes of protists in the termite gut.

We investigated the phylogenetic diversity, localisation and metabolism of an uncultured bacterial clade, Termite Group 2 (TG2), or ZB3, in the termite gut, which belongs to the candidate phylum 'Margulisbacteria'. We performed 16S rRNA amplicon sequencing analysis and detected TG2/ZB3 sequences in 40 out of 72 termite and cockroach species, which exclusively constituted a monophyletic cluster in the TG2/ZB3 clade. Fluorescence in situ hybridisation analysis in lower termites revealed that these bacteria are specifically attached to ectosymbiotic spirochetes of oxymonad gut protists. Draft genomes of four TG2/ZB3 phylotypes from a small number of bacterial cells were reconstructed, and functional genome analysis suggested that these bacteria hydrolyse and ferment cellulose/cellobiose to H2, CO2, acetate and ethanol. We also assembled a draft genome for a partner Treponema spirochete and found that it encoded genes for reductive acetogenesis from H2 and CO2. We hypothesise that the TG2/ZB3 bacteria we report here are commensal or mutualistic symbionts of the spirochetes, exploiting the spirochetes as H2 sinks. For these bacteria, we propose a novel genus, 'Candidatus Termititenax', which represents a hitherto uncharacterised class-level clade in 'Margulisbacteria'. Our findings add another layer, i.e., cellular association between bacteria, to the multi-layered symbiotic system in the termite gut.

Ectosymbiotic bacterial microbiota densely colonize the surface of thelastomatid nematodes in the gut of the wood-feeding cockroach Panesthia angustipennis.

Cockroaches generally harbor thelastomatid nematodes (pinworms) in their gut. In this study, we discovered that the surfaces of two undescribed thelastomatid species in the hindgut of the wood-feeding cockroach Panesthia angustipennis were consistently and densely colonized by bacteria. Epifluorescence microscopy using 4',6-diamidino-2-phenylindole and transmission electron microscopy revealed that several distinct morphotypes of bacteria covered almost the entire body surface of the nematodes in single or multiple layers. Sequencing analysis of 16S rRNA amplicons of either entire nematodes or sections of nematode body surfaces indicated that the associated bacterial microbiota consisted of several dominant phylotypes belonging to either Dysgonomonadaceae (Bacteroidales termite cluster V), Rikennellaceae or Ruminococcaceae. These phylotypes formed clades with sequences previously obtained from cockroach and/or termite guts. Comparisons of the bacterial community structure of the entire cockroach hindgut microbiota vs the nematode-associated microbiota suggested that these dominant bacterial phylotypes preferentially colonized the nematode surface. The two nematode species shared most of the dominant bacterial phylotypes, but the bacterial community structures differed significantly. Colonization by five predominant phylotypes was confirmed by fluorescence in situ hybridization analysis using phylotype-specific probes. Our study provides fundamental information on this previously unknown ectosymbiosis between gut bacteria and thelastomatid pinworms.

A High Burden of Asymptomatic Gastrointestinal Infections in Traditional Communities in Papua New Guinea.

Stool samples were collected from 148 healthy adults living a traditional subsistence lifestyle in Papua New Guinea and screened for enteric pathogens using real-time RT-PCR/PCR assays. Enteric pathogens were detected in a high proportion (41%) of individuals. Clear differences were observed in the detection of pathogens between highland and lowland communities. In particular, there was a marked difference in detection rates of norovirus GII (20% and 0%, respectively) and Shigella sp. (15% and 0%, respectively). Analysis of the relationship between enteric pathogen carriage and microbial community composition of participants, using box plots to compare specific normal flora population numbers, did not suggest that gut microbial composition was directly associated with pathogen carriage. This study suggests that enteric pathogens are common in healthy individuals in Papua New Guinean highland communities, presumably acting as a reservoir of infection and thus contributing to a high burden of gastrointestinal illnesses.

Nitrogen fixation and nifH diversity in human gut microbiota.

It has been hypothesized that nitrogen fixation occurs in the human gut. However, whether the gut microbiota truly has this potential remains unclear. We investigated the nitrogen-fixing activity and diversity of the nitrogenase reductase (NifH) genes in the faecal microbiota of humans, focusing on Papua New Guinean and Japanese individuals with low to high habitual nitrogen intake. A (15)N2 incorporation assay showed significant enrichment of (15)N in all faecal samples, irrespective of the host nitrogen intake, which was also supported by an acetylene reduction assay. The fixed nitrogen corresponded to 0.01% of the standard nitrogen requirement for humans, although our data implied that the contribution in the gut in vivo might be higher than this value. The nifH genes recovered in cloning and metagenomic analyses were classified in two clusters: one comprising sequences almost identical to Klebsiella sequences and the other related to sequences of Clostridiales members. These results are consistent with an analysis of databases of faecal metagenomes from other human populations. Collectively, the human gut microbiota has a potential for nitrogen fixation, which may be attributable to Klebsiella and Clostridiales strains, although no evidence was found that the nitrogen-fixing activity substantially contributes to the host nitrogen balance.

Association of protein intakes and variation of diet-scalp hair nitrogen isotopic discrimination factor in Papua New Guinea highlanders.

OBJECTIVES: We present new nitrogen isotopic discrimination factor between diets and scalp hairs (Δ(15) NHair-Diet : δ(15) NHair - δ(15) NDiet ) for indigenous residents in three communities in the Papua New Guinea Highlands who consumed various amounts and qualities of protein. The Δ(15) N is important for precise evaluation of the dietary habits of human populations; in both contemporary and traditional lifestyles. Several hypotheses have been proposed regarding factors that affect Δ(15) N values, based largely on observations from animal feeding experiments. However, variations and factors controlling Δ(15) N in humans are not well understood, mainly due to the difficulty of controlling the diets of participants. MATERIALS AND METHODS: These residents were studied because they have maintained relatively traditional dietary habits, which allow quantitative recording of diets. Δ(15) N was estimated by comparing hair δ(15) N values to mean dietary δ(15) N values calculated from the recorded intake of each food item and their δ(15) N values. RESULTS: The results showed that: i) there was a significant difference in Δ(15) N among study locations (3.9 ± 0.9‰ for most urbanized, 5.2 ± 1.0‰ for medium and 5.0 ± 0.9‰ for least urbanized communities; range = 1.2-7.3‰ for all participants); and ii) estimated Δ(15) N values were negatively correlated with several indicators of animal protein intake (% nitrogen in diet: range = 0.9-7.6%). DISCUSSION: We hypothesize that a combination of several factors, which presumably included urea recycling and amino acid and protein recycling and/or de novo synthesis during metabolic processes, altered the Δ(15) N values of the participants.

Development, validation, and use of a semi-quantitative food frequency questionnaire for assessing protein intake in Papua New Guinean Highlanders.

OBJECTIVES: The aim of this article was to develop a semi-quantitative food frequency questionnaire (FFQ) and evaluate its validity to estimate habitual protein intake, and investigate current dietary protein intakes of Papua New Guinea (PNG) Highlanders. METHODS: A 32-item FFQ was developed and tested among 135 healthy male and female volunteers. The FFQ-estimated daily total and animal protein intakes were compared with biomarkers and 3-day Weighed Food Records (WFR) by correlation analyses, Bland-Altman plot analyses and joint classification analyses. RESULTS: The FFQ-estimated total protein intake significantly correlated with urinary nitrogen in the first morning void after adjusting urinary creatinine concentration (r = 0.28, P < 0.01) and the FFQ-estimated animal protein intake significantly correlated with the hair δ(15) N (Spearman's r = 0.34, P < 0.001). The limits of agreement were ±2.39 Z-score residuals for total protein intake and ±2.19 Z-score for animal protein intake, and intra-individual differences increased as protein intake increased. The classification into the same and adjacent quartiles was 66.0% for total protein intake and 73.6% for animal protein intake. Median daily total and animal protein intake estimates from the FFQ and the 3-day WFR showed a good agreement with differences of 0.2 and 4.9 g, respectively. None of the studied communities in the PNG Highlands met the biologically required protein intake; although the community closer to an urban center showed higher protein intake than the more remote communities. CONCLUSIONS: The newly developed 32-item FFQ for PNG Highlanders is applicable for evaluation of protein intake at the individual level. Am. J. Hum. Biol. 27:349-357, 2015. © 2014 Wiley Periodicals, Inc.

A multivalent peptide library approach identifies a novel Shiga toxin inhibitor that induces aberrant cellular transport of the toxin.

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 causes bloody diarrhea and hemorrhagic colitis in humans, sometimes resulting in fatal systemic complications. Among the known Stx family members, Stx2 is responsible for the most severe forms of disease. Stx2 binds to target cells via multivalent interactions between its B-subunit pentamer and globotriaosyl ceramide. After binding, it is first retrogradely transported to the Golgi and then to the endoplasmic reticulum (ER). Using a multivalent peptide library approach, we identified a tetravalent peptide that exhibits a high affinity for the Stx2 B-subunit pentamer (KD = 0.13 microM) and markedly inhibits Stx2 cytotoxicity. The tetravalent peptide exerted its inhibitory effects by inducing aberrant cellular transport of Stx2. Although the tetravalent peptide/Stx2 complex was incorporated into cells and translocated to the Golgi, this process was followed by the effective degradation of Stx2 in an acidic compartment rather than by its transfer to the ER. This peptide thoroughly protected mice from a fatal dose of E. coli O157:H7 even when administered after an established infection. Thus, the multivalent peptide library approach enabled the identification of a peptide-based Stx2 inhibitor that has remarkable therapeutic potency and appears to function by inducing aberrant cellular transport and degradation of Stx2.

Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin.

The dialyzer for Shiga-toxin elimination was developed and its performance was established. The dialyzer was prepared by immobilization of multivalent ligands. Glycoconjugate polymers having oligosaccharides and amino groups were synthesized to function as Shiga-toxin adsorbents. The amino group was utilized to immobilize the polymer inside the cellulose hollow fiber of the dialyzer. Cellulose hollow fibers packed in the dialyzer were carboxymethylated under moderate conditions. The glycoconjugate polymers were bound covalently to the hollow fibers of the dialyzer by condensation reaction between the amino group of the polymer and the carboxyl group of the cellulose hollow fiber. Shiga-toxin eliminabilities of the prepared dialyzers were evaluated at various conditions. Even at high concentration of protein such as FCS, the dialyzer showed an excellent performance for Shiga-toxin adsorption.

Structural analysis of the interaction between Shiga toxin B subunits and linear polymers bearing clustered globotriose residues.

We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

Identification of the optimal structure required for a Shiga toxin neutralizer with oriented carbohydrates to function in the circulation.

Shiga toxin (Stx) is a major virulence factor of Stx-producing Escherichia coli. Recently, we developed a therapeutic Stx neutralizer with 6 trisaccharides of globotriaosyl ceramide, a receptor for Stx, in its dendrimer structure (referred to as "SUPER TWIG [1]6") to function in the circulation. Here, we determined the optimal structure of SUPER TWIG for it to function in the circulation and identified a SUPER TWIG with 18 trisaccharides, SUPER TWIG (2)18, as another potent Stx neutralizer. SUPER TWIGs (1)6 and (2)18 shared a structural similarity, a dumbbell shape in which 2 clusters of trisaccharides were connected via a linkage with a hydrophobic chain. The dumbbell shape was found to be required for formation of a complex with Stx that enables efficient uptake and degradation of Stx by macrophages and, consequently, for potent Stx-neutralizing activity in the circulation. We also determined the binding site of the SUPER TWIGs on Stx.

Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections.

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.