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bioRxiv2014

Horizontal Transfers and Gene Losses in the phospholipid pathway of Bartonella reveal clues about early ecological niches

Bartonellae are mammalian pathogens vectored by blood-feeding arthropods. Although of increasing medical importance, little is known about their ecological past, and host associations are underexplored. Previous studies suggest an influence of horizontal gene transfers in ecological niche colonization by acquisition of host pathogenicity genes. We here expand these analyses to metabolic pathways of 28 Bartonella genomes, and experimentally explore the distribution of bartonellae in 21 species of blood-feeding arthropods. Across genomes, repeated gene losses and horizontal gains in the phospholipid pathway were found. The evolutionary timing of these patterns suggests functional consequences likely leading to an early intracellular lifestyle for stem bartonellae. Comparative phylogenomic analyses discover three independent lineage-specific reacquisitions of a core metabolic gene - NAD(P)H-dependent glycerol-3-phosphate dehydrogenase (gpsA) - from Gammaproteobacteria and Epsilonproteobacteria. Transferred genes are significantly closely related to invertebrate Arsenophonus-, and Serratia-like endosymbionts, and mammalian Helicobacter-like pathogens, supporting a cellular association with arthropods and mammals at the base of extant bartonellae. Our studies suggest that the horizontal re-aquisitions had a key impact on bartonellae lineage specific ecological and functional evolution.

Microbiology↗

bioRxiv2014

The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin

Influenza is notable for its evolutionary capacity to escape immunity targeting the viral hemagglutinin. We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability. We created mutant viruses that incorporate most of the {approx} 104 amino-acid mutations to hemagglutinin from A/WSN/1933 (H1N1) influenza. After passaging these viruses in tissue culture to select for functional variants, we used deep sequencing to quantify mutation frequencies before and after selection. These data enable us to infer the preference for each amino acid at each site in hemagglutinin. These inferences are consistent with existing knowledge about the proteins structure and function, and can be used to create a model that describes hemagglutinins evolution far better than existing phylogenetic models. We show that hemagglutinin has a high inherent tolerance for mutations at antigenic sites, suggesting that this is one factor contributing to influenzas antigenic evolution.

Microbiology↗

bioRxiv2014

A putative antiviral role of plant cytidine deaminases

A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade. Host cytidine deaminases (e.g., APOBEC3 proteins) edit viral genomes giving raise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis. By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity. To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation. The model plant Arabidopsis thaliana encodes for nine cytidine deaminases (AtCDAs), raising the question of whether deamination is an antiviral mechanism in plants as well. Here we tested the effects of AtCDAs expression on the pararetrovirus Cauliflower mosaic virus (CaMV). We show that A. thaliana AtCDA1 gene product exerts a mutagenic activity, which indeed generates a negative correlation between the level of AtCDA1 expression and CaMV accumulation in the plant, suggesting that deamination may also work as an antiviral mechanism in plants.

Microbiology↗

bioRxiv2014

Bacillus Calmette-Guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation

Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M.bovis BCG was most commonly recovered (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and NOX2 knock-out mice. In addition we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality ([~] 50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-, IFN-{gamma}, IL-17 and IL-12) to BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and impaired granuloma formation.

Microbiology↗

bioRxiv2014

A New Perspective on HIV Cure

Introduction Introduction Mucosal effects of tenofovir An emerging hypothesis ART and anatomic sites... Cure without ART Conclusion and outlook References Current antiretroviral treatment (ART) is highly effective in controlling HIV replication and in many patients suppresses the number of virions measurable in peripheral blood, i.e., the HIV viral load, to undetectable levels. Nevertheless, whenever ART is stopped, HIV levels rebound and the disease returns. This lack of eradication is attributed to a stable latent reservoir of HIV-1 in resting CD4+ T lymphocytes and perhaps other susceptible cell types such as macrophages1. These cells harbor HIV in the form of proviruses that are integrated into the host chromosomal genome. During effective ART the de ...

Microbiology↗

bioRxiv2014

Induced sensitivity of Bacillus subtilis colony morphology to mechanical media compression

Bacterial from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly) or merely physical.\n\nHere we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained) state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells.\n\nThese results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus is a physical rather than biological phenomenon dependent on a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof.

Microbiology↗

bioRxiv2014

Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes

Comprehensive genome-wide analyses of bacterial DNA methylation have not been possible until the recent advent of single molecule, real-time (SMRT) sequencing. This technology enables the direct detection of N6-methyladenine (6mA) and 4-methylcytosine (4mC) at single nucleotide resolution on a genome-wide scale. The distributions of these two major types of DNA methylation, along with 5-methylcytosine (5mC), comprise the bacterial methylome, some rare exceptions notwithstanding. SMRT sequencing has already revealed marked diversity in bacterial methylomes as well as the existence of heterogeneity of methylation in cells in single bacterial colonies, where such epigenetic variation can enable bacterial populations to rapidly adapt to changing conditions. However, current methods for studying bacterial methylomes using SMRT sequencing mainly rely on population-level summaries that do not provide the single-cell resolution necessary for dissecting the epigenetic heterogeneity in bacterial populations. Here, we present a novel SMRT sequencing-based framework, consisting of two complementary methods, for single molecule-level detection of DNA methylation and assessment of methyltransferase activity through single molecule-level long read-based epigenetic phasing. Using seven bacterial strains and integrating data from SMRT and Illumina sequencing, we show that our method yields significantly improved resolution compared to existing population-level methods, and reveals several distinct types of epigenetic heterogeneity. Our approach enables new investigations of the complex architecture and dynamics of bacterial methylomes and provides a powerful new tool for the study of bacterial epigenetic control.

Microbiology↗

bioRxiv2014

Mucosal effects of tenofovir 1% gel

BACKGROUNDTenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against sexual HIV transmission. Because this is a new prevention strategy targeting large numbers of healthy people, we broadly assessed its effects on the mucosa.\n\nMETHODS AND FINDINGSIn MTN-007, a phase 1, randomized, double-blinded rectal microbicide trial, we used systems genomics/proteomics to determine the effect of tenofovir 1% gel, nonoxynol-9 2% gel, placebo gel or no treatment on rectal biopsies taken at baseline, after one application or after seven daily applications (15 subjects/arm). Experiments were repeated using primary vaginal epithelial cells from four healthy women. After seven days of administration, tenofovir 1% gel had broad-ranging biological effects on the rectal mucosa, which were much more pronounced than--but different from--those caused by the detergent nonoxynol-9. Tenofovir profoundly suppressed anti-inflammatory mediators such as interleukin 10; increased T cell densities; caused mitochondrial dysfunction, possibly by blocking PNPT1 expression; and altered regulatory pathways of cell differentiation and survival. Except for leukocyte-derived factors, all these effects were replicated in primary vaginal epithelial cells, which also proliferated significantly faster in tenofovir's presence.\n\nCONCLUSIONSTenofovir's suppression of anti-inflammatory activity could diminish its prophylactic efficacy over time. The breadth of mucosal changes, including mitochondrial dysfunction and epithelial proliferation, raises questions about its safety for long-term topical use. These findings suggest that a systems biology evaluation of mucosal effects may be beneficial before advancing to large-scale efficacy trials with topical HIV prevention agents that achieve high, long-lasting local drug concentrations.

Microbiology↗

bioRxiv2014

A simple method for constructing magnetic Escherichia coli

Magnetic force can serve as an ideal way to control the spatial behavior of microorganisms, because of its flexibility and penetrability. By incubation with the biocompatible compound, ammonium ferric citrate, as an iron source, we magnetized Escherichia coli, the most programmable chassis in synthetic biology. To enhance the magnetization efficiency, the ferritin protein, FtnA, from E. coli was cloned and overexpressed in strain BL21(DE3). The magnetization effect was observed within 30 min after harvest of bacteria, and the concentration of ammonium ferric acid used could be as low as 0.5 mM. Using different shapes of magnetic fields, different patterns could be generated easily. Our method may set up the foundation for a rational design of spatial structure of cell communities, which is important for their actual application.

Microbiology↗

bioRxiv2014

Independent Co-option of a Tailed Bacteriophage into a Killing Complex in Pseudomonas

Competition between microbes is widespread in nature, especially among those that are closely related. To combat competitors, bacteria have evolved numerous protein-based systems (bacteriocins) that kill strains closely related to the producer. In characterizing the bacteriocin complement and killing spectra for the model strain P. syringae B728a, we discovered its activity was not linked to any predicted bacteriocin, but is derived from a prophage. Instead of encoding an active prophage, this region encodes a bacteriophage-derived bacteriocin, termed an R-type syringacin. The R-type syringacin is striking in its convergence with the well-studied R-type pyocin of P. aeruginosa in both chromosomal synteny and molecular function. Genomic alignment, amino acid percent sequence identity, and phylogenetic inference all support a scenario where the R-type syringacin has been co-opted independently of the R-type pyocin. Moreover, the presence of this region is conserved among several other Pseudomonas species, and thus is likely important for intermicrobial interactions throughout this important genus.

Microbiology↗

bioRxiv2014

Genetic relateness of Haemophilus parasuis among reference strains and Chinese epidemic isolates

Haemophilus parasuis is the causative agent of Glassers disease and a commensal coloniser of the porcine upper respiratory tract. Multiple complex factors, including the early weaning of piglets and the management of high health status farms, make it a re-emerging agent, responsible for a recent increase in the prevalence and severity of disease in pigs in China. However, little genetic information is known about Chinese epidemic isolates. In this study, a phylogenetic method for genotyping the H. parasuis population with available Chinese epidemic isolates and reference strains from different origins is presented. Phylogenetic analysis confirmed that there are at least two different genotypes in H. parasuis population and a unique Chinese lineage with virulence results in the previous epidemics.

Microbiology↗

bioRxiv2015

Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation.

1Biological soil crusts (BSC) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N2-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N2-fixation is largely attributed to heterocystous cyanobacteria, however, early successional crusts possess few N2-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N2-fixation during the critical early stages of BSC development. DNA stable isotope probing (DNA-SIP) with 15N2 revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate 15N2 in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N2fixation has previously been observed in this family. The Proteobacteria identified share >98.5 %SSU rRNA gene sequence identity with isolates from genera known to possess diazotrophs (e.g. Pseudomonas, Klebsiella, Shigella, and Ideonella). The low abundance of these heterotrophic diazotrophs in BSC may explain why they have not been characterized previously. Diazotrophs play a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSC in arid ecosystems.

Microbiology↗

bioRxiv2015

Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection

Strangles, the most frequently diagnosed infectious disease of horses worldwide, is caused by Streptococcus equi. Despite its prevalence, the global diversity and mechanisms underlying the evolution of S. equi as a host-restricted pathogen remain poorly understood. Here we define the global population structure of this important pathogen and reveal a population replacement in the late 19th or early 20th century, contemporaneous with a spate of global conflicts. Our data reveal a dynamic genome that continues to mutate and decay, but also to amplify and acquire genes despite the organism having lost its natural competence and become host-restricted.\n\nThe lifestyle of S. equi within the horse is defined by short-term acute disease, strangles, followed by long-term carriage. Population analysis reveals evidence of convergent evolution in isolates from post-acute disease samples, as a result of niche adaptation to persistent carriage within a host. Mutations that lead to metabolic streamlining and the loss of virulence determinants are more frequently found in carriage isolates, suggesting that the pathogenic potential of S. equi reduces as a consequence of long term residency within the horse post acute disease. An example of this is the deletion of the equibactin siderophore locus that is associated with iron acquisition, which occurs exclusively in carrier isolates, and renders S. equi significantly less able to cause acute disease in the natural host. We identify several loci that may similarly be required for the full virulence of S. equi, directing future research towards the development of new vaccines against this host-restricted pathogen.

Microbiology↗

bioRxiv2015

Intragenomic homogeneity on Liberibacter 16S rDNA confirms phylogeny and explains ecological strategy

Three of the five currently recognized \"Candidatus Liberibacter\" spp., \"Ca. L. asiaticus\" (Las), \"Ca. L. americanus\" (Lam) and \"Ca. L. solanacearum\", and the newly erected genus Liberibacter species, L. crescens (Lcr), have had their genomes sequenced. In all four cases there are three homogeneous copies of the 16S rRNA gene, one present as the reverse complement of the other two. 16S intragenic homogeneity is common within the -Proteobacteria. The presence of three 16S rRNA copies indicates an advantage for a rapid response of population increase to favourable growth conditions. The metabolic cost of carrying multiple copies is avoided during periods of low cellular activity as this situation occurs at low temperatures, for example overwintering in deciduous plants or in a dormant insect host.\n\nA large insertion in the 16S rDNA sequence of three species compared to the other three species indicates a dichotomy in the Liberibacter genus and provides a phylogenetic signal of closeness to the proximal node within the Rhizobiaceae. In spite of similar symptoms in Citrus crops associated with Lam and Las infections, these species belong on either side of this dichotomy, thus confirming Lam as phylogenetically closer to the proximal node than Las.

Microbiology↗

bioRxiv2015

Cryptic haplotypes of "Candidatus Liberibacter africanus"

\"Candidatus Liberibacter africanus\" (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease.\n\nSomewhat similar symptoms in the Rutaceous tree Calodendrum capensis (Cape Chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of both commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in the citrus crop, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published.\n\nUntil fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. Resolving these nucleotide positions by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be reduced to that of a haplotype of Laf.\n\nFurther, the six 16S rRNA gene sequences currently available in GenBank identified as the species Laf separate into 2 haplotype groups. The 3 haplotypes of Laf are therefore LafA designated as the first accession sequenced (L22533), LafC for the former capensis subspecies and to recognise the prior use of this term, and LafB for the third haplotype not previously recognised. Thus the cryptic presence of 3 haplotypes is revealed by this review of the Laf 16S rDNA sequences.

Microbiology↗

bioRxiv2015

Variation in rural African gut microbiota is strongly correlated with colonization by Entamoeba and subsistence

The human gut microbiota is impacted by host nutrition and health status and therefore represents a potentially adaptive phenotype influenced by metabolic and immune constraints. Previous studies contrasting rural populations in developing countries to urban industrialized ones have shown that industrialization is strongly correlated with patterns in human gut microbiota; however, we know little about the relative contribution of factors such as climate, diet, medicine, hygiene practices, host genetics, and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiota of populations inhabiting similar environments but having different traditional subsistence modes and either shared or distinct genetic ancestry, and (ii) examine the relationship between gut parasites and bacterial communities. Characterizing the fecal microbiota of Pygmy hunter-gatherers as well as Bantu individuals from both farming and fishing populations in Southwest Cameroon, we found that the gut parasite Entamoeba is significantly correlated with microbiome composition and diversity. We show that across populations, colonization by this protozoa can be predicted with 79% accuracy based on the composition of an individual's gut microbiota, and that several of the taxa most important for distinguishing Entamoeba absence or presence are signature taxa for autoimmune disorders. We also found gut communities to vary significantly with subsistence mode, notably with some taxa previously shown to be enriched in other hunter-gatherers groups (in Tanzania and Peru) also discriminating hunter-gatherers from neighboring farming or fishing populations in Cameroon.\n\nAuthor SummaryThe community of microorganisms inhabiting the gastrointestinal tract plays a critical role in determining human health. Its been hypothesized that the industrialized lifestyle, marked by a diet rich in processed foods, higher use of antibiotics, increased hygiene, and exposure to various chemicals, has altered microbiota in ways that are harmful. Studies have addressed this by comparing rural and industrialized populations, and have found that they systematically vary in their gut microbiome composition. Nevertheless, the relative influence of host genetics, diet, climate, medication, hygiene practices, and parasitism is still not clear. In addition, microbial variation between nearby human populations has not been explored in depth. Moreover, The World Health Organization estimates that 24% of the worlds population, concentrated in developing countries, is infected with gut parasites. Despite this, and evidence for direct interactions between the immune system and both gut parasites and bacteria, we know relatively little about the relationship between gut helminths, protozoa, and bacteria. In our study, we aimed to address some of this complexity. To do so, we characterized the gut microbial communities and parasites from Pygmy hunter-gatherer and Bantu farming and fishing populations from seven locations in the rainforest of Southwest Cameroon. We found that both subsistence mode and the presence of the gut protozoa, Entamoeba, were significantly correlated with microbiome composition. These findings support previous studies demonstrating diet is an important determinant of gut microbiota, and further show that this pattern holds true at a local scale, in traditional societies inhabiting a similar environment. Additionally, we show a significant relationship between a common human parasite (Entamoeba) and gut bacterial community composition, suggesting potential important interactions between the immune system, gut bacteria, and gut parasites, highlighting the need for more hierarchical cross population studies that include parasitism as potential factor influencing gut microbiota dynamics.

Microbiology↗

bioRxiv2015

External Microbiota of Western United States Bats: Does It Matter Where You Are From?

White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans1, has spread west from New York to Missouri and has killed more than six million bats2. In bat hibernacula where WNS is present, mass mortality has been observed and there is a high potential for population collapse or extinction of some species at a regional level. Although WNS is not yet present in the western U.S., the high diversity ofbat species3 and appropriate conditions for P. destructans in area caves may put these populations at risk. The absence of WNS in western caves provides a unique opportunity to ask questions about how bat species, geographic location, and habitat shape pre-WNS bat microbiota. The importance of microbiota is shown in many organisms, including amphibians, where individuals that survive a chytrid infection carry a higher prevalence of Janthinobacterium lividum4. The establishment of a pre-WNS baseline microbiota of western bats is critical to understanding how P. destructans may impact the native microbiota of the bats. Previous studies5,6 that identified the microbiota of bats have focused on gut and fecal microbiota, with little attention given to the external microbiota. Here we show for the first time that habitat and geography influence differences in the abundance and diversity of external bat microbiota. From our 202 (62 cave-netted, 140 surface-netted) bat samples belonging to 13 species of western bats uninfected with WNS, we identified differences in microbiota diversity among sites, and between cave-netted versus surface-netted bats, regardless of sex and species. These results present novel information about the factors that shape external microbiota of bats providing new insights into patterns of diversity in a pre-WNS bat population.

Microbiology↗

bioRxiv2015

Multiple new species of Ophiocordyceps on ants

In tropical forests, one of the most common relationships between parasites and insects is that between the fungus Ophiocordyceps (Ophiocordycipitaceae, Hypocreales, Ascomycota) and ants, especially within the tribe Camponotini. These fungi have the ability to penetrate the exoskeleton of the ant and to manipulate the behavior of the host, making it leave the nest and ascend understorey shrubs, to die biting onto the vegetation: hence, the term zombie-ant fungi to describe this behavioral changes on the host. It is posited that this behavioral change aids spore dispersal and thus increases the chances of infection. Despite their undoubted importance for ecosystem functioning, these fungal pathogens are still poorly documented, especially regarding their diversity, ecology and evolutionary relationships. Here, we describe multiple new and host-specific species of the genus Ophiocordyceps on Camponotus and Polyrhachis ants from the central Amazonian region of Brazil, USA, Australia and Japan, which can readily be separated using classic taxonomic criteria, in particular ascospore morphology.

Microbiology↗