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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

Inferring time-derivatives, including cell growth rates, using Gaussian processes

Often the time-derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the population's growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time-derivatives as a function of time from time-series data. Our approach is based on established properties of Gaussian processes and therefore applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, allows interpolation with the corresponding error estimation, and can be applied to any number of experimental replicates. As illustrations, we infer growth rate from measurements of the optical density of populations of microbial cells and estimate the rate of in vitro assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies in a single yeast cell. Being accessible through both a GUI and from scripts, our algorithm should have broad application across the sciences.

Microbiology↗

bioRxiv2016

Rapid emergence of HCMV drug resistance in immunocompromised paediatric patients detected using target enrichment and deep sequencing

BackgroundCytomegalovirus can cause fatal disease in immunocompromised patients. With the advent of new anti-HCMV drugs there is interest in using virus sequence data to monitor resistance and identify new mutations.\n\nMethodsWe used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed paediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analysed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored.\n\nResultsTargeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient.\n\nConclusionsDeep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of eleven subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.

Microbiology↗

bioRxiv2016

The PAS domain-containing protein HeuR regulates heme uptake in Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial-derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu) is required for full colonization of the chicken gastrointestinal tract. Subsequent work found identified a PAS domain-containing regulator, termed HeuR, as required for chicken colonization. Here we confirmthat both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the heuR mutant being particularlyaffected during competition with wild-type C. jejuni. Transcriptomic analysis identified the chu genes-and those encoding other iron uptake systems-as likely regulatory targets of HeuR. Purified HeuR specifically bound the chuZA promoter region in electrophoretic mobility shift assays. Consistentwith a role forHeuR in chu expression, heuR mutants wereunable to efficiently use heme asa source of iron in iron-limitingconditions and, mutants exhibited decreased levels of cell-associated ironby massspectrometry.Finally, we demonstrate that a heuR mutant of C. jejuni isresistant to hydrogen peroxide, and that this resistance correlates to elevated levels ofcatalase activity.\n\nAuthor SummaryCampylobacter jejuni causes millions of gastrointestinal infection every year. This is primarily due to the its ability to reside in the gastrointestinal tract of chickens. C.jejuni contaminates chicken meat during harvesting and processing. Following consumption of undercooked chicken or uncooked food that was contaminated with raw chicken juice, humans develop a debilitating illness that is characterized by diarrhea and abdominal cramps. As chickens are the source of most human infections, there is a need to understand how C. jejuni colonizes chickens so we can develop ways to reduce its presence in chickens and thereby improve food safety. Most organisms require iron to thrive and that some bacteria steal iron from host molecules, including hemoglobin. Here we demonstrate that C. jejuni may need to get iron from hemoglobin in order to colonize the chicken and that aregulatory protein, HeuR, controls the ability ofthe bacteria to do this. If we can understand how this protein works, we may be able to develop ways to inhibit its function and reduce the ability of C. jejuni to get iron during chicken colonization. This would limit theamount of C. jejuni in the chicken and make food safer.

Microbiology↗

bioRxiv2016

Looking for a Signal in the Noise: Revisiting Obesity and the Microbiome

Two recent studies have re-analyzed published data and found that when datasets are analyzed independently there was limited support for the widely accepted hypothesis that changes in the microbiome are associated with obesity. This hypothesis was reconsidered by increasing the number of datasets and pooling the results across the individual datasets. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were applied to identify 10 studies for an updated and more synthetic analysis. Alpha diversity metrics and the relative risk of obesity based on those metrics were used to identify a limited number of significant associations with obesity; however, when the results of the studies were pooled using a random effects model significant associations were observed between Shannon diversity, number of observed OTUs, and Shannon evenness and obesity status. They were not observed for the ratio of Bacteroidetes and Firmicutes or their individual relative abundances. Although these tests yielded small P-values, the difference between the Shannon diversity index of non-obese and obese individuals was 2.07%. A power analysis demonstrated that only one of the studies had sufficient power to detect a 5% difference in diversity. When Random Forest machine learning models were trained on one dataset and then tested using the other 9 datasets, the median accuracy varied between 33.01 and 64.77% (median=56.67%). Although there was support for a relationship between the microbial communities found in human feces and obesity status, this association is relatively weak and its detection is confounded by large interpersonal variation and insufficient sample sizes.

Microbiology↗

bioRxiv2016

Total RNA Sequencing reveals microbial communities in human blood and disease specific effects

The role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain: schizophrenia, amyotrophic lateral sclerosis and bipolar disorder. By using high quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity and schizophrenia.

Microbiology↗

bioRxiv2016

Synergistic elimination of bacteria by phage and the immune system

Phage therapy has been viewed as a potential treatment for bacterial infections for over a century. Yet, the year 2016 marks the first phase I/II human trial of a phage therapeutic - to treat burn wound patients in Europe. The slow progress in realizing clinical therapeutics is matched by a similar dearth in principled understanding of phage therapy. Theoretical models and in vitro experiments find that combining phage and bacteria often leads to coexistence of both phage and bacteria or phage elimination altogether. Both outcomes stand in contrast to the stated goals of phage therapy. A potential resolution to the gap between models, experiments, and therapeutic use of phage is the hypothesis that the combined effect of phage and host immune system can synergistically eliminate bacterial pathogens. Here, we propose a phage therapy model that considers the nonlinear dynamics arising from interactions between bacteria, phage and the host innate immune system. The model builds upon earlier efforts by incorporating a maximum capacity of the immune response and density-dependent immune evasion by bacteria. We analytically identify a synergistic regime in this model in which phage and the innate immune response jointly contribute to the elimination of the target bacteria. Crucially, we find that in this synergistic regime, neither phage alone nor the innate immune system alone can eliminate the bacteria. We confirm these findings using numerical simulations in biologically plausible scenarios. We utilize our numerical simulations to explore the synergistic effect and its significance for guiding the use of phage therapy in clinically relevant applications.

Microbiology↗

bioRxiv2016

Characterizing and predicting cyanobacterial blooms in an 8-year amplicon sequencing time-course

Cyanobacterial blooms occur in lakes worldwide, producing toxins that pose a serious public health threat. Eutrophication caused by human activities and warmer temperatures both contribute to blooms, but it is still difficult to predict precisely when and where blooms will occur. One reason that prediction is so difficult is that blooms can be caused by different species or genera of cyanobacteria, which may interact with other bacteria and respond to a variety of environmental cues. Here we used a deep 16S amplicon sequencing approach to profile the bacterial community in eutrophic Lake Champlain over time, to characterize the composition and repeatability of cyanobacterial blooms, and to determine the potential for blooms to be predicted based on time-course sequence data. Our analysis, based on 135 samples between 2006 and 2013, spans multiple bloom events. We found that bloom events significantly alter the bacterial community without reducing overall diversity, suggesting that a distinct microbial community - including non-cyanobacteria - prospers during the bloom. We also observed that the community changes cyclically over the course of a year, with a repeatable pattern from year to year. This suggests that, in principle, bloom events are predictable. We used probabilistic assemblages of OTUs to characterize the bloom-associated community, and to classify samples into bloom or non-bloom categories, achieving up to 92% classification accuracy (86% after excluding cyanobacterial sequences). Finally, using symbolic regression, we were able to predict the start date of a bloom with 78-92% accuracy (depending on the data used for model training), and found that sequence data was a better predictor than environmental variables.

Microbiology↗

bioRxiv2016

Atypical Salmonella enterica serovars in murine and human infection models: Is it time to reassess our approach to the study of salmonellosis?

Nontyphoidal Salmonella species are globally disseminated pathogens and the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using in vivo murine models and cell lines typically challenged with Salmonella Typhimurium. Although serovars Enteritidis and Typhimurium are responsible for the most of human infections reported to the CDC, several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical S. enterica serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage model of infection is unsuitable for inferring human relevant differences in nontyphoidal Salmonella infections whereas differentiated human THP-1 macrophages allowed these isolates to be further characterised in a more relevant, human context.

Microbiology↗

bioRxiv2016

Linking comparative genomics and environmental distribution patterns of microbial populations through metagenomics

Combining well-established practices from comparative genomics and the emerging opportunities from assembly-based metagenomics can enhance the utility of increasing number of metagenome-assembled genomes (MAGs). Here we used protein clustering to characterize 48 MAGs and 10 cultivars based on their entire gene content, and linked this information to their environmental distribution patterns to better understand the microbial response to the 2010 Deepwater Horizon oil spill in the Gulf of Mexico coastline. Our results suggest that while most oil-associated bacterial populations originated from the ocean, a few actually emerged from the sand rare biosphere. These new findings suggest that there are considerable benefits to employ approaches from comparative genomics to study the whole content of newly identified genomes, and the investigation of emerging patterns in the environmental context can augment the efficacy of assembly-based metagenomic surveys.

Microbiology↗

bioRxiv2016

Refutation: Spontaneous secondary mutations confound analysis of the essential two component system WalKR in Staphylococcus aureus

Ji et al.,1 recently detailed the structure of the extracytoplasmic Per-Arnt-Sim (PAS) domain of WalK (WalKEC-PAS), the sensor kinase of the essential two-component system WalKR in Staphylococcus aureus. The authors made two independent walK mutants in S. aureus, each with a single amino acid alteration in WalKEC-PAS. They postulated from comparative structural analysis and primary sequence comparisons that these residues might be important for extra cellular signal transduction. We have also been exploring the function of WalKR and were surprised by the striking phenotypic impact of a single amino acid substitutions in the WalK sensor, which were contrary to our own unpublished observations.\n\nThe authors subjected their WalKEC-PAS mutants (WalKD119A and WalKVl49A) to a series of phenotypic screens to probe the function of this domain. Compared to the parental methicillin sensiti ...

Microbiology↗

bioRxiv2016

Modelling the effects of bacterial cell state and spatial location on tuberculosis treatment: Insights from a hybrid multiscale cellular automaton model

If improvements are to be made in tuberculosis (TB) treatment, an increased understanding of disease in the lung is needed. Studies have shown that bacteria in a less metabolically active state, associated with the presence of lipid bodies, are less susceptible to antibiotics, and recent results have highlighted the disparity in concentration of different compounds into lesions. Treatment success therefore depends critically on the responses of the individual bacteria that constitute the infection.\n\nWe propose a hybrid, individual-based approach that analyses spatio-temporal dynamics at the cellular level, linking the behaviour of individual bacteria and host cells with the macroscopic behaviour of the microenvironment. The individual elements (bacteria, macrophages and T cells) are modelled using cellular automaton (CA) rules, and the evolution of oxygen, drugs and chemokine dynamics are incorporated in order to study the effects of the microenvironment in the pathological lesion. We allow bacteria to switch states depending on oxygen concentration, which affects how they respond to treatment. This is the first multiscale model of its type to consider both oxygen-driven phenotypic switching of the Mycobacterium tuberculosis and antibiotic treatment. Using this model, we investigate the role of bacterial cell state and of initial bacterial location on treatment outcome. We demonstrate that when bacteria are located further away from blood vessels, less favourable outcomes are more likely, i.e. longer time before infection is contained/cleared, treatment failure or later relapse. We also show that in cases where bacteria remain at the end of simulations, the organisms tend to be slower-growing and are often located within granulomas, surrounded by caseous material.

Microbiology↗

bioRxiv2016

Metabolic Reconstruction and Modeling Microbial Electrosynthesis

Microbial electrosynthesis is a renewable energy and chemical production platform that relies on microbial taxa to capture electrons from a cathode and fix carbon. Yet the metabolic capacity of multispecies microbial communities on electrosynthetic biocathodes remains unknown. We assembled 13 genomes from a high-performing electroacetogenic culture, and mapped their transcriptional activity from a range of conditions. This allowed us to create a metabolic model of the primary community members (Acetobacterium, Sulfurospirillum, and Desulfovibrio). Acetobacterium was the primary carbon fixer, and a keystone member of the community. Based on transcripts upregulated near the electrode surface, soluble hydrogenases and ferredoxins from Acetobacterium and hydrogenases, formate dehydrogenase, and cytochromes of Desulfovibrio were essential conduits for electron flow from the electrode into the electrosynthetic community. A nitrogenase gene cluster with an adjacent ferredoxin and one of two Rnf complexes within the genome of the Acetobacterium were also upregulated on the electrode. Nitrogenase is known to serve as a hydrogenase, thereby it would contribute to hydrogen production by the biocathode. Oxygenases of microaerobic members of the community throughout the cathode chamber, including Sulfurospirillum and Rhodobacteraceae, were expressed. While the reactors were maintained anaerobically, this gene expression would support anaerobic growth and thus electrosynthesis by scrubbing small amounts of O2 out of the reactor. These molecular discoveries and metabolic modeling now serve as a foundation for future examination and development of electrosynthetic microbial communities.

Microbiology↗

bioRxiv2016

Novel Partitivirus Infection of Bat White-nose Syndrome (WNS) Fungal Pathogen Pseudogymnoascus destructans Links Eurasian and North American Isolates

Bat White-nose Syndrome (WNS) fungus Pseudogymnoascus destructans had caused mass mortality in the North American bats. A single clone of the pathogen (Hap_1) was likely introduced in the United States while Eurasian population comprised of several haplotypes. The origin and spread of P. destructans remain enigmatic due in part to a lack of precise population markers. We searched for P. destructans mycoviruses as they are highly host-specific, and their spread could provide a window on the origin of the host fungus. We discovered a P. destructans bipartite virus PdPV-1 with two double-stranded RNA (dsRNA) segments - LS (1,683 bp) and SS (1,524 bp) with motifs similar to viral RNA-dependent RNA polymerase (RdRp) and putative capsid proteins (CPs), respectively. Both LS and SS ORFs were embedded only in the positive strand of each dsRNA segment. Sequence alignments and phylogenetic analysis suggested that both segments constitute the genome of a new virus similar to the mycoviruses in the family Partitiviridae genus Gammapartitivirus. Purified viral particles appeared as isometric virions with approximately 33 nm diameters typical of partitiviruses. A newly developed RT-PCR assay revealed that all US isolates and only a few Eurasian isolates were infected with PdPV-1. PdPV-1 was P. destructans - specific as closely related non-pathogenic fungi P. appendiculatus and P. roseus tested negative. Thus, PdPV-1 establishes a link between the Eurasian and North American P. destructans. PdPV-1 could be used as an experimental tool to further investigate fungal biogeography, and the host - pathogen interactions.

Microbiology↗

bioRxiv2016

Optimization of an anti Staphylococcus antibiotic produced by tropical soil dwelling Streptomyces parvulus

An antibiotic produced by strain Streptomyces parvulus showing activity against Staphylococcus citreus was subjected to various optimization parameters for enhancing its production. Nutritional and physiological parameters produced by S. parvulus under shaken flask conditions were determined. Optimization of these parameters led to 11% increase in antibiotic activity with a mean zone of inhibition of 42 mm.\n\nHighest antibiotic production was obtained at 250 rpm for 14 days with optimum temperature of 28{degrees}C and pH 7. Kuster#x2019;s modified medium containing glycerol 0.7% (v/v), casein 0.03% (w/v), NaCl 0% (w/v), phosphate 0.25% (w/v), KNO3 0.1% (w/v) and CaCO3 0.0015% (w/v) concentration was found ideal.

Microbiology↗

bioRxiv2016

Comparative analysis highlights variable genome content of wheat rusts and divergence of the mating loci.

Three members of the Puccini genus, P. triticina (Pt), P. striiformis f.sp. tritici(Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; by comparison repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst (5.97 SNPs/kb) nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1,358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate enzyme degradation. Two allelic homeodomain, HD1 and HD2, pairs and three pheromone receptor (STE3) mating-type genes were identified in each dikaryotic Puccinia species. The HD proteins were active in a heterologous Ustilago maydis mating assay and host induced gene silencing of the HD and STE3 alleles reduced wheat host infection.

Microbiology↗

bioRxiv2016

Norovirus-mediated modification of the translational landscape via virus and host-induced cleavage of translation initiation factors.

Noroviruses produce viral RNAs lacking a 5 cap structure and instead use a virus-encoded VPg protein covalently linked to viral RNA to interact with translation initiation factors and drive viral protein synthesis. Norovirus infection results in the induction of the innate response leading to interferon stimulated gene (ISG) transcription. However the translation of the induced ISG mRNAs is suppressed. Using a novel mass spectrometry approach we demonstrate that diminished host mRNA translation correlates with changes to the composition of the eukaryotic initiation factor complex. The suppression of host ISG translation correlates with the activity of the viral protease (NS6) and the activation of cellular caspases leading to the establishment of an apoptotic environment. These results indicate that noroviruses exploit the differences between viral VPg-dependent and cellular cap-dependent translation in order to diminish the host response to infection.

Microbiology↗

bioRxiv2016

Characterization of Methicillin-resistant Staphylococcus aureus Isolates from Fitness Centers in Memphis Metropolitan Area, USA

Indoor skin-contact surfaces of public fitness centers may serve as reservoirs of potential human transmission of methicillin-resistant Staphylococcus aureus (MRSA). We found a high prevalence of multi-drug resistant (MDR)-MRSA of CC59 lineage harboring a variety of extracellular toxin genes from surface swab samples collected from inanimate surfaces of fitness centers in Memphis metropolitan area, USA. Our findings underscore the role of inanimate surfaces as potential sources of transmission of MDR-MRSA strains with considerable genetic diversity.

Microbiology↗

bioRxiv2016

When is a bacterial "virulence factor" really virulent?

Bacterial traits that contribute to disease are termed virulence factors and there is much interest in therapeutic approaches that disrupt such traits. However, ecological theory predicts disease severity to be multifactorial and context dependent, which might complicate our efforts to identify the most generally important virulence factors. Here, we use meta-analysis to quantify disease outcomes associated with one well-studied virulence factor - pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa. Consistent with ecological theory, we found that the effect of pyoverdine, albeit frequently contributing to disease, varied considerably across infection models. In many cases its effect was relatively minor, suggesting that pyoverdine is rarely essential for infections. Our work demonstrates the utility of meta-analysis as a tool to quantify variation and overall effects of purported virulence factors across different infection models. This standardised approach will help us to evaluate promising targets for anti-virulence approaches.

Microbiology↗