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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 1,153 records · Page 64Linked to original sources
bioRxiv2017

Annihilation of Methicillin-resistant Staphylococcus aureus via Photobleaching of Staphyloxanthin

Given that the dearth of new antibiotic development loads an existential burden on successful infectious disease therapy1, health organizations are calling for alternative approaches to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report a drug-free photonic approach to eliminate MRSA through photobleaching of staphyloxanthin, an indispensable membrane-bound antioxidant of S. aureus2-5. The photobleaching process, uncovered through a transient absorption imaging study and quantitated by absorption spectroscopy and mass spectrometry, decomposes staphyloxanthin and sensitizes MRSA to reactive oxygen species attack. Consequently, staphyloxanthin bleaching by low-level blue light eradicates MRSA synergistically with external or internal reactive oxygen species. The effectiveness of this synergistic therapy is validated in MRSA culture, MRSA-infected macrophage cells, S. aureus biofilms, and a mouse wound infection model. Collectively, these findings highlight broad applications of staphyloxanthin photobleaching for treatment of MRSA infections.

microbiology↗
bioRxiv2017

The physiological regulation of macropinocytosis during Dictyostelium growth and development

Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium as a model for macropinocytosis, we developed a high-throughput flow cytometry assay for macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. Dictyostelium has two feeding states: phagocytic and macropinocytic. When cells are switched from phagocytic growth on bacteria to liquid media, the rate of macropinocytosis slowly increases, due to increased size and frequency of macropinosomes. Upregulation is triggered by a minimal medium of 3 amino acids plus glucose and likely depends on macropinocytosis itself. Bacteria suppress macropinocytosis while their product, folate, partially suppresses upregulation of macropinocytosis. Starvation, which initiates development, does not of itself suppress macropinocytosis: this can continue in isolated cells, but is shut down by a conditioned-medium factor or activation of PKA signalling. Thus macropinocytosis is a facultative ability of Dictyostelium cells, regulated by environmental conditions that are identified here.\n\nSummaryA high-throughput flow cytometry assay shows that macropinocytosis in D. discoideum is upregulated in the presence of nutrients and absence of bacteria. Development and bacteria induce cells to downregulate macropinocytosis.

microbiology↗
bioRxiv2018

Fe oxidation by a fused cytochrome-porin common to diverse Fe-oxidizing bacteria

Iron (Fe) oxidation is one of Earths major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron-oxidizers. The genomes of many, diverse iron-oxidizers encode a homolog to an outer-membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron-oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 {+/-} 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. Sequence analysis and modeling reveal the entire Cyc2 family share a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. Based on structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron-oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism. ImportanceIron is practically ubiquitous across Earths environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or "rusting," can occur abiotically at near neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems. FeOB produce highly reactive Fe(III) oxyhydroxides that bind a variety of nutrients and toxins, thus these microbes are likely a controlling force in iron and other biogeochemical cycles. There has been mounting evidence that Cyc2 functions as an iron oxidase in neutrophiles, but definitive proof of its function has long eluded us. This work provides conclusive biochemical evidence of iron oxidation by Cyc2 from neutrophiles. Cyc2 is common to a wide variety of iron-oxidizers, including acidophilic and phototrophic iron-oxidizers, suggesting that this fused cytochrome-porin structure is especially well-adapted for iron oxidation.

microbiology↗
bioRxiv2017

Accurate Determination of Bacterial Abundances in Human Metagenomes Using Full-length 16S Sequencing Reads

DNA sequencing of PCR-amplified marker genes, especially but not limited to the 16S rRNA gene, is perhaps the most common approach for profiling microbial communities. Due to technological constraints of commonly available DNA sequencing, these approaches usually take the form of short reads sequenced from a narrow, targeted variable region, with a corresponding loss of taxonomic resolution relative to the full length marker gene. We use Pacific Biosciences single-molecule, real-time circular consensus sequencing to sequence amplicons spanning the entire length of the 16S rRNA gene. However, this sequencing technology suffers from high sequencing error rate that needs to be addressed in order to take full advantage of the longer sequence. Here, we present a method to model the sequencing error process using a generalized pair hidden Markov chain model and estimate bacterial abundances in microbial samples. We demonstrate, with simulated and real data, that our model and its associated estimation procedure are able to give accurate estimates at the species (or subspecies) level, and is more flexible than existing methods like SImple Non-Bayesian TAXonomy (SINTAX).

microbiology↗
bioRxiv2017

Pneumococcal vaccine impacts on the population genomics of non-typeable Haemophilus influenzae

Between 2008/09 and 2012/13 the molecular epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage in children <5 years of age was determined; a period that included pneumococcal conjugate vaccine (PCV) 13 introduction. Significantly increased carriage in post-PCV13 years was observed and lineage-specific associations with S. pneumoniae were observed before and after PCV13 introduction. NTHi were characterised into eleven discrete, temporally stable lineages, congruent with current knowledge regarding the clonality of NTHi. This increase could not be linked to the expansion of a particular clone and demonstrates different dynamics to before PCV13 implementation during which time NTHi co-carried with vaccine serotype pneumococci.

microbiology↗
bioRxiv2017

Electron microscopy reveals unique nano forms of bacterial spores

Endospore formation under environmental stress conditions is a well-established phenomenon for members of bacterial phylum Firmicutes, among which the most well studied ones belong to genus Bacillus and Clostridium. So far, known sizes of the spores are all larger than 500 nm. Nano-forms of bacteria have been reported but the notion still remains controversial. In this study, we provide visual evidences of living nano-entities (named here as nano-spores) formed by a bacterial species Bacillus cereus under prolonged stress, which are capable of escaping though standard sterile filtration procedure. The existence of nano-forms of bacteria was initially identified in a yeast ribosome preparation. We further demonstrate the transformation of the nano-spores into mature cells upon nutrient supply. Our study not only demonstrates the ability of bacteria to get transformed into yet-unknown form in order to survive under harsh environment, but also brings to light the existence of the smallest possible form of life.

microbiology↗
bioRxiv2017

Optogenetics reprogramming of planktonic cells for biofilm formation

Single-cell behaviors play essential roles during early-stage biofilms formation. In this study, we evaluated whether biofilm formation could be guided by precisely manipulating single cells behaviors. Thus, we established an illumination method to precisely manipulate the type IV pili (TFP) mediated motility and microcolony formation of Pseudomonas aeruginosa by using a combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation and adaptive microscopy. We termed this method as Adaptive Tracking Illumination (ATI). We reported that ATI enables the precise manipulation of TFP mediated motility and microcolony formation during biofilm formation by manipulating bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms can be controlled using ATI. Thus, the established method (i.e., ATI) can markedly promote ongoing studies of biofilms.

microbiology↗
bioRxiv2017

Automated detection of Mycobacterial growth on 96-well plates for rapid and accurate Tuberculosis drug susceptibility testing

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the minimum inhibitory concentrations (MICs) to be elucidated. The two participating laboratories each inoculated ten 96-well plates with the standard H37Rv reference strain and, after two weeks incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (> 30,000) of samples of M. tuberculosis from patients over the next few years.

microbiology↗
bioRxiv2017

Molecular Identification and Characterization of Two Rubber Dandelion Amalgaviruses

The Amalgaviridae family comprise persistent viruses that share the genome architecture of Totiviridae and gene evolutionary resemblance to Partitiviridae. Two genera have been assigned to this family, including genus Amalgavirus consisting in nine recognized species, corresponding to plant infecting viruses with dsRNA monosegmented genomes of ca. 3.4 kb. Here, we present the molecular characterization of two novel viruses detected in rubber dandelion (Taraxacum kok-saghyz). The sequenced viruses are 3,409 and 3,413 nt long, including two partially overlapping ORFs encoding a putative coat protein and an RNA-dependent RNA polymerase (RdRP). Phylogenetic insights based on the RdRP suggest them to be members of two new species within the Amalgavirus genus. Multiple independent RNAseq data suggest that the identified viruses have a dynamic distribution and low relative RNA levels in infected plants. Virus presence was not associated with any apparent symptoms on the plant hosts. We propose the names rubber dandelion latent virus 1 & 2 to the detected amalgaviruses; the first viruses to be associated to this emergent and sustainable natural rubber crop.

microbiology↗
bioRxiv2017

Timing of host feeding drives rhythms in parasite replication

Circadian rhythms enable organisms to synchronise the processes underpinning survival and reproduction to anticipate daily changes in the external environment. Recent work shows that daily (circadian) rhythms also enable parasites to maximise fitness in the context of ecological interactions with their hosts. Because parasite rhythms matter for their fitness, understanding how they are regulated could lead to innovative ways to reduce the severity and spread of diseases. Here, we examine how host circadian rhythms influence rhythms in the asexual replication of malaria parasites. Asexual replication is responsible for the severity of malaria and fuels transmission of the disease, yet, how parasite rhythms are driven remains a mystery. We perturbed feeding rhythms of hosts by 12 hours (i.e. diurnal feeding in nocturnal mice) to desynchronise the hosts peripheral oscillators from the central, light-entrained oscillator in the brain and their rhythmic outputs. We demonstrate that the rhythms of rodent malaria parasites in day-fed hosts become inverted relative to the rhythms of parasites in night-fed hosts. Our results reveal that the hosts peripheral rhythms (associated with the timing of feeding and metabolism), but not rhythms driven by the central, light-entrained circadian oscillator in the brain, determine the timing (phase) of parasite rhythms. Further investigation reveals that parasite rhythms correlate closely with blood glucose rhythms. In addition, we show that parasite rhythms resynchronise to the altered host feeding rhythms when food availability is shifted, which is not mediated through rhythms in the host immune system. Our observations suggest that parasites actively control their developmental rhythms. Finally, counter to expectation, the severity of disease symptoms expressed by hosts was not affected by desynchronisation of their central and peripheral rhythms. Our study at the intersection of disease ecology and chronobiology opens up a new arena for studying host-parasite-vector coevolution and has broad implications for applied bioscience.\n\nAuthor summaryHow cycles of asexual replication by malaria parasites are coordinated to occur in synchrony with the circadian rhythms of the host is a long-standing mystery. We reveal that rhythms associated with the time-of-day that hosts feed are responsible for the timing of rhythms in parasite development. Specifically, we altered host feeding time to phase-shift peripheral rhythms, whilst leaving rhythms driven by the central circadian oscillator in the brain unchanged. We found that parasite developmental rhythms remained synchronous but changed their phase, by 12 hours, to follow the timing of host feeding. Furthermore, our results suggest that parasites themselves schedule rhythms in their replication to coordinate with rhythms in glucose in the hosts blood, rather than have rhythms imposed upon them by, for example, host immune responses. Our findings reveal a novel relationship between hosts and parasites that if disrupted, could reduce both the severity and transmission of malaria infection.

microbiology↗
bioRxiv2017

A simple mung bean infection model for studying the virulence of Pseudomonas aeruginosa

Here we highlight the development of a simple and high throughput mung bean model to study virulence in the opportunistic pathogen Pseudomonas aeruginosa. The model is easy to setup and infection and virulence can be monitored for up to 10 days. In a first test of the model, we found that mung bean seedlings infected with PAO1 showed poor development of roots and high mortality rates compared to un-infected controls. We also found that a quorum sensing (QS) mutant was significantly less virulent when compared with the PAO1 wild type. Our work introduces a new tool for studying virulence in P. aeruginosa, that will allow for high throughput virulence studies of mutants, and for testing the in vivo efficacy of new therapies at a time when new antimicrobial drugs are desperately needed.

microbiology↗
bioRxiv2017

Light assisted antibiotics

The overuse of antibiotics is accelerating the bacterial resistance, and therefore there is a need to reduce the amount of antibiotics used for treatment. Here, we demonstrate that specific wavelengths in a narrow range around 296 nm are able to eradicate bacteria in the biofilm state more effectively, than antibiotics and the combination of irradiation and antibiotics is even better, introducing a novel concept light assisted antibiotics. The investigated wavelength range was 249 nm to 338 nm with an approximate step of 5 nm. The novel concept can significantly reduce the amount of antibiotics needed for eradicating mature bacterial biofilms. The irradiation treatment was combined with tobramycin and its efficiency was compared to combinatory antibiotic treatment and highly concentrated antibiotic monotherapy. The eradication efficacies, on mature biofilms, achieved by light assisted antibiotic and by the antibiotic monotherapy at 10-fold higher concentration, were equivalent. The present achievement could motivate the development of light assisted antibiotic treatments for treating infections.

microbiology↗
bioRxiv2017

The rust fungus Melampsora larici-populina expresses a conserved genetic program and distinct sets of secreted protein genes during infection of its two host plants, larch and poplar

Mechanims required for broad spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycle. Melampsora larici-populina infects two taxonomically unrelated plants, larch on which sexual reproduction is achieved and poplar on which clonal multiplication occurs leading to severe epidemics in plantations. High-depth RNA sequencing was applied to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia and aecia. Comparative transcriptomics of infection on poplar and larch hosts was performed using available expression data. Secreted protein was the only significantly over-represented category among differentially expressed M. larici-populina genes in basidia, pycnia and aecia compared together, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes commonly expressed on the two hosts and a fraction exhibiting a host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

microbiology↗
bioRxiv2017

ANTISEPTIC AGENTS ELICIT SHORT-TERM, PERSONALIZED AND BODY SITE-SPECIFIC SHIFTS IN RESIDENT SKIN BACTERIAL COMMUNITIES

Despite critical functions in cutaneous health and disease, it is unclear how resident skin microbial communities are altered by topical antimicrobial interventions commonly used in personal and clinical settings. Here we show that acute exposure to antiseptic treatments elicits rapid but short-term depletion of microbial community diversity and membership. Thirteen subjects were enrolled in a longitudinal treatment study to analyze the effects of topical treatments (ethanol, povidone-iodine, chlorhexidine, water) on the skin microbiome at two skin sites of disparate microenvironment: forearm and back. Treatment effects were highly dependent on personalized and body site-specific colonization signatures, which concealed community dynamics at the population level when not accounted for in this analysis. The magnitude of disruption was influenced by the identity and abundance of particular bacterial inhabitants. Lowly abundant members of the skin microbiota were more likely to be displaced, and subsequently replaced by the most abundant taxa prior to treatment. Members of the skin commensal family Propionibactericeae were particularly resilient to treatment, suggesting a distinct competitive advantage in the face of disturbance. These results provide insight into the stability and resilience of the skin microbiome, while establishing the impact of topical antiseptic treatment on skin bacterial dynamics and community ecology.

microbiology↗
bioRxiv2017

Folic acid instability and bacterial metabolism combine to impact C. elegans development and ageing

Supplementation with the synthetic oxidised folate, folic acid is used to prevent neural tube defects and other symptoms of folate deficiency. However, several unanswered questions remain over folic acid efficacy, safety and interactions with gut microbes. Prevention of a development defect caused by folate deficiency in the nematode worm Caenorhabditis elegans requires > 10 fold higher concentrations of folic acid compared to folinic acid, a reduced folate. Here we show that the major route for folic acid to restore normal development is indirect via the Escherichia coli used to feed C. elegans. This route occurs mainly via the E. coli transporter AbgT, which takes up the folic acid breakdown product para-aminobenzoate-glutamate (PABA-glu). We found that folic acid preparations, including a commercial supplement, contain 0.3- 4.0 % of this breakdown product. Previously, we have shown that inhibiting bacterial folate synthesis increases C. elegans lifespan by removing a life-shortening bacterial activity. Here, we show that folic acid restores bacterial folate synthesis and reverses this lifespan increase. It is still to be determined whether this bacterial route increases host folate levels in humans and if there are situations where increased bacterial folate synthesis has negative health complications.

microbiology↗
bioRxiv2017

The Combined activities of FtsW and MurJ are required for lipid II translocation during cell division in Escherichia coli

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in E. coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of how MurJ functions during cell division, which gives possibilities for future investigations and further antibiotics developments.

microbiology↗
bioRxiv2017

Diversity and evolution of the emerging Pandoraviridae family

With DNA genomes up to 2.5 Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting Pandoraviruses remained the most spectacular viruses since their description in 2013. Our isolation of three new strains from distant locations and environments allowed us to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses, led to the discovery of many non-coding transcripts while significantly reducing the former set of predicted protein-coding genes. We found that the Pandoraviridae exhibit an open pan genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggests that de novo gene creation is a strong component in the evolution of the giant Pandoravirus genomes.

microbiology↗
bioRxiv2017

Programmable transport of micro- and nanoparticles by Paramecium caudatum

We exploit chemo- and galvanotactic behaviour of Paramecium caudatum to design a hybrid device that allows for controlled uptake, transport and deposition of environmental micro- and nanoparticulates in an aqueous medium. Manipulation of these objects is specific, programmable and parallel. We demonstrate how device operation and output interpretation may be automated via a DIY low-cost fluorescence spectrometer, driven by a microprocessor board. The applications of the device presented range from collection and detoxification of environmental contaminants (e.g. nanoparticles), to micromixing, to natural expressions of computer logic.

microbiology↗