dATP/ATP, a Multifunctional Nucleotide, Stimulates Bacterial Cell Lysis, Extracellular DNA Release and Biofilm Development

Background: Signaling by extracellular adenosine 59-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a ‘‘danger signal’’, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this ‘‘danger signal’’. Methods and Principal Findings: Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells. Conclusions and Significance: These data suggest that bacteria may sense extracellular dATP/ATP as a signal of ‘‘danger’’ and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an ‘‘inter-domain’ ’ warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells

Evaluation of Microfluidic Biosensor Development Using Microscopic Analysis of Molecular Beacon Hybridization

Abstract. Molecular beacons, oligonucleotide probes that fluoresce upon hybridization to a target nucleic acid, can be used in microfluidic devices to detect and quantify nucleic acids in solution as well as inside bacterial cells. Three essential steps towards the development of such devices as integrated microfluidic biosensors using molecular beacons were investigated in the present study. First, experiments using real-time confocal microscopy indicated that diffusion of DNA molecular beacons across a 100-µm diameter microfluidic channel took less than one minute after the flow of reagents was stopped. Second, experiments to evaluate hybridization kinetics of DNA molecular beacons with target nucleic acids in solution showed that DNA molecular beacons can be used to characterize hybridization kinetics in real time in microfluidic channels and that hybridization signals approached their maximum in approximately three minutes. Finally, it was demonstrated that peptide nucleic acid molecular beacons can be used to detect bacterial cells in microfluidic devices. These results suggest that the use of microfluidic devices to detect nucleic acids in solution and in bacterial cells is promising and that further development of an integrated microfluidic biosensor for bacterial detection based on this concept is warranted

Separation of the bacterial species, Escherichia coli, from mixed-species microbial communities for transcriptome analysis

Abstract Background The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis. Results E. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved. Conclusions A method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.http://deepblue.lib.umich.edu/bitstream/2027.42/112838/1/12866_2011_Article_1346.pd

Conceptualizing Human Microbiota: From Multicelled Organ to Ecological Community

The microbiota of a typical, healthy human contains 10 times as many cells as the human body and incorporates bacteria, viruses, archea, protozoans, and fungi. This diverse microbiome (the collective genomes of the microbial symbionts that inhabit a human host) is essential for human functioning. We discuss the unstated assumptions and implications of current conceptualizations of human microbiota: (1) a single unit that interacts with the host and the external environment; a multicelled organ; (2) an assemblage of multiple taxa, but considered as a single unit in its interactions with the host; (3) an assemblage of multiple taxa, which each interacts with the host and the environment independently; and (4) a dynamic ecological community consisting of multiple taxa each potentially interacting with each other, the host, and the environment. Each conceptualization leads to different predictions, methodologies, and research strategies

Animal Model Reveals Potential Waterborne Transmission of Helicobacter pylori Infection

BackgroundHelicobacter pylori infection has been consistently associated with lack of access to clean water and proper sanitation, but no studies have demonstrated that the transmission of H. pylori can occur from drinking contaminated water. In this study, we used a laboratory mouse model to test whether waterborne H. pylori could cause gastric infection.Materials and MethodsGroups of immunocompetent C57/BL6 Helicobacter‐free mice were exposed to static concentrations (1.29 × 105, 106, 107, 108, and 109 CFU/L) of H. pylori in their drinking water for 4 weeks. One group of Helicobacter‐free mice was exposed to uncontaminated water as a negative control. H. pylori morphology changes in water were examined using microscopy Live/Dead staining. Following exposure, H. pylori infection and inflammation status in the stomach were evaluated using quantitative culture, PCR, the rapid urease test, and histology.ResultsNone of the mice in the negative control or 105 groups were infected. One of 20 cages (one of 40 mice) of the 106 group, three of 19 cages (four of 38 mice) of the 107 CFU/L group, 19 of 20 cages (33 of 40 mice) of the 108 group, and 20 of 20 cages (39 of 40 mice) of the 109 CFU/L group were infected. Infected mice had significantly higher gastric inflammation than uninfected mice (27.86% higher inflammation, p < .0001).ConclusionsWe offer proof that H. pylori in water is infectious in mice, suggesting that humans drinking contaminated water may be at risk of contracting H. pylori infection. Much work needs to be performed to better understand the risk of infection from drinking H. pylori‐contaminated water.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113676/1/hel12216.pd

Bayesian Network Expansion Identifies New ROS and Biofilm Regulators

Signaling and regulatory pathways that guide gene expression have only been partially defined for most organisms. However, given the increasing number of microarray measurements, it may be possible to reconstruct such pathways and uncover missing connections directly from experimental data. Using a compendium of microarray gene expression data obtained from Escherichia coli, we constructed a series of Bayesian network models for the reactive oxygen species (ROS) pathway as defined by EcoCyc. A consensus Bayesian network model was generated using those networks sharing the top recovered score. This microarray-based network only partially agreed with the known ROS pathway curated from the literature and databases. A top network was then expanded to predict genes that could enhance the Bayesian network model using an algorithm we termed ‘BN+1’. This expansion procedure predicted many stress-related genes (e.g., dusB and uspE), and their possible interactions with other ROS pathway genes. A term enrichment method discovered that biofilm-associated microarray data usually contained high expression levels of both uspE and gadX. The predicted involvement of gene uspE in the ROS pathway and interactions between uspE and gadX were confirmed experimentally using E. coli reporter strains. Genes gadX and uspE showed a feedback relationship in regulating each other's expression. Both genes were verified to regulate biofilm formation through gene knockout experiments. These data suggest that the BN+1 expansion method can faithfully uncover hidden or unknown genes for a selected pathway with significant biological roles. The presently reported BN+1 expansion method is a generalized approach applicable to the characterization and expansion of other biological pathways and living systems

The Activity of Small Urea‐γ‐AApeptides Toward Gram‐Positive Bacteria

Host Defense Peptides (HDPs) have gained considerable interest due to the omnipresent threat of bacterial infection as a serious public health concern. However, development of HDPs is impeded by several drawbacks, such as poor selectivity, susceptibility to proteolytic degradation, low‐to‐moderate activity and requiring complex syntheses. Herein we report a class of lipo‐linear α/urea‐γ‐AApeptides with a hybrid backbone and low molecular weight. The heterogeneous backbone not only enhances chemodiversity, but also shows effective antimicrobial activity against Gram‐positive bacteria and is capable of disrupting bacterial membranes and killing bacteria rapidly. Given their low molecular weight and ease of access via facile synthesis, they could be practical antibiotic agents.Double‐AA peptides: We investigated a new class of small linear molecules as potential antibiotic agents against Gram‐positive bacteria. Our studies suggest that these compounds can disrupt bacterial membranes and kill bacteria rapidly. Given their low molecular weight and ease of accessibility through a facile synthesis approach, they are good candidates for development into antibiotic agents.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152544/1/cmdc201900520-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152544/2/cmdc201900520.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152544/3/cmdc201900520_am.pd

Multilevel Nitrogen Additions Alter Chemical Composition and Turnover of the Labile Fraction Soil Organic Matter via Effects on Vegetation and Microorganisms

Global nitrogen (N) deposition greatly impacts soil carbon sequestration. A 2- yr multiple N addition (0, 10, 20, 40, 80, and 160 kg N·ha- 1·yr- 1) experiment was conducted in alpine grassland to illustrate the mechanisms underlying the observed soil organic matter (SOM) dynamics on the Qinghai- Tibet Plateau (QTP). Labile fraction SOM (LF- SOM) fingerprints were characterized by pyrolysis- gas chromatography/tandem- mass spectrometry, and microbial functional genes (GeoChip 4.6) were analyzed in conjunction with LF- SOM fingerprints to decipher the responses of LF- SOM transformation to N additions. The significant correlations between LF- SOM and microbial biomass, between organic compounds in LF- SOM and compound degradation- related genes, and between LF- SOM and net ecosystem exchange implied LF- SOM were the main fraction utilized by microorganisms and the most sensitive fraction to N additions. The LF- SOM increased at the lowest N addition levels (10 and 20 kg N·ha- 1·yr- 1) and decreased at higher N addition levels (40 to 160 kg N·ha- 1·yr- 1), but the decrease of LF- SOM was weakened at 160 kg N·ha- 1·yr- 1 addition. The nonlinear response of LF- SOM to N additions was due to the mass balance between plant inputs and microbial degradation. Plant- derived compounds in LF- SOM were more sensitive to N addition than microbial- derived and aromatic compounds. It is predicted that when the N deposition rate increased by 10 kg N·ha- 1·yr- 1 on the QTP, carbon sequestration in the labile fraction may increase by nearly 170% compared with that under the current N deposition rate. These findings provide insight into future N deposition impacts on LF- SOM preservation on the QTP.Key PointsThe LF- SOM quantity increased at the lowest N additions (N10 and N20) and decreased from N40 to N160, but the decrease was weakened at the highest N addition (N160)Plant- derived compounds in LF- SOM were more sensitive to N addition than microbial- derived and aromatic compoundsThe organic compounds in LF- SOM were significantly correlated with compound degradation- related genesPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154963/1/jgrg21637_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154963/2/jgrg21637.pd

A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit–Activating Enzyme CahJ

Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.Breaking the barrier: Biofilm formation is employed by pathogenic microbes to defend against antibiotic action. This study probes both structurally and functionally CahJ, a key biosynthetic adenylation enzyme involved in generation of the cahuitamycin biofilm inhibitors, and lays a foundation for the development of effective new analogues.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145379/1/cbic201800233_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145379/2/cbic201800233.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145379/3/cbic201800233-sup-0001-misc_information.pd

An assessment of drinking water contamination with Helicobacter pylori in Lima, Peru

BackgroundHelicobacter pylori is a gut bacterium that is the primary cause of gastric cancer. H. pylori infection has been consistently associated with lack of access to sanitation and clean drinking water. In this study, we conducted time‐series sampling of drinking water in Lima, Peru, to examine trends of H. pylori contamination and other water characteristics.Materials and methodsDrinking water samples were collected from a single faucet in Lima’s Lince district 5 days per week from June 2015 to May 2016, and pH, temperature, free available chlorine, and conductivity were measured. Quantities of H. pylori in all water samples were measured using quantitative polymerase chain reaction. Relationships between the presence/absence and quantity of H. pylori and water characteristics in the 2015‐2016 period were examined using regression methods accounting for the time‐series design.ResultsForty‐nine of 241 (20.3%) of drinking water samples were contaminated with H. pylori. Statistical analyses identified no associations between sampling date and the likelihood of contamination with H. pylori. Statistically significant relationships were found between lower temperatures and a lower likelihood of the presence of H. pylori (P < .05), as well as between higher pH and higher quantities of H. pylori (P < .05).ConclusionsThis study has provided evidence of the presence of H. pylori DNA in the drinking water of a single drinking water faucet in the Lince district of Lima. However, no seasonal trends were observed. Further studies are needed to determine the presence of H. pylori in other drinking water sources in other districts in Lima, as well as to determine the viability of H. pylori in these water sources. Such studies would potentially allow for better understanding and estimates of the risk of infection due to exposure to H. pylori in drinking water.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142894/1/hel12462.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142894/2/hel12462_am.pd