Profiling the circulating miRNAs in mice exposed to gram-positive and gram-negative bacteria by Illumina small RNA deep sequencing
ABSTRACT: We profiled the expression of circulating microRNAs (miRNAs) in mice exposed to gram-positive and gram-negative bacteria using Illumina small RNA deep sequencing. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. Male C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain body temperature at 37°C. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) purchased from Caliper (Caliper, USA) were used to induce bacterial infection in the mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). To create mixed gram-negative and gram-positive bacterial infection, 1 × 108 Xen14 bacteria and 1 × 108 Xen29 bacteria/100 μL of PBS were used for wound contamination. Three animal models were used to create bacterial infection routes: subcutaneous injection (hereafter referred to as (I)), cut wound (hereafter referred to as (C)), and skin grafting (hereafter referred to as (S)). In the (I) model, E. coli and/or S. aureus suspensions were injected subcutaneously into the backs of the mice using an Fr. 25 needle. In the (C) model, a 1 cm incision wound was created in the midline of the back, smeared with E. coli and/or S. aureus suspension, and the wound was closed directly with a 4-0 nylon suture. In the (S) model, a 1×1 cm rectangular full- thickness skin graft was lifted from the backs of the mice, E. coli and/or S. aureus suspensions were spread over the wound bed, and the skin graft was reattached and closed with a 4-0 nylon suture. An additional group of animals in each of these three models was inoculated with PBS to serve as a negative control. Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer.
Project description:To explore the circulating miRNA expression after subcutaneous injection of Gram negative and positive bacteria in the mice The recombinant specific Gram negative pathogens Escherichia coli (xen14) and Gram positive pathogens Staphyllococcus aureus (xen29) were purchased from the Caliper (Caliper, Princeton, NJ, USA). 1×108 Escherichia coli or Staphyllococcus aureus pathogen in 100 μl PBS was injected subcutaneously with Fr. 25 needle into the back of the mice to cause bacterial infection of the mice. An extra group of animals was inoculated with PBS to serve as a negative control. The mice had access to food and water ad libitum both before and after bacteria injection. The mice were killed at the indicated time points (4, 8, and 24 h) after the bacteria injection, and whole blood was drawn.
Project description:The rise of multi-drug resistance in bacterial pathogens imposes the need to study these organisms from new angles. A little explored outset is to scrutinize bacterial niche adaptations and interactions among pathogenic and commensal bacteria, because they can provide a better understanding of the fitness of pathogens in their human host. We have previously shown that co-culturing of the pathogen Staphylococcus aureus with co-resident Klebsiella oxytoca or Bacillus thuringiensis wound isolates resulted in reduced levels of virulence factor secretion, suggesting that the presence of these co-resident bacteria would modulate S. aureus virulence. In the present study, we performed an in-depth investigation of changes in S. aureus gene expression upon co-cultivation with K. oxytoca and B. thuringiensis under infection-mimicking conditions. To this end, we profiled the cellular proteomes of the co-existing bacteria with special focus on S. aureus. In parallel, we employed RNA sequencing to highlight global changes in staphylococcal behaviour. The results imply that co-colonizing bacteria from chronic wounds can pacify S. aureus, and this conclusion was verified in a Galleria mellonella infection model. Altogether, our findings show that the presence of K. oxytoca and B. thuringiensis leads to massive rearrangements in S. aureus physiology and substantial reduction in virulence.
Project description:Purpose: The goal of this study is to explore the role of miRNAs in dairy cow response to E. coli and S. aureus, mastitis causing pathogens, is not well understood. Results: The global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing. A total of 231 known bovine miRNAs were identified with more than 10 counts per million (CPM) in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the total reads of known bovine miRNAs. One hundred and fifty novel miRNAs were identified and half of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P<0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to one for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (Bta-miR184, miR-24-3p, miR-148, miR-486 and bta-let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG (Kyoto encyclopedia of genes and genomes) pathways of the immune system, signal transduction, cellular process, nervous system, development and pathways in human diseases, especially cancer. Conclusion: Using next-generation sequencing, our study identified 150 novel bovine miRNAs and revealed a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. E. coli elicited an earlier differential regulation of miRNAs as opposed to a delayed regulation by S. aureus. Furthermore, target gene prediction showed significant enrichments for functions in different biological and cellular processes as well as KEGG pathways in immunity, development and human diseases. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis of mastitis and development of control measures. Bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing, no replicates, using illumina HiScanSQ platform.
Project description:Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions. Transcriptional profiling of two independent biological replicates of Pseudomonas aeruginosa biofilms, as grown to 72 hours and used as inocula applied to the murine wounds, was performed. A principle components analysis (PCA) was used to provide an overview of the transcriptome data from the 28-day mouse wound scab, comparing the data to the biofilm inoculum, and to published reports of P. aeruginosa biofilm and planktonic samples. The analysis shows that the transcriptome of the mouse wound scab was distinct from the biofilm inoculum that was applied to the wound, demonstrating a shift in biofilm gene expression following 28 days of infection. We sought to characterize P. aeruginosa activity within biofilms in the mouse wound model by isolating and identifying mRNA from the biofilms used as inocula and from the wound scabs 28 days post infection.
Project description:Oxaliplatin (oxPt) resistance in colorectal cancers (CRC) is a major unsolved problem. Consequently, predictive markers and a better understanding of resistance mechanisms are urgently needed. To investigate if the recently identified predictive miR-625-3p is functionally involved in oxPt resistance, stable and inducible models of miR-625-3p dysregulation were analyzed. Ectopic expression of miR-625-3p in CRC cells led to increased resistance towards oxPt. The mitogen-activated protein kinase (MAPK) kinase 6 (MAP2K6/MKK6) – an activator of p38 MAPK - was identified as a functional target of miR-625-3p, and, in agreement, was down-regulated in patients not responding to oxPt therapy. The miR-625-3p resistance phenotype could be reversed by anti-miR-625-3p treatment and by ectopic expression of a miR-625-3p insensitive MAP2K6 variant. Transcriptome, proteome and phosphoproteome profiles revealed inactivation of MAP2K6-p38 signaling as a possible driving force behind oxPt resistance. We conclude that miR-625-3p induces oxPt resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks.
Project description:The bacterial cell wall has been a celebrated target for antibiotics and holds real promise as a target for the discovery of new chemical matter to surmount pervasive multi-drug resistance among pathogenic bacteria. While the walls of Gram-negative bacteria are composed primarily of peptidoglycan, those of Gram-positives are more substantial and contain, in addition, large amounts of the polymer teichoic acid, covalently attached to peptidoglycan. Wall teichoic acids are a diverse group of phosphate-rich, extracellular polysaccharides that have been largely regarded as ancillary cell surface components. Recently, wall teichoic acid was shown to be essential to the proper rod-shaped cell morphology of the prototype Gram-positive bacterium Bacillus subtilis and an important virulence factor for the human pathogen Staphylococcus aureus. Thus wall teichoic acid synthesis is an intriguing target for the development of new cell wall-active antibiotics. Nevertheless, recent studies have shown that the dispensability of genes encoding teichoic acid biosynthetic enzymes in both B. subtilis and S. aureus is paradoxical and complex. Here, we report here on the discovery of a promoter (PywaC), which is sensitive to lesions in teichoic acid synthesis. Using this promoter we developed a luminescent, cell-based, reporter system to take a chemical-genetic approach to understanding the complexity of wall teichoic acid biogenesis using a large collection of antibiotics of well characterized biological activity. Our results reveal surprising interactions among undecaprenol, peptidoglycan and teichoic acid biosynthesis that help explain the complexity of teichoic acid gene dispensability. Furthermore, the new reporter assay represents an exciting avenue for the discovery of novel antibacterial molecules that impinge broadly on Gram-positive bacterial cell wall biogenesis. Keywords: comparison between depleted and repleted tagD mutant Overall design: A conditional xylose-inducible tagD mutant of B. subtilis was generated. Cells were grown in the presence and absence of xylose. Cells grown in the absence of xylose stopped growing after tagD depletion. Two time points were analysed. Time point 1: early depletion. Time point 2: late depletion, i.e. close to death.
Project description:We evaluated the responses of attack phase Bdellvobrio bacteriovorus HD100 when they are exposed to S. aureus biofilm. Overall design: Experiment design of RNA seqeuncing was done as described at 'Bdellovibrio bacteriovorus HD100, a predator of Gram-negative bacteria, benefits energetically from Staphylococcus aureus biofilms without predation'
Project description:A comprehensive –omic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of IAS smooth muscle contractile phenotype and basal tone. MicroRNA profiling, genome wide expression, validation and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a and rno-miR-206 were found to be up-regulated in aging IAS. qRT-PCR confirmed the up-regulated expression of these miRNAs and down regulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, SRF, Smad1, Smad2, RhoA/ROCK2, Fn1, Sm22-v2, Klf4, and Acta2) involved in regulation of SM contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist (U-46619 (thromboxane A2 analog))-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Lastly, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone, and suggests miR-133a is feasible therapeutic target in aging-associated rectoanal incontinence. Overall design: Three rats (Fischer 344) provided by the National Institutes of Aging of three different age groups (6 months, 18 months, and 26 months of age) were used for these studies. The smooth muscle cells (SMCs) from the internal anal sphincter (IAS) from above different age groups were isolated using collagenase dispersion method. The cells were subjected to RNA collection; and RNA from these samples were used for mRNA microarray analysis. For another sets of studies, from another batch of Fischer 344 rats (of 6 months, 18 months, and 26 months of age), the IAS SMCs were isolated and their miRNA fractions using miRVana miRNA Isolation kit following the manufacturer’s protocol were collected for miRNA microarray analysis.
Project description:Impaired cutaneous wound healing is a major complication in elderly people and patients suffering from diabetes with raising rates in industrialized countries. Heterogeneity of clinical manifestations hampers effective molecular diagnostics and decisions for appropriate therapeutic regimens. Using a customized positional quantitative proteomics workflow, we have established a time-resolved proteome and N-terminome resource from wound exudates in a clinical pig wound model that we exploited as robust template to interpret a heterogeneous dataset from patients undergoing the same wound treatment. With Zyxin, IQGAP1 and HtrA1, this analysis and validation by targeted proteomics identified differential abundances and proteolytic processing of proteins of epidermal and dermal origin as prospective biomarker candidates for assessment of critical turning points in wound progression. Thus, we demonstrate the power of a fine-tuned animal wound model to bridge the translational gap as prerequisite for future extended clinical studies with large cohorts of individuals affected by healing impairments.
Project description:Initial transcriptional response of human peripheral monocytes infected with a set of three gram-positive bacterial pathogens (Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae). Monocytes were isolated from five probands.