Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

BACKGROUND: Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE and also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. RESULTS: We find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency. CONCLUSIONS: These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

Genome sequence of Listeria monocytogenes 07PF0776, a cardiotropic serovar 4b strain.

Listeria monocytogenes is a food-borne bacterial pathogen commonly associated with serious invasive infections of the central nervous system or of the developing fetus. We present the genome sequence of Listeria monocytogenes 07PF0776, a serovar 4b isolate from a human myocardial abscess that exhibits enhanced invasion of cardiac tissue.

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses.

BACKGROUND: Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae. RESULTS: The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor. CONCLUSIONS: Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.

Loss of Interleukin-6 Influences Transcriptional Immune Signatures and Alters Bacterial Colonization in the Skin.

The skin functions as a protective barrier to inhibit the entry of foreign pathogens, all the while hosting a diverse milieu of microorganisms. Over time, skin cells, immune cells, cytokines, and microbes interact to integrate the processes of maintaining the skin's physical and immune barrier. In the present study, the basal expression of two immunologically divergent mouse strains C57BL/6 and BALB/c, as well as a strain on the C57 background lacking IL-6, was characterized. Additionally, cutaneous antimicrobial gene expression profiles and skin bacterial microbiome were assessed between strains. Total RNA sequencing was performed on untreated C57BL/6 (control), BALB/c, and IL-6-deficient skin samples and found over 3,400 genes differentially modulated between strains. It was found that each strain modulated its own transcriptional "profile" associated with skin homeostasis and also influenced the overall bacterial colonization as indicated by the differential phyla present on each strain. Together, these data not only provide a comprehensive view of the transcriptional changes in homeostatic skin of different mouse strains but also highlight the possible influence of the strain differences (e.g., Th1/Th2 balance) as well as a role for IL-6 in overall skin immunity and resident microbial populations.

Transcriptional profiling of irritant contact dermatitis (ICD) in a mouse model identifies specific patterns of gene expression and immune-regulation.

BACKGROUND: Irritant contact dermatitis (ICD) is a cutaneous inflammatory response to a variety of triggers that requires no sensitization and accounts for up to 80% of occupational dermatitis cases. IL-6 has been alternately associated with both allergic and irritant dermatitis and is closely linked to skin wound healing, therefore making it an ideal candidate to investigate in the mechanism of ICD. RESULTS: Despite being a well-known pro-inflammatory cytokine, IL-6 deficient (IL-6KO) mice show much more severe ICD than controls. Transcriptome analysis was employed to examine irritant-exposed and control skin samples from C57BL/6 and IL-6KO mice. Over 1900 transcripts were found differentially modulated between C57 (1184 total) and IL-6KO (802 total) mice with the magnitude of expression significantly disparate. Overall gene ontology revealed metabolic and cellular enriched functional processes but numerous pro-inflammatory and immune associated genes (Cxcl2, Cxcl3, Cxcl5, Acod, Hamp, c-Lectins, for example), keratin associated genes (Krt6b and various Krtaps), and members of the Sprr and Lce family, which promote skin barrier integrity and keratinocyte functions, were also differentially modulated. CONCLUSIONS: The altered expression of these genes may provide a potential mechanism to explain the increased ICD severity in IL-6-deficient mice. Overall, this study offers new insight into the pathogenesis of ICD, indicates new mediators/biomarkers that may influence the variability of responses to irritants and provides potential targets for therapeutic development.

Neuroinvasive Listeria monocytogenes Infection Triggers IFN-Activation of Microglia and Upregulates Microglial miR-155.

MicroRNA (miR) miR-155 modulates microglial activation and polarization, but its role in activation of microglia during bacterial brain infection is unclear. We studied miR-155 expression in brains of C57BL/6 (B6.WT) mice infected i.p. with the neuro-invasive bacterial pathogen Listeria monocytogenes (L. monocytogenes). Infected mice were treated with ampicillin starting 2 days (d) post-infection (p.i.) and analyzed 3d, 7d, and 14d p.i. Virulent L. monocytogenes strains EGD and 10403s upregulated miR-155 in whole brain 7 d p.i. whereas infection with avirulent, non-neurotropic Δhly or ΔactA L. monocytogenes mutants did not. Similarly, infection with virulent but not mutated bacteria upregulated IFN-γ mRNA in the brain at 7 d p.i. Upregulation of miR-155 in microglia was confirmed by qPCR of flow cytometry-sorted CD45intCD11bpos brain cells. Subsequently, brain leukocyte influxes and gene expression in sorted microglia were compared in L. monocytogenes-infected B6.WT and B6.Cg-Mir155tm1.1Rsky/J (B6.miR-155-/-) mice. Brain influxes of Ly-6Chigh monocytes and upregulation of IFN-related genes in microglia were similar to B6.WT mice at 3 d p.i. In contrast, by d 7 p.i. expressions of microglial IFN-related genes, including markers of M1 polarization, were significantly lower in B6.miR-155-/- mice and by 14 d p.i., influxes of activated T-lymphocytes were markedly reduced. Notably, CD45highCD11bpos brain cells from B6.miR-155-/- mice isolated at 7 d p.i. expressed 2-fold fewer IFN-γ transcripts than did cells from B6.WT mice suggesting reduced IFN-γ stimulation contributed to dampened gene expression in B6.miR-155-/- microglia. Lastly, in vitro stimulation of 7 d p.i. brain cells with heat-killed L. monocytogenes induced greater production of TNF in B6.miR-155-/- microglia than in B6.WT microglia. Thus, miR-155 affects brain inflammation by multiple mechanisms during neuroinvasive L. monocytogenes infection. Peripheral miR-155 promotes brain inflammation through its required role in optimal development of IFN-γ-secreting lymphocytes that enter the brain and activate microglia. Microglial miR-155 promotes M1 polarization, and also inhibits inflammatory responses to stimulation by heat-killed L. monocytogenes, perhaps by targeting Tab2.

Pretreatment with troglitazone decreases lethality during endotoxemia in mice.

Troglitazone is an oral antidiabetic drug that is a ligand for peroxisome proliferator activated receptor gamma (PPARgamma). Based on other studies that have implicated an immunosuppressive role for PPARgamma during inflammatory responses, we hypothesized that troglitazone treatment would improve survival in a murine model of endotoxemia and that the protective effect would be mediated by decreased expression of inflammatory mediators. C57Bl/6N x Sv/129 (wild-type [WT]) or PPARalpha null mice treated for 2 weeks with dietary troglitazone (0.1%) had significantly fewer deaths and a higher LD(50) value compared to control-fed mice when challenged with lipopolysaccharide (LPS). PPARalpha null mice were more sensitive to the lethal effects of LPS as evidenced by a 2-fold lower LD(50) (6.6 mg/kg) compared to WT mice (14.6 mg/kg). Troglitazone treatment had no significant effect on LPS-induced plasma TNF, glucose, or nitric oxide levels in WT or PPARalpha null mice at any of the time points examined. However, troglitazone treatment significantly reduced LPS-induced plasma IL-6 levels in both WT and PPARalpha null mice. The results of these studies suggest that troglitazone treatment protects mice against a lethal challenge of LPS, but whether or not this effect is mediated through decreased expression of inflammatory mediators remains unclear.

Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20.

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.

Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates.

In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.