YbeY:n, RfaH:n, and Hfq:n roolit Yersinia entrecolitan geenisäätelyssä ja virulenssissä

Y. enterocolitica is a human pathogen that cause mostly food-borne yersiniosis, usually a diarrheal disease sometimes followed by post-infectious reactive arthritis. Yersiniosis is considered to be the third most common cause of gastroenteritis in Europe. In Finland both Y. pseudotuberculosis and Y. enterocolitica cause hundreds of human infections annually. The aim of the study is to characterize the intricate regulatory networks of Yersinia especially those that control the expression of the virulence factors. To achieve that goal three regulators (YbeY, RfaH and Hfq) were initially selected. The absence of ybeY gene in Y. enterocolitica serotype O:3 resulted in misprocessing of 16S rRNA, severe decrease of growth rate, disturbed regulation of the Yersinia virulence plasmid genes expression and affected the expression of small RNAs. Furthermore, the ybeY mutant displayed impairment of many virulence-related features, and decreased infectivity in the cell infection model. The study revealed that RfaH of Y. enterocolitica O:3 acts as a highly specific regulator that enhances the transcription of selected operons involved in biosynthesis of lipopolysaccharide. Furthermore, the transcriptomics study indicated that some changes seen in the rfaH mutant strain were actually due to indirect responses to the loss of O-antigen. Moreover, the lack of RfaH resulted in attenuated stress response and lower resistance to selected chemical compounds. The loss of Hfq caused impairment in growth, elongation of the bacterial cells, and decreased the resistance of bacteria to heat, acid and oxidative stresses, as well as attenuation in mouse infection experiments. Moreover, this study revealed that several alterations typical for the hfq-negative phenotype were due to derepression of the transcriptional factor RovM. In conclusion, all the studied mutations caused different alterations in gene regulatory networks and eventually led to a compromise in the virulence of Y. enterocolitica O:3. The studied mutants showed significant decrease in resistance to different environmental conditions and alterations in the bacterial physiology that contribute to vitality and ability to establish infection in host organism.Yersinia enterocolitica on bakteerilaji, joka on lähisukua Y. pestikselle, paiseruton aiheuttavalle bakteerille. Moni villi- ja kotieläin kantaa Yersinia enterocoliticaa. Koti- ja tuotantoeläinten bakteerit voivat saastuttaa maidon ja ruoan, joka voi kypsentämättömänä tartuttaa ihmisen. Yersiniat voivat aiheuttaa ihmiselle suolisto- ja yleisinfektioita. Yleisimmät oireet ovat kuume, ripuli ja vatsakipu, joka voi muistuttaa umpilisäkkeen tulehdusta. Yleensä oireet kestävät muutamasta päivästä kolmeen viikkoon, mutta osalle sairastuneista voi kehittyä reaktiivinen niveltulehdus. Yersinioosi on salmonellojen ja kampylobakteerien jälkeen kolmanneksi yleisin ruokaperäinen bakteerin aiheuttama ripulitauti. Suomessa Yersinian esiintyvyys on yksi Euroopan unionin korkeimmista, noin 500-700 tapausta vuodessa (suunnilleen 10 tapausta per 100 000 asukasta). Lisäksi Yersinia on toiminut jo vuosia tutkimuksessa malliesimerkkinä, miten bakteerit aiheuttavat infektioita. Infektioprosessin ymmärtämisestä on useita käytännön implikaatioita: se voi toimia ensimmäisenä askeleena uusien-lääkkeiden ja rokotteiden kehitykselle, sekä parantaa tautien diagnostiikkaa. Tässä tutkimuksessa tutkittiin kolmea eri proteiinia (YbeY, RfaH ja Hfq) tavoitteena karakterisoida niiden rooli Yersinian fysiologiassa ja virulenssissa. Tutkimustulokset osoittivat, että nämä proteiinit ovat tärkeitä bakteerin taudinaiheuttamiskyvylle, normaalille kasvulle ja erilaisista ympäristöistä selviytymiselle. Tämä tutkimus laajentaa tietoa, miten bakteerit ilmentävät geeninsä ollakseen tehokkaita aiheuttamaan infektion

Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+thymocyte stage

The natural CD25 + FOXP3 + regulatory T cell (Treg) population is generated as a distinct lineage in the thymus, but the details of Treg development in humans remain unclear, and the timing of Treg commitment is also contested. Here we have analyzed the emergence of CD25 + cells at the CD4 + CD8 + double positive (DP) stage in the human thymus. We show that these cells share T cell receptor repertoire with CD25 + CD4 single-positive thymocytes, believed to be committed Tregs. They already have a fully demethylated FOXP3 enhancer region and thus display stable expression of FOXP3 and the associated Treg phenotype. Transcriptome analysis also grouped the DP CD25 + and CD4 CD25 + thymocytes apart from the CD25 - subsets. Together with earlier studies, our data are consistent with human Treg commitment already at the DP thymocyte stage. We suggest that the most important antigens and signals necessary for human Treg differentiation may be found in the thymic cortex.Peer reviewe

Characterization of vB_SauM-fRuSau02, a twort-like bacteriophage isolated from a therapeutic phage cocktail

Funding Information: Acknowledgments: Helsinki University Hospital special state subsidy for health science research grants, the Academy of Finland (project 1288701) and Jane & Aatos Erkko Foundation are acknowledged for funding (to MS). KL was supported by the Emil Aaltonen Foundation. Annamari Heikinheimo is thanked for providing the porcine S. aureus strain collection and Taeok Bae for sharing the TB4 and tagO strains. Arnab Bhattacharjee is acknowledged for his help with structural modelling and Joseph Michael Ochieng’ Oduor for critically reading the manuscript. Publisher Copyright: © 2017 by the authors.Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+ C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.Peer reviewe

RNA-Sequencing Reveals the Progression of Phage-Host Interactions between phi R1-37 and Yersinia enterocolitica

Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_phi R1-37 (phi R1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of phi R1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins.Peer reviewe

Deciphering the Antibacterial Mode of Action of Alpha-Mangostin on Staphylococcus epidermidis RP62A Through an Integrated Transcriptomic and Proteomic Approach

Background: Alpha-mangostin (alpha-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of alpha-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 mu g/ml) of alpha-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of alpha-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of alpha-MG treated cells indicated that genes/proteins affected by alpha-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabl, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of alpha-MG. The obtained results suggest that alpha-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.Peer reviewe

YerA41, a Yersinia ruckeri Bacteriophage: Determination of a Non-Sequencable DNA Bacteriophage Genome via RNA-Sequencing

YerA41 is a Myoviridae bacteriophage that was originally isolated due its ability to infect Yersinia ruckeri bacteria, the causative agent of enteric redmouth disease of salmonid fish. Several attempts to determine its genomic DNA sequence using traditional and next generation sequencing technologies failed, indicating that the phage genome is modified in such a way that it is an unsuitable template for PCR amplification and for conventional sequencing. To determine the YerA41 genome sequence, we performed RNA-sequencing from phage-infected Y. ruckeri cells at different time points post-infection. The host-genome specific reads were subtracted and de novo assembly was performed on the remaining unaligned reads. This resulted in nine phage-specific scaffolds with a total length of 143 kb that shared only low level and scattered identity to known sequences deposited in DNA databases. Annotation of the sequences revealed 201 predicted genes, most of which found no homologs in the databases. Proteome studies identified altogether 63 phage particle-associated proteins. The RNA-sequencing data were used to characterize the transcriptional control of YerA41 and to investigate its impact on the bacterial gene expression. Overall, our results indicate that RNA-sequencing can be successfully used to obtain the genomic sequence of non-sequencable phages, providing simultaneous information about the phage–host interactions during the process of infection

Prevalence, Cell Tropism, and Clinical Impact of Human Parvovirus Persistence in Adenomatous, Cancerous, Inflamed, and Healthy Intestinal Mucosa

Parvoviruses are single-stranded DNA viruses, infecting many animals from insects to humans. Human parvovirus B19 (B19V) causes erythema infectiosum, arthropathy, anemia, and fetal death, and human bocavirus (HBoV) 1 causes respiratory tract infections, while HBoV2-4 are enteric. Parvoviral genomes can persist in diverse non-permissive tissues after acute infection, but the host-cell tropism and the impact of their tissue persistence are poorly studied. We searched for parvoviral DNA in a total of 427 intestinal biopsy specimens, as paired disease-affected and healthy mucosa, obtained from 130 patients with malignancy, ulcerative colitis (UC), or adenomas, and in similar intestinal segments from 55 healthy subjects. Only three (1.6%) individuals exhibited intestinal HBoV DNA (one each of HBoV1, 2, and 3). Conversely, B19V DNA persisted frequently in the intestine, with 50, 47, 31, and 27% detection rates in the patients with malignancy, UC, or adenomas, and in the healthy subjects, respectively. Intra-individually, B19V DNA persisted significantly more often in the healthy intestinal segments than in the inflamed colons of UC patients. The highest loads of B19V DNA were seen in the ileum and colon specimens of two healthy individuals. With dual-RNAscope in situ hybridization and immunohistochemistry assays, we located the B19V persistence sites of these intestines in mucosal B cells of lymphoid follicles and vascular endothelial cells. Viral messenger RNA transcription remained, however, undetected. RNA sequencing (RNA-seq) identified 272 differentially expressed cellular genes between B19V DNA-positive and -negative healthy ileum biopsy specimens. Pathway enrichment analysis revealed that B19V persistence activated the intestinal cell viability and inhibited apoptosis. Lifelong B19V DNA persistence thus modulates host gene expression, which may lead to clinical outcomes.Peer reviewe

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix

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Identifying components required for OMP biogenesis as novel targets for antiinfective drugs

The emergence of multiresistant Gram-negative bacteria requires new therapies for combating bacterial infections. Targeting the biogenesis of virulence factors could be an alternative strategy instead of killing bacteria with antibiotics. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier. At the same time it facilitates the exchange of molecules and harbors a multitude of proteins associated with virulence. In order to insert proteins into the OM, an essential oligomeric membrane-associated protein complex, the ss-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones may serve as attractive targets to develop novel antiinfective agents. Herein, we aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, OM barrier function, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, we assume that both BamB and SurA are promising targets for the development of new antiinfective drugs in the future.Peer reviewe