Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e σB regulon

<p>Abstract</p> <p>Background</p> <p>The opportunistic food-borne gram-positive pathogen <it>Listeria monocytogenes </it>can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σ^B, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σ^Bexpression during growth of <it>L. monocytogenes </it>EGD-e. Whole genome-based transcriptional profiling was used to identify σ^B-dependent genes at different growth phases.</p> <p>Results</p> <p>We detected 105 σ^B-positively regulated genes and 111 genes which appeared to be under negative control of σ^Band validated 36 σ^B-positively regulated genes <it>in vivo </it>using a reporter gene fusion system.</p> <p>Conclusion</p> <p>Genes comprising the σ^Bregulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σ^B-positively regulated genes of <it>L. monocytogenes </it>are absent in <it>L. innocua</it>. A total of 75 σ^B-positively regulated listerial genes had homologs in <it>B. subtilis</it>, but only 33 have been previously described as being σ^B-regulated in <it>B. subtilis </it>even though both species share a highly conserved σ^B-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.</p

Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes

BACKGROUND: Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. RESULTS: The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. CONCLUSION: Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages

Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen

New insights into the pathogenesis and molecular understanding of cutaneous T-cell lymphomas

Stadler R, Hain C. Neues zu Pathogenese und molekularem Verständnis bei kutanen T-Zell-Lymphomen. Dermatologie. 2022.The pathogenesis of cutaneous T‑cell lymphomas (CTCL) is still an enigma. Therefore, extensive translational research efforts have been undertaken in recent years to gain further clinical and molecular insights. There is increasing evidence that the different clinical appearance of the CTCL subtypes derives from the assumption that they develop from different skin subpopulations of Tcells. Detection and quantification of the malignant T‑cell clones is crucial for the diagnosis and prognosis of CTCL. Numerous recurrent mutant cellular signalling pathways have been found in recent years. This includes the JAK-STAT, NFkappaB, T‑cell receptor and MAP kinase signalling pathways, as well as cell cycle control and epigenetics. The most recent analyses imply atumour evolution model with initial copy number variation, like amplification or deletions of specific DNA fragments (CNVs) and only subsequent later single nucleotide variations (SNVs). The crucial question, however, is which CNVs are sufficient to initiate general tumourigenesis? The challenge is to identify possible driver genes. Increasing molecular understanding in CTCL will include new breakthrough therapeutic options in the near future. © 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.Die Pathogenese der kutanen T‑Zell-Lymphome (KTZL) ist nach wie vor ein Enigma. Daher wurden gerade in den letzten Jahren umfangreiche translationale Forschungsanstrengungen unternommen, um zu weitergehenden klinischen und molekularen Erkenntnissen zu gelangen. Es gibt zunehmend Hinweise darauf, dass das unterschiedliche klinische Erscheinungsbild der KTZL-Subtypen darauf zurückzuführen ist, dass sie von verschiedenen hautständigen Subpopulationen von T‑Zellen abstammen. Der Nachweis und die Quantifizierung der bösartigen T‑Zell-Klone sind entscheidend für die Diagnose und Prognose von KTZL. Es wurden in den letzten Jahren zahlreiche wiederkehrend mutierte zelluläre Signalwege gefunden. Das beinhaltet JAK(Januskinase)-STAT(„signal transducers and activators of transcription“)-, NF-κB(„nuclear factor kappa B“)-, T‑Zell-Rezeptor- und MAP(„mitogen-activated protein“)-Kinase-Signalwege sowie die Zellzykluskontrolle und Epigenetik. Die jüngsten Analysen implizieren ein Tumorevolutionsmodell mit anfänglichen Kopienzahlvariationen wie Amplifikationen oder Deletionen bestimmter DNA(Desoxyribonukleinsäure)-Abschnitte („copy number variations“ [CNVs]) und erst darauffolgenden späteren Einzelnukleotidvariationen („single nucleotide variations“ [SNVs]). Die entscheidende Frage ist jedoch, welche CNVs ausreichen, um eine generelle Tumorgenese zu beginnen. Die Herausforderung ist, mögliche Treibergene zu identifizieren. Das zunehmende molekulare Verständnis bei KTZL wird in naher Zukunft neue bahnbrechende Therapieoptionen beinhalten

Unraveling the Structural Variations of Early-Stage Mycosis Fungoides—CD3 Based Purification and Third Generation Sequencing as Novel Tools for the Genomic Landscape in CTCL

Hain C, Stadler R, Kalinowski J. Unraveling the Structural Variations of Early-Stage Mycosis Fungoides—CD3 Based Purification and Third Generation Sequencing as Novel Tools for the Genomic Landscape in CTCL. Cancers. 2022;14(18): 4466.Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). At present, knowledge of genetic changes in early-stage MF is insufficient. Additionally, low tumor cell fraction renders calling of copy-number variations as the predominant mutations in MF challenging, thereby impeding further investigations. We show that enrichment of T cells from a biopsy of a stage I MF patient greatly increases tumor fraction. This improvement enables accurate calling of recurrent MF copy-number variants such as ARID1A and CDKN2A deletion and STAT5 amplification, undetected in the unprocessed biopsy. Furthermore, we demonstrate that application of long-read nanopore sequencing is especially useful for the structural variant rich CTCL. We detect the structural variants underlying recurrent MF copy-number variants and show phasing of multiple breakpoints into complex structural variant haplotypes. Additionally, we record multiple occurrences of templated insertion structural variants in this sample. Taken together, this study suggests a workflow to make the early stages of MF accessible for genetic analysis, and indicates long-read sequencing as a major tool for genetic analysis for MF

Sezary syndrome shows whole genome duplication as a late event in tumor evolution

Hain C, Stadler R, Kalinowski J. Sezary syndrome shows whole genome duplication as a late event in tumor evolution. The Journal of investigative dermatology. 2021: 02500-8

Molecular pathogenesis of cutaneous lymphoma-Future directions

Stadler R, Hain C, Cieslak C, Stranzenbach R. Molecular pathogenesis of cutaneous lymphoma-Future directions. Experimental Dermatology . 2020.The pathogenesis of cutaneous T-cell lymphomas is not clear. In recent years, the genetic changes in CTCL were explored. The detected mutations showed a great deal of heterogeneity between individual patients. The studies documented various copy number variations (CNV) and single nucleotide variations (SNV) in multiple genes involved in multiple signalling pathways. Recurrently mutated signalling pathways include JAK-STAT, MAPK, T-cell receptor, TNF receptor and NF kappa B signalling. In the period between 2018 and today, additional studies towards the genetic changes in CTCL were carried out. Genetic changes in gamma delta T-cell lymphoma are also shown in genes of the JAK-STAT, MAPK, MYC and chromatin signalling pathways. These studies might indicate a shift away from targeted sequencing approaches towards whole-genome sequencing. This approach demands additional resources in terms of funding but has the advantage of finding mutations in non-coding regions. These mutations were neglected for a long time, but as shown in contemporary research these regions harbour highly recurrent mutations affecting gene expression and regulation. Nevertheless, the detection of specific molecular changes in known pathways enables considerations for targeted therapies

Structural variations in low tumor fraction mycosis fungoides unraveled by nanopore sequencing

Hain C, Cieslak C, Stadler R, Kalinowski J. Structural variations in low tumor fraction mycosis fungoides unraveled by nanopore sequencing. European Journal of Cancer. 2022;173(Suppl. 1):S31