Jan Oppelt

Human Treponema pallidum 11q/j isolate belongs to subsp. endemicum but contains two loci with a sequence in TP0548 and TP0488 similar to subsp. pertenue and subsp. pallidum, respectively

Background Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). An unusual human TEN 11q/j isolate was obtained from a syphilis-like primary genital lesion from a patient that returned to France from Pakistan. Methodology/Principal findings The TEN 11q/j isolate was characterized using nested PCR followed by Sanger sequencing and/or direct Illumina sequencing. Altogether, 44 chromosomal regions were analyzed. Overall, the 11q/j isolate clustered with TEN strains Bosnia A and Iraq B as expected from previous TEN classification of the 11q/j isolate. However, the 11q/j sequence in a 505 bp-long region at the TP0488 locus was similar to Treponema pallidum subsp. pallidum (TPA) strains, but not to TEN Bosnia A and Iraq B sequences, suggesting a recombination event at this locus. Similarly, the 11q/j sequence in a 613 bp-long region at the TP0548 locus was similar to Treponema pallidum subsp. pertenue (TPE) strains, but not to TEN sequences. Conclusions/Significance A detailed analysis of two recombinant loci found in the 11q/j clinical isolate revealed that the recombination event occurred just once, in the TP0488, with the donor sequence originating from a TPA strain. Since TEN Bosnia A and Iraq B were found to contain TPA-like sequences at the TP0548 locus, the recombination at TP0548 took place in a treponeme that was an ancestor to both TEN Bosnia A and Iraq B. The sequence of 11q/j isolate in TP0548 represents an ancestral TEN sequence that is similar to yaws-causing treponemes. In addition to the importance of the 11q/j isolate for reconstruction of the TEN phylogeny, this case emphasizes the possible role of TEN strains in development of syphilis-like lesions.

Formation of Staphylococcus aureus biofilm in the presence of sub-lethal concentrations of disinfectants: a transcriptomic analysis using RNA-seq

ABSTRACT Staphylococcus aureus is a common biofilm-forming pathogen. Low doses of disinfectants have previously been reported to promote biofilm formation and to increase virulence. The aim of this study was to use transcriptome sequencing (RNA-seq) analysis to investigate global transcriptional changes in S. aureus in response to sublethal concentrations of the commonly used food industry disinfectants ethanol (EtOH) and chloramine T (ChT) and their combination (EtOH_ChT) in order to better understand the effects of these agents on biofilm formation. Treatment with EtOH and EtOH_ChT resulted in more significantly altered expression profiles than treatment with ChT. Our results revealed that EtOH and EtOH_ChT treatments enhanced the expression of genes responsible for regulation of gene expression (sigB), cell surface factors (clfAB), adhesins (sdrDE), and capsular polysaccharides (cap8EFGL), resulting in more intact biofilm. In addition, in this study we were able to identify the pathways involved in the adaptation of S. aureus to the stress of ChT treatment. Further, EtOH suppressed the effect of ChT on gene expression when these agents were used together at sublethal concentrations. These data show that in the presence of sublethal concentrations of tested disinfectants, S. aureus cells trigger protective mechanisms and try to cope with them. IMPORTANCE So far, the effect of disinfectants is not satisfactorily explained. The presented data will allow a better understanding of the mode of disinfectant action with regard to biofilm formation and the ability of bacteria to survive the treatment. Such an understanding could contribute to the effort to eliminate possible sources of bacteria, making disinfectant application as efficient as possible. Biofilm formation plays significant role in the spread and pathogenesis of bacterial species.

Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N‐alpha‐terminal acetyltransferase Nat4 and loss of its associated H4 N‐terminal acetylation (N‐acH4) extend yeast replicative lifespan. Notably, nat4Δ‐induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N‐acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N‐acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress‐response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ‐mediated longevity. Collectively, these findings establish histone N‐acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.

Reanalysis of Chinese Treponema pallidum samples: all Chinese samples cluster with SS14-like group of syphilis-causing treponemes

ObjectiveTreponema pallidum subsp. pallidum (TPA) is the causative agent of syphilis. Genetic analyses of TPA reference strains and human clinical isolates have revealed two genetically distinct groups of syphilis-causing treponemes, called Nichols-like and SS14-like groups. So far, no genetic intermediates, i.e. strains containing a mixed pattern of Nichols-like and SS14-like genomic sequences, have been identified. Recently, Sun et al. (Oncotarget 2016. https://doi.org/10.18632/oncotarget.10154) described a new “phylogenetic group” (called Lineage 2) among Chinese TPA strains. This lineage exhibited a “mosaic genomic structure” of Nichols-like and SS14-like lineages.ResultsWe reanalyzed the primary sequencing data (Project Number PRJNA305961) from the Sun et al. publication with respect to the molecular basis of Lineage 2. While Sun et al. based the analysis on several selected genomic single nucleotide variants (SNVs) and a subset of highly variable but phylogenetically poorly informative genes, which may confound the phylogenetic analysis, our reanalysis primarily focused on a complete set of whole genomic SNVs. Based on our reanalysis, only two separate TPA clusters were identified: one consisted of Nichols-like TPA strains, the other was formed by the SS14-like TPA strains, including all Chinese strains.

Sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment: First complete whole genome sequence obtained directly from human clinical material

Treponema pallidum subsp. pallidum (TPA) is the infectious agent of syphilis, a disease that infects more than 5 million people annually. Since TPA is an uncultivable bacterium, most of the information on TPA genetics comes from genome sequencing and molecular typing studies. This study presents the first complete TPA genome (without sequencing gaps) of clinical isolate (UZ1974), which was obtained directly from clinical material, without multiplication in rabbits. Whole genome sequencing was performed using a newly developed Anti-Treponemal Antibody Enrichment technique combined with previously reported Pooled Segment Genome Sequencing. We identified the UW074B genome, isolated from a sample previously propagated in rabbits, to be the closest relative of the UZ1974 genome and calculated the TPA mutation rate as 2.8 x 10−10 per site per generation.

Complete genome sequences of two strains of Treponema pallidum subsp. pertenue from Indonesia: Modular structure of several treponemal genes

Background Treponema pallidum subsp. pertenue (TPE) is the causative agent of yaws, a multistage disease endemic in tropical regions in Africa, Asia, Oceania, and South America. To date, seven TPE strains have been completely sequenced and analyzed including five TPE strains of human origin (CDC-2, CDC 2575, Gauthier, Ghana-051, and Samoa D) and two TPE strains isolated from the baboons (Fribourg-Blanc and LMNP-1). This study revealed the complete genome sequences of two TPE strains, Kampung Dalan K363 and Sei Geringging K403, isolated in 1990 from villages in the Pariaman region of Sumatra, Indonesia and compared these genome sequences with other known TPE genomes. Methodology/principal findings The genomes were determined using the pooled segment genome sequencing method combined with the Illumina sequencing platform resulting in an average coverage depth of 1,021x and 644x for the TPE Kampung Dalan K363 and TPE Sei Geringging K403 genomes, respectively. Both Indonesian TPE strains were genetically related to each other and were more distantly related to other, previously characterized TPE strains. The modular character of several genes, including TP0136 and TP0858 gene orthologs, was identified by analysis of the corresponding sequences. To systematically detect genes potentially having a modular genetic structure, we performed a whole genome analysis-of-occurrence of direct or inverted repeats of 17 or more nucleotides in length. Besides in tpr genes, a frequent presence of repeats was found in the genetic regions spanning TP0126–TP0136, TP0856–TP0858, and TP0896 genes. Conclusions/significance Comparisons of genome sequences of TPE Kampung Dalan K363 and Sei Geringging K403 with other TPE strains revealed a modular structure of several genomic loci including the TP0136, TP0856, and TP0858 genes. Diversification of TPE genomes appears to be facilitated by intra-strain genome recombination events.

Liver regeneration during the associating liver partition and portal vein ligation for staged hepatectomy procedure in Sus scrofa is positively modulated by stem cells

This present study investigated the impact of the application of stem cells to liver regeneration following the first stage of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). The experiment was conducted on a pig model (n=6, 3 that did not receive application of stem cells, 3 that received application stem cells). Collected samples of liver (day 0 and 9 following surgery) were subjected to complete transcriptome sequencing. In total, 39 differentially expressed genes were found in the group without the application of the stem cells (genes of unwanted processes such as fibrosis and inflammation). In the group that did receive application of stem cells, no significantly differentially expressed genes were found, indicating a properly regenerated liver remnant. The present study therefore demonstrated, to the best of our knowledge for the first time, the positive effect of stem cells application in the liver regeneration process during ALPPS procedure in the pig model.

MicroRNA miR-34a downregulates FOXP1 during DNA damage response to limit BCR signalling in chronic lymphocytic leukaemia B cells

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3′-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.

Rituximab primarily targets an intra-clonal BCR signaling proficient CLL subpopulation characterized by high CD20 levels

The use of the anti-CD20 antibody rituximab has improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab was shown to act via various mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct apoptosis, or sensitization to chemotherapy (reviewed in ref. [1]). However, the biological function of CD20 and the reasons for the impressive activity of rituximab and other anti-CD20 antibodies remain elusive. It has been suggested, but remains controversial, whether CD20 functions as a calcium channel, couples with CD40 and MHCII, or B-cell receptor (BCR), and whether it has a role in T cell-dependent and -independent immunity [1,2,3,4]. This is also of great clinical interest for the design of combinatorial treatment of rituximab with BCR inhibitors, DNA damaging or immunomodulatory drugs, or CAR T cells. We have previously shown that microenvironmental interactions upregulate the CD20 levels on CLL cells through the CXCR4/SDF-1 axis [5]. Here we describe that higher CD20 expression has a direct role in the BCR signaling in CLL cells, and a BCR-proficient intra-clonal CLL cell subpopulation is more efficiently eliminated by rituximab in vivo due to higher CD20 levels.

Abstract 1012: CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo

CD20 supports BCR signaling in an intra clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR proficient B cells in vivo, CD20 supports BCR signaling in an intra clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR proficient B cells in vivo.