Publications by authors named "Christian Berens"

75 Publications

Newly Isolated Animal Pathogen Is Cytotoxic to Human Epithelial Cells.

Int J Mol Sci 2021 Mar 29;22(7). Epub 2021 Mar 29.

Microbiology Division, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen and the widely distributed animal pathogen . In this study, strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of to different human epithelial cell lines and to an invertebrate animal model, larvae, comparable to diphtheria toxin-secreting Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.
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http://dx.doi.org/10.3390/ijms22073549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037504PMC
March 2021

Shiga Toxin-Producing E. coli in Animals: Detection, Characterization, and Virulence Assessment.

Methods Mol Biol 2021 ;2291:19-86

Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany.

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.
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http://dx.doi.org/10.1007/978-1-0716-1339-9_2DOI Listing
April 2021

The T4SS Effector AnkF Is Important for Intracellular Replication.

Front Cell Infect Microbiol 2020 13;10:559915. Epub 2020 Nov 13.

Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

is an obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Following uptake by alveolar macrophages, the pathogen replicates in an acidic phagolysosomal vacuole, the -containing vacuole (CCV). Effector proteins translocated into the host cell by the type IV secretion system (T4SS) are important for the establishment of the CCV. Here we focus on the effector protein AnkF and its role in establishing the CCV. The AnkF knock out mutant invades host cells as efficiently as wild-type , but this mutant is hampered in its ability to replicate intracellularly, indicating that AnkF might be involved in the development of a replicative CCV. To unravel the underlying reason(s), we searched for AnkF interactors in host cells and identified vimentin through a yeast two-hybrid approach. While AnkF does not alter vimentin expression at the mRNA or protein levels, the presence of AnkF results in structural reorganization and vesicular co-localization with recombinant vimentin. Ectopically expressed AnkF partially accumulates around the established CCV and endogenous vimentin is recruited to the CCV in a time-dependent manner, suggesting that AnkF might attract vimentin to the CCV. However, knocking-down endogenous vimentin does not affect intracellular replication of . Other cytoskeletal components are recruited to the CCV and might compensate for the lack of vimentin. Taken together, AnkF is essential for the establishment of the replicative CCV, however, its mode of action is still elusive.
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http://dx.doi.org/10.3389/fcimb.2020.559915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691251PMC
November 2020

The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor.

Sci Rep 2020 09 21;10(1):15396. Epub 2020 Sep 21.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Wasserturmstraße 3/5, 91054, Erlangen, Germany.

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.
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http://dx.doi.org/10.1038/s41598-020-72340-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506536PMC
September 2020

Metabolic Traits of Bovine Shiga Toxin-Producing (STEC) Strains with Different Colonization Properties.

Toxins (Basel) 2020 06 22;12(6). Epub 2020 Jun 22.

Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Str. 96a, 07743 Jena, Germany.

Cattle harbor Shiga toxin-producing (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STEC) or only sporadically (STEC) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STEC strains produced significantly more biofilm than STEC when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STEC, but not or only by some STEC. Genomic sequences of the respective and operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STEC. These findings suggest that STEC conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.
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http://dx.doi.org/10.3390/toxins12060414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354573PMC
June 2020

Dynamic Interplay of Host and Pathogens in an Avian Whole-Blood Model.

Front Immunol 2020 31;11:500. Epub 2020 Mar 31.

Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institut, Jena, Germany.

Microbial survival in blood is an essential step toward the development of disseminated diseases and blood stream infections. For poultry, however, little is known about the interactions of host cells and pathogens in blood. We established an chicken whole-blood infection assay as a tool to analyze interactions between host cells and three model pathogens, , and . Following a systems biology approach, we complemented the experimental measurements with functional and quantitative immune characteristics by virtual infection modeling. All three pathogens were killed in whole blood, but each to a different extent and with different kinetics. Monocytes, and to a lesser extent heterophils, associated with pathogens. Both association with host cells and transcriptional activation of genes encoding immune-associated functions differed depending on both the pathogen and the genetic background of the chickens. Our results provide first insights into quantitative interactions of three model pathogens with different immune cell populations in avian blood, demonstrating a broad spectrum of different characteristics during the immune response that depends on the pathogen and the chicken line.
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http://dx.doi.org/10.3389/fimmu.2020.00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136455PMC
March 2021

Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt.

Vet Microbiol 2020 Jan 29;240:108539. Epub 2019 Nov 29.

Leibniz Institute of Photonic Technology (IPHT), 07745, Jena, Germany; INFECTOGNOSTICS Research Campus, 07745, Jena, Germany. Electronic address:

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.
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http://dx.doi.org/10.1016/j.vetmic.2019.108539DOI Listing
January 2020

Microarray-based detection of resistance and virulence factors in commensal Escherichia coli from livestock and farmers in Egypt.

Vet Microbiol 2020 Jan 29;240:108539. Epub 2019 Nov 29.

Leibniz Institute of Photonic Technology (IPHT), 07745, Jena, Germany; INFECTOGNOSTICS Research Campus, 07745, Jena, Germany. Electronic address:

The objective of our study was to provide a molecular analysis using DNA-microarray based assays of commensal E. coli populations from apparently healthy livestock and their attendants to assess the virulence potential as well as multidrug resistance (MDR) genotypes. We randomly collected 132 fecal samples from seemingly healthy smallholder´s food producing animals [buffalo (n = 32) and cattle (n = 50)] as well as from contacting farmers (n = 50). Bacterial isolation and identification were performed using standard protocols, while E. coli isolates were characterized using a DNA microarray system targeting 60 different virulence and 47 antibiotic resistance genes of clinical importance and allowing assignment to most common H and O types. From the fecal samples examined, 47 E. coli isolates were obtained. The array predicted serotypes for 14 out of the 47 E. coli isolates. Six E. coli isolates were identified as STEC since Shiga toxin genes were detected. In summary, 36 different virulence genes were identified; of which, hemL, lpfA and iss were most prevalent. Thirty-four E. coli isolates were found to carry at least one antimicrobial resistance gene. Of these, 20 did exhibit genes allowing strain classification as MDR. More than half of the isolates contained antimicrobial resistance genes associated with beta lactam resistance 27/47 (57.5 %). The 13 remaining isolates did not contain any resistance gene tested with the array. Our study demonstrated the presence of antimicrobial resistance genes and virulence genotypes among commensal E. coli of human and animal sources.
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http://dx.doi.org/10.1016/j.vetmic.2019.108539DOI Listing
January 2020

Modulation of host cell metabolism by T4SS-encoding intracellular pathogens.

Curr Opin Microbiol 2019 02 11;47:59-65. Epub 2019 Jan 11.

Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität (FAU) Erlangen-Nürnberg, Wasserturmstraße 3/5, D-91054 Erlangen, Germany. Electronic address:

Intracellular bacterial pathogens intimately interact with the infected host cell to prevent elimination and to ensure survival. One group of intracellular pathogens, including Coxiella burnetii, Legionella pneumophila, Brucella spp., Anaplasma phagocytophilum, and Ehrlichia chaffeensis, utilizes a type IV secretion system (T4SS) that injects effectors to modulate host cell signalling, vesicular trafficking, autophagy, cell death and transcription to ensure survival [1]. So far, little emphasis has been directed towards understanding how these bacteria manipulate host cell metabolism. This manipulation is not only important for gaining access to nutrients, but also for regulating specific virulence programs [2,3]. Here, we will summarize recent progress made in characterizing the manipulation of host cell metabolism by C. burnetii and other intracellular pathogens utilizing a T4SS.
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http://dx.doi.org/10.1016/j.mib.2018.11.010DOI Listing
February 2019

Spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle as revealed by a genetic-insulators-containing dual-fluorescence HIV-1-based vector.

Sci Rep 2018 07 5;8(1):10204. Epub 2018 Jul 5.

Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.
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http://dx.doi.org/10.1038/s41598-018-28161-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033903PMC
July 2018

Genetic instability associated with loop or stem-loop structures within transcription units can be independent of nucleotide excision repair.

Nucleic Acids Res 2018 04;46(7):3498-3516

Department of Biology, New York University, New York, NY 10003, USA.

Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem-loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them.
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http://dx.doi.org/10.1093/nar/gky110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909459PMC
April 2018

Complete Annotated Genome Sequences of Two Shiga Toxin-Producing Strains and One Atypical Enteropathogenic Strain, Isolated from Naturally Colonized Cattle of German Origin.

Genome Announc 2017 May 11;5(19). Epub 2017 May 11.

Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany.

Shiga toxin-producing (STEC) strains are important zoonotic enteric pathogens with the main reservoir in cattle. Here, we present the genomes of two STEC strains and one atypical enteropathogenic strain from cattle origin, obtained during a longitudinal study in German cattle herds.
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http://dx.doi.org/10.1128/genomeA.00321-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427212PMC
May 2017

Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Strains Colonizing Cattle.

Front Microbiol 2017 21;8:424. Epub 2017 Mar 21.

Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis Jena, Germany.

Shiga toxin-producing (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 ( = 21) and O182:H25 ( = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for , identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between and and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates ( = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5' and 3' flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain's pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.
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http://dx.doi.org/10.3389/fmicb.2017.00424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359238PMC
March 2017

Modulation of host cell apoptotic pathways by intracellular pathogens.

Curr Opin Microbiol 2017 02 19;35:88-99. Epub 2017 Mar 19.

Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Wasserturmstraße 3/5, D-91054 Erlangen, Germany. Electronic address:

Nearly all steps of the host cell apoptotic cascade can be subverted by intracellular microorganisms. Some pathogens modulate early steps and interfere with sensing of extracellular signals, cellular stress or signal transduction; others target Bcl-2 proteins, caspases, or inhibitor of apoptosis proteins (IAPs). In many cases the exact molecular mechanisms leading to interference with the host cell apoptotic cascade are still unknown. However, there are some examples where bacterial factors that modulate host cell death have been identified. In this review we will summarize recent findings on how intracellular pathogens or their secreted proteins alter the intrinsic and/or extrinsic host cell apoptotic pathways.
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http://dx.doi.org/10.1016/j.mib.2017.03.001DOI Listing
February 2017

The Novel Aminomethylcycline Omadacycline Has High Specificity for the Primary Tetracycline-Binding Site on the Bacterial Ribosome.

Antibiotics (Basel) 2016 Sep 22;5(4). Epub 2016 Sep 22.

Microbiology, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.

Omadacycline is an aminomethylcycline antibiotic with potent activity against many Gram-positive and Gram-negative pathogens, including strains carrying the major efflux and ribosome protection resistance determinants. This makes it a promising candidate for therapy of severe infectious diseases. Omadacycline inhibits bacterial protein biosynthesis and competes with tetracycline for binding to the ribosome. Its interactions with the 70S ribosome were, therefore, analyzed in great detail and compared with tigecycline and tetracycline. All three antibiotics are inhibited by mutations in the 16S rRNA that mediate resistance to tetracycline in Brachyspira hyodysenteriae, Helicobacter pylori, Mycoplasma hominis, and Propionibacterium acnes. Chemical probing with dimethyl sulfate and Fenton cleavage with iron(II)-complexes of the tetracycline derivatives revealed that each antibiotic interacts in an idiosyncratic manner with the ribosome. X-ray crystallography had previously revealed one primary binding site for tetracycline on the ribosome and up to five secondary sites. All tetracyclines analyzed here interact with the primary site and tetracycline also with two secondary sites. In addition, each derivative displays a unique set of non-specific interactions with the 16S rRNA.
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http://dx.doi.org/10.3390/antibiotics5040032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187513PMC
September 2016

The Accessory Genome of Shiga Toxin-Producing Escherichia coli Defines a Persistent Colonization Type in Cattle.

Appl Environ Microbiol 2016 09 15;82(17):5455-64. Epub 2016 Aug 15.

Friedrich-Loeffler-Institut/Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany

Unlabelled: Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STEC(per)) (shedding for ≥4 months) and 74 sporadically colonizing STEC (STEC(spo)) (shedding for ≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STEC(per) isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STEC(spo) isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STEC(per) isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STEC(spo) isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STEC(per) from STEC(spo) isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level.

Importance: Ruminants, especially cattle, are sources of food-borne infections by Shiga toxin-producing Escherichia coli (STEC) in humans. Some STEC strains persist in cattle for longer periods of time, while others are detected only sporadically. Persisting strains can serve as gene reservoirs that supply E. coli with virulence factors, thereby generating new outbreak strains. Attempts to reduce the human risk for acquiring STEC infections should therefore include strategies to control such persisting STEC strains. By analyzing representative genes of their core and accessory genomes, we show that bovine STEC with a persistent colonization type emerged independently from sporadically colonizing isolates and evolved in parallel evolutionary branches. However, persistent colonizing strains share similar sets of accessory genes. Defining the genetic patterns that distinguish persistent from sporadically colonizing STEC isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level.
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http://dx.doi.org/10.1128/AEM.00909-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988194PMC
September 2016

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

J Biol Chem 2016 Jan 11;291(2):848-61. Epub 2015 Nov 11.

From the Departments of Biology and Chemistry, New York University, New York, New York 10003, the Division of Science, New York University Abu Dhabi, Post Office Box 129188, Abu Dhabi, United Arab Emirates

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.
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http://dx.doi.org/10.1074/jbc.M115.685271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705403PMC
January 2016

Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus.

Fungal Genet Biol 2016 Apr 10;89:72-83. Epub 2015 Nov 10.

Institute of Biotechnology, Department of Applied and Molecular Microbiology, Berlin University of Technology, Gustav-Meyer-Allee 25, 13355 Berlin, Germany. Electronic address:

In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilising codon optimised cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racA(G18V) dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations. In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.
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http://dx.doi.org/10.1016/j.fgb.2015.11.003DOI Listing
April 2016

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling.

J Vis Exp 2015 Jun 25(100):e52903. Epub 2015 Jun 25.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen;

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.
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http://dx.doi.org/10.3791/52903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544420PMC
June 2015

Apoptotic cell clearance and its role in the origin and resolution of chronic inflammation.

Front Immunol 2015 25;6:139. Epub 2015 Mar 25.

Department of Internal Medicine 3, Rheumatology and Immunology, Friedrich-Alexander University of Erlangen-Nürnberg , Erlangen , Germany.

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http://dx.doi.org/10.3389/fimmu.2015.00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373391PMC
April 2015

RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression.

Biotechnol J 2015 Feb;10(2):246-57

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany.

RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so-called "aptamer domain" with a conformational change in the downstream "expression platform" which then determines system output. The modular organization of riboswitches and the relative ease with which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression.
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http://dx.doi.org/10.1002/biot.201300498DOI Listing
February 2015

The progression of cell death affects the rejection of allogeneic tumors in immune-competent mice - implications for cancer therapy.

Front Immunol 2014 11;5:560. Epub 2014 Nov 11.

Department of Biology, Friedrich-Alexander University of Erlangen-Nuremberg , Erlangen , Germany.

Large amounts of dead and dying cells are produced during cancer therapy and allograft rejection. Depending on the death pathway and stimuli involved, dying cells exhibit diverse features, resulting in defined physiological consequences for the host. It is not fully understood how dying and dead cells modulate the immune response of the host. To address this problem, different death stimuli were studied in B16F10 melanoma cells by regulated inducible transgene expression of the pro-apoptotic active forms of caspase-3 (revCasp-3), Bid (tBid), and the Mycobacterium tuberculosis-necrosis inducing toxin (CpnTCTD). The immune outcome elicited for each death stimulus was assessed by evaluating the allograft rejection of melanoma tumors implanted subcutaneously in BALB/c mice immunized with dying cells. Expression of all proteins efficiently killed cells in vitro (>90%) and displayed distinctive morphological and physiological features as assessed by multiparametric flow cytometry analysis. BALB/c mice immunized with allogeneic dying melanoma cells expressing revCasp-3 or CpnTCTD showed strong rejection of the allogeneic challenge. In contrast, mice immunized with cells dying either after expression of tBid or irradiation with UVB did not, suggesting an immunologically silent cell death. Surprisingly, immunogenic cell death induced by expression of revCasp-3 or CpnTCTD correlated with elevated intracellular reactive oxygen species (ROS) levels at the time point of immunization. Conversely, early mitochondrial dysfunction induced by tBid expression or UVB irradiation accounted for the absence of intracellular ROS accumulation at the time point of immunization. Although ROS inhibition in vitro was not sufficient to abrogate the immunogenicity in our allo-immunization model, we suggest that the point of ROS generation and its intracellular accumulation may be an important factor for its role as damage associated molecular pattern in the development of allogeneic responses.
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http://dx.doi.org/10.3389/fimmu.2014.00560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227513PMC
November 2014

Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa.

RNA 2014 Dec 21;20(12):1963-76. Epub 2014 Oct 21.

Dipartimento di Bioscienze, Università degli Studi di Milano, 20133 Milano, Italy

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37 °C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL(+)/rpsL31 Escherichia coli strain in which the dominant rpsL(+) allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA-TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.
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http://dx.doi.org/10.1261/rna.044354.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238360PMC
December 2014

Riboswitch engineering - making the all-important second and third steps.

Curr Opin Biotechnol 2015 Feb 17;31:10-5. Epub 2014 Aug 17.

Fachbereich Biologie, Technische Universität Darmstadt, Schnittspahnstraße 10, 64287 Darmstadt, Germany. Electronic address:

Synthetic biology uses our understanding of biological systems to develop innovative solutions for challenges in fields as diverse as genetic control and logic devices, bioremediation, materials production or diagnostics and therapy in medicine by designing new biological components. RNA-based elements are key components of these engineered systems. Their structural and functional diversity is ideal for generating regulatory riboswitches that react with many different types of output to molecular and environmental signals. Recent advances have added new sensor and output domains to the existing toolbox, and demonstrated the portability of riboswitches to many different organisms. Improvements in riboswitch design and screens for selecting in vivo active switches provide the means to isolate riboswitches with regulatory properties more like their natural counterparts.
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http://dx.doi.org/10.1016/j.copbio.2014.07.014DOI Listing
February 2015

The role of dead cell clearance in the etiology and pathogenesis of systemic lupus erythematosus: dendritic cells as potential targets.

Expert Rev Clin Immunol 2014 Sep 1;10(9):1151-64. Epub 2014 Aug 1.

Friedrich-Alexander Universität, Department of Internal Medicine 3-Rheumatology and Immunology, D-91054 Erlangen, Germany.

Overwhelming apoptosis combined with a deficiency in clearing apoptotic cells is thought to be an important etiopathogenic event in the autoimmune disease systemic lupus erythematosus (SLE). Lazy macrophages, complement or DNase I deficiency as well as insufficient natural IgM might be important factors leading to such a clearance deficiency. A defective clearance of apoptotic cells leads to the activation and maturation of plasmacytoid and myeloid dendritic cells (DCs) by material derived from secondary necrotic cells carrying modified autoantigens. This results in the presentation of autoantigens to autoreactive T and B cells in an immunogenic manner, thereby leading to autoantibody production, chronic inflammation and severe tissue damage. Since DC activation and IFN-α production by plasmacytoid dendritic cells play a critical role in the course of SLE pathogenesis, therapeutic intervention to end this vicious cycle might be a promising approach for treating the disease.
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http://dx.doi.org/10.1586/1744666X.2014.944162DOI Listing
September 2014

tLivin displays flexibility by promoting alternative cell death mechanisms.

PLoS One 2014 24;9(6):e101075. Epub 2014 Jun 24.

Division of Hematology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Livin is a member of the Inhibitor of Apoptosis (IAP) protein family that inhibits apoptosis triggered by a variety of stimuli. We previously demonstrated that while Livin inhibits caspase activity, caspases can cleave Livin to produce a truncated protein, tLivin and that this newly formed tLivin paradoxically induces cell death. However to date, the mechanism of tLivin-induced cell death is not fully understood. In this study, we set out to characterize the form of cell death mediated by tLivin. Here we demonstrate that, unlike most death-promoting proteins, tLivin is a flexible inducer of cell death capable of promoting necrosis or apoptosis in different cell lines. The unusual flexibility of tLivin is displayed by its ability to activate an alternative form of cell death when apoptosis is inhibited. Thus, tLivin can promote more than one form of cell death in the same cell type. Interestingly, in cells where tLivin induces necrosis, deletion of the caspase binding BIR domain results in tLivin-induced apoptosis, suggesting the BIR domain can potentially hamper the ability of tLivin to induce apoptosis. We further elucidate that tLivin activates the JNK pathway and both tLivin-induced apoptosis and necrosis are partially mediated by JNK activity. Acquired resistance to apoptosis, common in many tumors, impinges on the efficiency of conventional anti-cancer agents that function primarily by inducing apoptosis. The ability of tLivin to induce death of apoptosis-compromised cells makes it an attractive candidate for targeted cancer therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101075PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069184PMC
November 2015

Tumor immunotherapy: lessons from autoimmunity.

Front Immunol 2014 13;5:212. Epub 2014 May 13.

Department of Biology, University of Erlangen-Nuremberg , Erlangen , Germany.

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http://dx.doi.org/10.3389/fimmu.2014.00212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026709PMC
June 2014

A novel TetR-regulating peptide turns off rtTA-mediated activation of gene expression.

PLoS One 2014 8;9(5):e96546. Epub 2014 May 8.

Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Conditional regulation of gene expression is a powerful and indispensable method for analyzing gene function. The "Tet-On" system is a tool widely used for that purpose. Here, the transregulator rtTA mediates expression of a gene of interest after addition of the small molecule effector doxycycline. Although very effective in rapidly turning on gene expression, the system is hampered by the long half-life of doxycycline which makes shutting down gene expression rapidly very difficult to achieve. We isolated an rtTA-binding peptide by in vivo selection that acts as a doxycycline antagonist and leads to rapid and efficient shut down of rtTA-mediated reporter gene expression in a human cell line. This peptide represents the basis for novel effector molecules which complement the "Tet-system" by enabling the investigator to rapidly turn gene expression not just on at will, but now also off.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096546PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014509PMC
January 2015

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity.

Proc Natl Acad Sci U S A 2014 May 21;111(18):6750-5. Epub 2014 Apr 21.

Departments of Microbiology and Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL 35294.

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.
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http://dx.doi.org/10.1073/pnas.1400136111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020113PMC
May 2014

Antiapoptotic activity of Coxiella burnetii effector protein AnkG is controlled by p32-dependent trafficking.

Infect Immun 2014 Jul 14;82(7):2763-71. Epub 2014 Apr 14.

Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

Intracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogen Coxiella burnetii displays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR(22/23S)) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.
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http://dx.doi.org/10.1128/IAI.01204-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097630PMC
July 2014