Virulence-related comparative transcriptomics of infectious and non-infectious chlamydial particles.

BACKGROUND: Members of the phylum Chlamydiae are obligate intracellular pathogens of humans and animals and have a serious impact on host health. They comprise several zoonotic species with varying disease outcomes and prevalence. To investigate differences in virulence, we focused on Chlamydia psittaci, C. abortus and Waddlia chondrophila. Most threatening is C. psittaci, which frequently infects humans and causes psittacosis associated with severe pneumonia. The closest relative of C. psittaci is C. abortus, which shares the vast majority of genes but less frequently infects humans, and causes stillbirth and sepsis. W. chondrophila is more distantly related, and occasional human infections are associated with respiratory diseases or miscarriage. One possible explanation for differences in virulence originate from species-specific genes as well as differentially expressed homologous virulence factors. RESULTS: RNA-sequencing (RNA-Seq) was applied to purified infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs) in order to elucidate the transcriptome of the infectious and replicative chlamydial states. The results showed that approximately half of all genes were differentially expressed. For a descriptive comparison, genes were categorised according to their function in the RAST database. This list was extended by the inclusion of inclusion membrane proteins, outer membrane proteins, polymorphic membrane proteins and type III secretion system effectors. In addition, the expression of fifty-six known and a variety of predicted virulence and immunogenic factors with homologs in C. psittaci, C. abortus and W. chondrophila was analysed. To confirm the RNA-Seq results, the expression of nine factors was validated using real-time quantitative polymerase chain reaction (RT-qPCR). Comparison of RNA-Seq and RT-qPCR results showed a high mean Pearson correlation coefficient of 0.95. CONCLUSIONS: It was shown that both the replicative and infectious chlamydial state contained distinctive transcriptomes and the cellular processes emphasised in EBs and RBs differed substantially based on the chlamydial species. In addition, the very first interspecies transcriptome comparison is presented here, and the considerable differences in expression of homologous virulence factors might contribute to the differing infection rates and disease outcomes of the pathogens. The RNA-Seq results were confirmed by RT-qPCR and demonstrate the feasibility of interspecies transcriptome comparisons in chlamydia.

Gramibactin is a bacterial siderophore with a diazeniumdiolate ligand system.

Genome mining and chemical analyses revealed that rhizosphere bacteria (Paraburkholderia graminis) produce a new type of siderophore, gramibactin, a lipodepsipeptide that efficiently binds iron with a logß value of 27.6. Complexation-induced proton NMR chemical shifts show that the unusual N-nitrosohydroxylamine (diazeniumdiolate) moieties participate in metal binding. Gramibactin biosynthesis genes are conserved in numerous plant-associated bacteria associated with rice, wheat, and maize, which may utilize iron from the complex.

High-intensity UV laser ChIP-seq for the study of protein-DNA interactions in living cells.

Genome-wide mapping of transcription factor binding is generally performed by chemical protein-DNA crosslinking, followed by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Here we present the ChIP-seq technique based on photochemical crosslinking of protein-DNA interactions by high-intensity ultraviolet (UV) laser irradiation in living mammalian cells (UV-ChIP-seq). UV laser irradiation induces an efficient and instant formation of covalent "zero-length" crosslinks exclusively between nucleic acids and proteins that are in immediate contact, thus resulting in a "snapshot" of direct protein-DNA interactions in their natural environment. Here we show that UV-ChIP-seq, applied for genome-wide profiling of the sequence-specific transcriptional repressor B-cell lymphoma 6 (BCL6) in human diffuse large B-cell lymphoma (DLBCL) cells, produces sensitive and precise protein-DNA binding profiles, highly enriched with canonical BCL6 DNA sequence motifs. Using this technique, we also found numerous previously undetectable direct BCL6 binding sites, particularly in condensed, inaccessible areas of chromatin.

Quantification of arthritic bone degradation by analysis of 3D micro-computed tomography data.

The use of animal models of arthritis is a key component in the evaluation of therapeutic strategies against the human disease rheumatoid arthritis (RA). Here we present quantitative measurements of bone degradation characterised by the cortical bone profile using glucose-6-phosphate isomerase (G6PI) induced arthritis. We applied micro-computed tomography (µCT) during three arthritis experiments and one control experiment to image the metatarsals of the hind paws and to investigate the effect of experimental arthritis on their cortical bone profile. For measurements of the cortical profile we automatically identified slices that are orthogonal to individual metatarsals, thereby making the measurements independent of animal placement in the scanner. We measured the average cortical thickness index (CTI) of the metatarsals, as well as the thickness changes along the metatarsal. In this study we introduced the cortical thickness gradient (CTG) as a new measure and we investigated how arthritis affects this measure. We found that in general both CTI and CTG are able to quantify arthritic progression, whilst CTG was found to be the more sensitive measure.

Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9.

Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases-metalloenzymes that have been found in all domains of life-enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2). Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9Δ mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi. IMPORTANCE: All living organisms have to cope with alternating CO2 concentrations as CO2 levels range from very low in the atmosphere (0.04%) to high (5% and more) in other niches, including the human body. In fungi, CO2 is sensed via two pathways. The first regulates virulence in pathogenic yeast by direct activation of adenylyl cyclase. The second pathway, although playing a fundamental role in fungal metabolism, is much less understood. Here the transcription factor Cst6/Rca1 controls carbon homeostasis by regulating carbonic anhydrase expression. Upstream signaling in this pathway remains elusive. We identify Sch9 as the kinase controlling Cst6/Rca1 activity in yeast and demonstrate that this pathway is conserved in pathogenic yeast species, which highlights identified key players as potential pharmacological targets. Furthermore, we provide a direct link between adaptation to changing CO2 conditions and lipid/Pkh1/2 signaling in yeast, thus establishing a new signaling cascade central to metabolic adaptation.

Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes.

Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.

Purification of infectious and non-infectious chlamydial particles using iodixanol for density gradient preparation.

Chlamydiae are obligate intracellular bacteria with two distinct morphological stages, the infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs). Here we describe a rapid and straightforward protocol for the purification of EBs and RBs involving special density gradients. It has been successfully applied to three chlamydial species.

Metagenomic analysis between free-living and cultured Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters.

In this study, we analyzed and compared feces of free-living and cultivated fish species, Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters. Metagenome analysis was performed using Illumina MiSeq sequencing of the whole metagenomic DNA isolated from fish feces samples. The analysis covered both prokaryotic and eukaryotic DNA. Feces samples from mariculture fish revealed a highly stable distribution of several orders of bacteria when compared to samples from free-living fish, which were highly diverse and dominated by Vibrionales, Pseudomonales, Rhizobiales and non-classifiable Alphaproteobacteria. The eukaryotic content of the samples was dominated by residues of the host and nine additional fish species that formed a portion of the diet. Investigations on functional annotations for predominant bacterial taxa, using Gene Ontology enrichment, revealed a number of functions related to DNA metabolic processes, especially DNA repair, as well as antibiotic response in the free-living fish species.

Microbiome analysis and detection of pathogenic bacteria of Penaeus monodon from Jakarta Bay and Bali.

Penaeus monodon, the Asian black tiger shrimp is one of the most widely consumed marine crustaceans worldwide. In this study, we examine and compare the fecal microbiota of P. monodon from highly polluted waters around Jakarta Bay, with those of less polluted waters of Bali. Using next generation sequencing techniques, we identified potential bacterial pathogens and common viral diseases of shrimp. Proteobacteria (96.08%) was found to be the most predominant phylum, followed by Bacteriodetes (2.32%), Fusobacteria (0.96%), and Firmicutes (0.53%). On the order level, Vibrionales (66.20%) and Pseudoaltermonadales (24.81%) were detected as predominant taxa. qPCR profiling was used as a confirmatory step and further revealed Vibrio alginolyticus and Photobacterium damselae as two potential pathogenic species present in most of the samples. In addition, viral diseases for shrimp were discovered among the samples, WSSV in Jakarta free-living samples, YHV in Bali free-living samples and IHHNV in both.

Microbial Diversity and Parasitic Load in Tropical Fish of Different Environmental Conditions.

In this study we analysed fecal bacterial communities and parasites of three important Indonesian fish species, Epinephelus fuscoguttatus, Epinephelus sexfasciatus and Atule mate. We then compared the biodiversity of bacterial communities and parasites of these three fish species collected in highly polluted Jakarta Bay with those collected in less polluted Indonesian areas of Cilacap (E. sexfasciatus, A. mate) and Thousand Islands (E. fuscoguttatus). In addition, E. fuscoguttatus from net cages in an open water mariculture facility was compared with free living E. fuscoguttatus from its surroundings. Both core and shared microbiomes were investigated. Our results reveal that, while the core microbiomes of all three fish species were composed of fairly the same classes of bacteria, the proportions of these bacterial classes strongly varied. The microbial composition of phylogenetically distant fish species, i.e. A. mate and E. sexfasciatus from Jakarta Bay and Cilacap were more closely related than the microbial composition of more phylogentically closer species, i.e. E. fuscoguttatus, E. sexfasciatus from Jakarta Bay, Cilacap and Thousand Islands. In addition, we detected a weak negative correlation between the load of selected bacterial pathogens, i.e. Vibrio sp. and Photobacterium sp. and the number of endoparasites. In the case of Flavobacterium sp. the opposite was observed, i.e. a weak positive correlation. Of the three recorded pathogenic bacterial genera, Vibrio sp. was commonly found in E. fuscoguttatus from mariculture, and lessly in the vicinity of the net cages and rarely in the fishes from the heavily polluted waters from Jakarta Bay. Flavobacterium sp. showed higher counts in mariculture fish and Photobacteria sp. was the most prominent in fish inside and close to the net cages.

Population structure of the Indonesian giant tiger shrimp Penaeus monodon: a window into evolutionary similarities between paralogous mitochondrial DNA sequences and their genomes.

Here we used both microsatellites and mtCR (mitochondrial DNA control region) sequences as genetic markers to examine the genetic diversity and population structure of Penaeus monodon shrimp from six Indonesian regions. The microsatellite data showed that shrimp from the Indian and the Pacific Ocean were genetically distinct from each other. It has been reported previously that P. monodon mtCR sequences from the Indo-Pacific group into two major paralogous clades of unclear origin. Here we show that the population structure inferred from mtCR sequences matches the microsatellite-based population structure for one of these clades. This is consistent with the notion that this mtCR clade shares evolutionary history with nuclear DNA and may thus represent nuclear mitochondrial pseudogenes (Numts).

Expression of the toxin-antitoxin genes yefM(Lrh), yoeB(Lrh) in human Lactobacillus rhamnosus isolates.

Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected.

Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

Chlamydia psittaci: new insights into genomic diversity, clinical pathology, host-pathogen interaction and anti-bacterial immunity.

The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen.

Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells.

Chlamydia psittaci inclusion membrane protein IncB associates with host protein Snapin.

Chlamydia (C.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. During a unique developmental cycle of this obligate intracellular pathogen, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. C. psittaci uses dynein motor proteins for optimal early development. Chlamydial proteins that mediate this process are unknown. Two-hybrid screening with the C. psittaci inclusion protein IncB as bait against a HeLa Yeast Two-hybrid (YTH) library revealed that the host protein Snapin interacts with IncB. Snapin is a cytoplasmic protein that plays a multivalent role in intracellular trafficking. Confocal fluorescence microscopy using an IncB-specific antibody demonstrated that IncB, Snapin, and dynein were co-localized near the inclusion of C. psittaci-infected HEp-2 cells. This co-localization was lost when Snapin was depleted by RNAi. The interaction of Snapin with both IncB and dynein has been shown in vitro and in vivo. We propose that Snapin connects chlamydial inclusions with the microtubule network by interacting with both IncB and dynein.

Automated Segmentation and Object Classification of CT Images: Application to In Vivo Molecular Imaging of Avian Embryos.

Background. Although chick embryogenesis has been studied extensively, there has been growing interest in the investigation of skeletogenesis. In addition to improved poultry health and minimized economic loss, a greater understanding of skeletal abnormalities can also have implications for human medicine. True in vivo studies require noninvasive imaging techniques such as high-resolution microCT. However, the manual analysis of acquired images is both time consuming and subjective. Methods. We have developed a system for automated image segmentation that entails object-based image analysis followed by the classification of the extracted image objects. For image segmentation, a rule set was developed using Definiens image analysis software. The classification engine was implemented using the WEKA machine learning tool. Results. Our system reduces analysis time and observer bias while maintaining high accuracy. Applying the system to the quantification of long bone growth has allowed us to present the first true in ovo data for bone length growth recorded in the same chick embryos. Conclusions. The procedures developed represent an innovative approach for the automated segmentation, classification, quantification, and visualization of microCT images. MicroCT offers the possibility of performing longitudinal studies and thereby provides unique insights into the morpho- and embryogenesis of live chick embryos.

The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat.

The complete nucleotide sequences of four plasmids hosted by a Salmonella enterica serovar. Derby strain 6MK1 isolated from pork were determined by shotgun Sanger sequencing. A 107,637 base pairs (bp) conjugative plasmid pSD107 containing 150 putative coding sequences (CDS) could be assigned to the narrow host range incompatibility group IncI1. A detailed annotation of all CDS was carried out, revealing the presence of genes needed for plasmid replication, conjugal transfer, plasmid partitioning and stability as well as resistance to antimicrobials. The resistance determinants dhfrA1, aadA1, qacEΔ1, sul1 (supplied by a class 1 integron), blaTEM-1b (carried by a truncated Tn2 flanked by IS26), sul2 and strAB confer multidrug resistance to the host bacterium. In addition to pSD107, three small cryptic plasmids pSD4.0, pSD4.6 and pSD5.6 were identified, showing significant sequence similarities to already known replicons of Escherichia coli and S. enterica. In conjugation experiments performed on solid medium, pSD107 was successfully transferred to a nalidixic acid resistant E. coli DH5α, mobilizing pSD4.0 and, more infrequently, also pSD4.6. All transferred plasmids were stably propagated in the recipient strain without selective pressure for approximately 66 generations. The absolute plasmid copy numbers were determined in real time PCR experiments, revealing an approximate 1:1:1:1 ratio of the four replicons compared to the chromosome. The evolutionary position of pSD107 within the IncI1 family of plasmids was inferred from a maximum likelihood phylogenetic tree and by comparison of genetic key elements in a set of 17 IncI1 reference plasmids.

Identification of gene expression profiles in HeLa cells and HepG2 cells infected with Coxsackievirus B3.

Viral infections of host cells cause multiple changes of cellular metabolism including immediate defense mechanisms as well as processes to support viral replication. Coxsackievirus B3 (CVB3) is a member of the Picornavirus family and is responsible for a wide variety of mild or severe infections including acute and chronic inflammations. Thereby, intracellular signaling can be changed very comprehensively. In order to compare the influence of CVB3 replication on gene expression pattern of two different cell lines, DNA microarray systems were used to study a set of 780 genes related to inflammation. Expression analysis of HeLa cells and HepG2 cells infected with CVB3 identified 34 genes whose mRNA levels were altered significantly upon infection. The expression of additional 16 genes in HepG2 cells and 31 genes in HeLa cells were found to be influenced during CVB3 replication as well. All genes expressed differentially were sorted with regard to their functions and interpreted in view of known contributors to the infection process. The activation of the tumor necrosis factor pathways by CVB3 represents one peculiar observation, including apoptosis, stress, and inflammation responses.