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Auto Tag: University of Lausanne

July 7, 2019  |  

Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence.

Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection.In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined.In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation).Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

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July 7, 2019  |  

Evolutionary dynamics of pathoadaptation revealed by three independent acquisitions of the VirB/D4 type IV secretion system in Bartonella.

The a-proteobacterial genus Bartonella comprises a group of ubiquitous mammalian pathogens that are studied as a model for the evolution of bacterial pathogenesis. Vast abundance of two particular phylogenetic lineages of Bartonella had been linked to enhanced host adaptability enabled by lineage-specific acquisition of a VirB/D4 type IV secretion system (T4SS) and parallel evolution of complex effector repertoires. However, the limited availability of genome sequences from one of those lineages as well as other, remote branches of Bartonella has so far hampered comprehensive understanding of how the VirB/D4 T4SS and its effectors called Beps have shaped Bartonella evolution. Here, we report the discovery of a third repertoire of Beps associated with the VirB/D4 T4SS of B. ancashensis, a novel human pathogen that lacks any signs of host adaptability and is only distantly related to the two species-rich lineages encoding a VirB/D4 T4SS. Furthermore, sequencing of ten new Bartonella isolates from under-sampled lineages enabled combined in silico analyses and wet lab experiments that suggest several parallel layers of functional diversification during evolution of the three Bep repertoires from a single ancestral effector. Our analyses show that the Beps of B. ancashensis share many features with the two other repertoires, but may represent a more ancestral state that has not yet unleashed the adaptive potential of such an effector set. We anticipate that the effectors of B. ancashensis will enable future studies to dissect the evolutionary history of Bartonella effectors and help unraveling the evolutionary forces underlying bacterial host adaptation.© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019  |  

Clostridium chauvoei, an evolutionary dead-end pathogen.

Full genome sequences of 20 strains of Clostridium chauvoei, the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951-2015) were determined and analyzed. The study reveals that the genome of the species C. chauvoei is highly homogeneous compared to the closely related species C. perfringens, a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most C. chauvoei strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 – 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of fliC1, fliC2 and fliC3 genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic C.chauvoei toxin A, the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of C. chauvoei. These data indicate that C. chauvoei is a strict ruminant-associated pathogen that has reached a dead end in its evolution.

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July 7, 2019  |  

One year genome evolution of Lausannevirus in allopatric versus sympatric conditions.

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5?Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346?kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12?months. Lausannevirus genome evolution in sympatric conditions was investigated by its co-culture with Estrella lausannensis, an obligate intracellular bacterium, in the amoeba A. castellanii during one year. Cultures were split every 3?months. Genome sequencing revealed that in these conditions both, Lausannevirus and E. lausannensis, show stable genome, presenting no major rearrangement. In fact, after one year they acquired from 2 to 7 and from 4 to 10 mutations per culture for Lausannevirus and E. lausannensis, respectively. Interestingly, different mutations in the endonuclease encoding genes of Lausannevirus were observed in different subcultures, highlighting the importance of this gene product in the replication of Lausannevirus. Conversely, mutations in E. lausannensis were mainly located in a gene encoding for a phosphoenolpyruvate-protein phosphotransferase (PtsI), implicated in sugar metabolism. Moreover, in our conditions and with our analyses we detected no horizontal gene transfer during one year of co-culture.© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019  |  

Insights from the genome sequence of Mycobacterium lepraemurium: Massive gene decay and reductive evolution.

Mycobacterium lepraemurium is the causative agent of murine leprosy, a chronic, granulomatous disease similar to human leprosy. Due to the similar clinical manifestations of human and murine leprosy and the difficulty of growing both bacilli axenically, Mycobacterium leprae and M. lepraemurium were once thought to be closely related, although it was later suggested that M. lepraemurium might be related to Mycobacterium avium In this study, the complete genome of M. lepraemurium was sequenced using a combination of PacBio and Illumina sequencing. Phylogenomic analyses confirmed that M. lepraemurium is a distinct species within the M. avium complex (MAC). The M. lepraemurium genome is 4.05 Mb in length, which is considerably smaller than other MAC genomes, and it comprises 2,682 functional genes and 1,139 pseudogenes, which indicates that M. lepraemurium has undergone genome reduction. An error-prone repair homologue of the DNA polymerase III a-subunit was found to be nonfunctional in M. lepraemurium, which might contribute to pseudogene formation due to the accumulation of mutations in nonessential genes. M. lepraemurium has retained the functionality of several genes thought to influence virulence among members of the MAC.IMPORTANCEMycobacterium lepraemurium seems to be evolving toward a minimal set of genes required for an obligatory intracellular lifestyle within its host, a niche seldom adopted by most mycobacteria, as they are free-living. M. lepraemurium could be used as a model to elucidate functions of genes shared with other members of the MAC. Its reduced gene set can be exploited for studying the essentiality of genes in related pathogenic species, which might lead to discovery of common virulence factors or clarify host-pathogen interactions. M. lepraemurium can be cultivated in vitro only under specific conditions and even then with difficulty. Elucidating the metabolic (in)capabilities of M. lepraemurium will help develop suitable axenic media and facilitate genetic studies. Copyright © 2017 Benjak et al.

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July 7, 2019  |  

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.© 2017 John Wiley & Sons Ltd.

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July 7, 2019  |  

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses.The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.

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July 7, 2019  |  

Complete genome sequences of 12 species of stable defined moderately diverse mouse microbiota 2.

We report here the complete genome sequences of 12 bacterial species of stable defined moderately diverse mouse microbiota 2 (sDMDMm2) used to colonize germ-free mice with defined microbes. Whole-genome sequencing of these species was performed using the PacBio sequencing platform yielding circularized genome sequences of all 12 species. Copyright © 2016 Uchimura et al.

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July 7, 2019  |  

Comparative genomics analysis of Streptococcus tigurinus strains identifies genetic elements specifically and uniquely present in highly virulent strains.

Streptococcus tigurinus is responsible for severe invasive infections such as infective endocarditis, spondylodiscitis and meningitis. As described, S. tigurinus isolates AZ_3aT and AZ_14 were highly virulent (HV phenotype) in an experimental model of infective endocarditis and showed enhanced adherence and invasion of human endothelial cells when compared to low virulent S. tigurinus isolate AZ_8 (LV phenotype). Here, we sought whether genetic determinants could explain the higher virulence of AZ_3aT and AZ_14 isolates. Several genetic determinants specific to the HV strains were identified through extensive comparative genomics amongst which some were thought to be highly relevant for the observed HV phenotype. These included i) an iron uptake and metabolism operon, ii) an ascorbate assimilation operon, iii) a newly acquired PI-2-like pilus islets described for the first time in S. tigurinus, iv) a hyaluronate metabolism operon, v) an Entner-Doudoroff pathway of carbohydrates metabolism, and vi) an alternate pathways for indole biosynthesis. We believe that the identified genomic features could largely explain the phenotype of high infectivity of the two HV S. tigurinus strains. Indeed, these features include determinants that could be involved at different stages of the disease such as survival of S. tigurinus in blood (iron uptake and ascorbate metabolism operons), initial attachment of bacterial pathogen to the damaged cardiac tissue and/or vegetation that formed on site (PI-2-like pilus islets), tissue invasion (hyaluronate operon and Entner-Doudoroff pathway) and regulation of pathogenicity (indole biosynthesis pathway).

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July 7, 2019  |  

Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).

The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ~3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3′ untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ~3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.© 2016 Gosselin et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019  |  

Decay of sexual trait genes in an asexual parasitoid wasp.

Trait loss is a widespread phenomenon with pervasive consequences for a species’ evolutionary potential. The genetic changes underlying trait loss have only been clarified in a small number of cases. None of these studies can identify whether the loss of the trait under study was a result of neutral mutation accumulation or negative selection. This distinction is relatively clear-cut in the loss of sexual traits in asexual organisms. Male-specific sexual traits are not expressed and can only decay through neutral mutations, whereas female-specific traits are expressed and subject to negative selection. We present the genome of an asexual parasitoid wasp and compare it to that of a sexual lineage of the same species. We identify a short-list of 16 genes for which the asexual lineage carries deleterious SNP or indel variants, whereas the sexual lineage does not. Using tissue-specific expression data from other insects, we show that fifteen of these are expressed in male-specific reproductive tissues. Only one deleterious variant was found that is expressed in the female-specific spermathecae, a trait that is heavily degraded and thought to be under negative selection in L. clavipes. Although the phenotypic decay of male-specific sexual traits in asexuals is generally slow compared with the decay of female-specific sexual traits, we show that male-specific traits do indeed accumulate deleterious mutations as expected by theory. Our results provide an excellent starting point for detailed study of the genomics of neutral and selected trait decay.

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July 7, 2019  |  

The genome of the toluene-degrading Pseudomonas veronii strain 1YdBTEX2 and its differential gene expression in contaminated sand.

The natural restoration of soils polluted by aromatic hydrocarbons such as benzene, toluene, ethylbenzene and m- and p-xylene (BTEX) may be accelerated by inoculation of specific biodegraders (bioaugmentation). Bioaugmentation mainly involves introducing bacteria that deploy their metabolic properties and adaptation potential to survive and propagate in the contaminated environment by degrading the pollutant. In order to better understand the adaptive response of cells during a transition to contaminated material, we analyzed here the genome and short-term (1 h) changes in genome-wide gene expression of the BTEX-degrading bacterium Pseudomonas veronii 1YdBTEX2 in non-sterile soil and liquid medium, both in presence or absence of toluene. We obtained a gapless genome sequence of P. veronii 1YdBTEX2 covering three individual replicons with a total size of 8 Mb, two of which are largely unrelated to current known bacterial replicons. One-hour exposure to toluene, both in soil and liquid, triggered massive transcription (up to 208-fold induction) of multiple gene clusters, such as toluene degradation pathway(s), chemotaxis and toluene efflux pumps. This clearly underlines their key role in the adaptive response to toluene. In comparison to liquid medium, cells in soil drastically changed expression of genes involved in membrane functioning (e.g., lipid composition, lipid metabolism, cell fatty acid synthesis), osmotic stress response (e.g., polyamine or trehalose synthesis, uptake of potassium) and putrescine metabolism, highlighting the immediate response mechanisms of P. veronii 1YdBTEX2 for successful establishment in polluted soil.

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July 7, 2019  |  

Cell cycle constraints and environmental control of local DNA hypomethylation in a-proteobacteria.

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of a-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5′-GANTC-3′ sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different a-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.

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