Diversity of the cell-wall associated genomic island of the archaeon Haloquadratum walsbyi

Background

Haloquadratum walsbyi represents up to 80 % of cells in NaCl-saturated brines worldwide, but is notoriously difficult to maintain under laboratory conditions. In order to establish the extent of genetic diversity in a natural population of this microbe, we screened a H. walsbyi enriched metagenomic fosmid library and recovered seven novel version of its cell-wall associated genomic island. The fosmid inserts were sequenced and analysed.

Results

The novel cell-wall associated islands delineated two major clades within H. walsbyi. The islands predominantly contained genes putatively involved in biosynthesis of surface layer, genes encoding cell surface glycoproteins and genes involved in envelope formation. We further found that these genes are maintained in the population and that the diversity of this region arises through homologous recombination but also through the action of mobile genetic elements, including viruses.

Conclusions

The population of H. walsbyi in the studied saltern brine is composed of numerous clonal lineages that differ in surface structures including the cell wall. This type of variation probably reflects a number of mechanisms that minimize the infection rate of predating viruses.

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Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

Background

Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.

Results

Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.

Conclusion

We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.

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The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

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RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior

Background

Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long.

Results

We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes.

Conclusions

We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

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Unveiling nonessential gene deletions that confer significant morphological phenotypes beyond natural yeast strains

Background

Phenotypes are variable within species, with high phenotypic variation in the fitness and cell morphology of natural yeast strains due to genetic variation. A gene deletion collection of yeast laboratory strains also contains phenotypic variations, demonstrating the involvement of each gene and its specific function. However, to date, no study has compared the phenotypic variations between natural strains and gene deletion mutants in yeast.

Results

The morphological variance was compared between 110 most distinct gene deletion strains and 36 typical natural yeast strains using a generalized linear model. The gene deletion strains had higher morphological variance than the natural strains. Thirty-six gene deletion mutants conferred significant morphological changes beyond that of the natural strains, revealing the importance of the genes with high genetic interaction and specific cellular functions for species conservation.

Conclusion

Based on the morphological analysis, we discovered gene deletion mutants whose morphologies were not seen in nature. Our multivariate approach to the morphological diversity provided a new insight into the evolution and species conservation of yeast.

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Pesticidal and pest repellency activities of rhizomes of Drynaria quercifolia (J. Smith) against Tribolium castaneum (Herbst)

Background

Tribolium castaneum (Herbst) is a harmful pest of stored grain and flour-based products in tropical and subtropical region. In the present study, rhizome of Drynaria quercifolia (J. Smith) was evaluated for pesticidal and pest repellency activities against T. castaneum, using surface film method and filter paper disc method, respectively. In addition, activity of the isolated compound 3,4-dihydroxybenzoic acid was evaluated against the pest.

Results

Chloroform soluble fraction of ethanol extract of rhizome of D. quercifolia showed significant pesticidal activity at doses 0.88 to 1.77 mg/cm² and significant pest repellency activity at doses 0.94 to 0.23 mg/cm². No pesticidal and pest repellency activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of ethanol extract as well as for 3,4-dihydroxybenzoic acid.

Conclusion

Considering our findings it can be concluded that chloroform soluble fraction of rhizome of D. quercifolia is useful in controlling T. castaneum of stored grain and flour-based products.

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Uniparental mitochondrial DNA inheritance is not affected in Ustilago maydis Δatg11 mutants blocked in mitophagy

Background

Maternal or uniparental inheritance (UPI) of mitochondria is generally observed in sexual eukaryotes, however, the underlying mechanisms are diverse and largely unknown. Recently, based on the use of mutants blocked in autophagy, it has been demonstrated that autophagy is required for strict maternal inheritance in the nematode Caenorhabditis elegans. Uniparental mitochondrial DNA (mtDNA) inheritance has been well documented for numerous fungal species, and in particular, has been shown to be genetically governed by the mating-type loci in the isogamous species Cryptococcus neoformans, Phycomyces blakesleeanus and Ustilago maydis. Previously, we have shown that the a2 mating-type locus gene lga2 is decisive for UPI during sexual development of U. maydis. In axenic culture, conditional overexpression of lga2 triggers efficient loss of mtDNA as well as mitophagy. To assess a functional relationship, we have investigated UPI in U. maydis Δatg11 mutants, which are blocked in mitophagy.

Results

This study has revealed that Δatg11 mutants are not affected in pathogenic development and this has allowed us to analyse UPI under comparable developmental conditions between mating-compatible wild-type and mutant strain combinations. Explicitly, we have examined two independent strain combinations that gave rise to different efficiencies of UPI. We demonstrate that in both cases UPI is atg11-independent, providing evidence that mitophagy is not critical for UPI in U. maydis, even under conditions of strict UPI.

Conclusions

Until now, analysis of a role of mitophagy in UPI has not been reported for microbial species. Our study suggests that selective autophagy does not contribute to UPI in U. maydis, but is rather a consequence of selective mtDNA elimination in response to mitochondrial damage.

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Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background

The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations.

Results

Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI.

Conclusions

In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

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Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

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A new platform for ultra-high density Staphylococcus aureus transposon libraries

Background

Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.

Results

Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.

Conclusions

The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

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