Complex imaging of phase domains by deep neural networks

The reconstruction of a single-particle image from the modulus of its Fourier transform, by phase-retrieval methods, has been extensively applied in X-ray structural science. Particularly for strong-phase objects, such as the phase domains found inside crystals by Bragg coherent diffraction imaging (BCDI), conventional iteration methods are time consuming and sensitive to their initial guess because of their iterative nature. Here, a deep-neural-network model is presented which gives a fast and accurate estimate of the complex single-particle image in the form of a universal approximator learned from synthetic data. A way to combine the deep-neural-network model with conventional iterative methods is then presented to refine the accuracy of the reconstructed results from the proposed deep-neural-network model. Improved convergence is also demonstrated with experimental BCDI data

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

Ultrafast x-ray diffraction study of melt-front dynamics in polycrystalline thin films

Melting is a fundamental process of matter that is still not fully understood at the microscopic level. Here, we use time-resolved x-ray diffraction to examine the ultrafast melting of polycrystalline gold thin films using an optical laser pump followed by a delayed hard x-ray probe pulse. We observe the formation of an intermediate new diffraction peak, which we attribute to material trapped between the solid and melted states, that forms 50 ps after laser excitation and persists beyond 500 ps. The peak width grows rapidly for 50 ps and then narrows distinctly at longer time scales. We attribute this to a melting band originating from the grain boundaries and propagating into the grains. Our observation of this intermediate state has implications for the use of ultrafast lasers for ablation during pulsed laser deposition

Mobilization of genomic islands of Staphylococcus aureus by temperate bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus

Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host

In-Vivo Expression Profiling of Pseudomonas aeruginosa Infections Reveals Niche-Specific and Strain-Independent Transcriptional Programs

Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies

Evidence of Transfer by Conjugation of Type IV Secretion System Genes between Bartonella Species and Rhizobium radiobacter in Amoeba

Background: Bartonella species cospeciate with mammals and live within erythrocytes. Even in these specific niches, it has been recently suggested by bioinformatic analysis of full genome sequences that Lateral Gene Transfer (LGT) may occur but this has never been demonstrated biologically. Here we describe the sequence of the B. rattaustraliani (AUST/NH4 T) circular plasmid (pNH4) that encodes the tra cluster of the Type IV secretion system (T4SS) and we eventually provide evidence that Bartonella species may conjugate and exchange this plasmid inside amoeba. Principal Findings: The T4SS of pNH4 is critical for intracellular viability of bacterial pathogens, exhibits bioinformatic evidence of LGT among bacteria living in phagocytic protists. For instance, 3 out of 4 T4SS encoding genes from pNH4 appear to be closely related to Rhizobiales, suggesting that gene exchange occurs between intracellular bacteria from mammals (bartonellae) and plants (Rhizobiales). We show that B. rattaustraliani and Rhizobium radiobacter both survived within the amoeba Acanthamoeba polyphaga and can conjugate together. Our findings further support the hypothesis that tra genes might also move into and out of bacterial communities by conjugation, which might be the primary means of genomic evolution for intracellular adaptation by cross-talk of interchangeable genes between Bartonella species and plant pathogens. Conclusions: Based on this, we speculate that amoeba favor the transfer of genes as phagocytic protists, which allows fo

Insight into cross-talk between intra-amoebal pathogens

Abstract: Background: Amoebae are phagocytic protists where genetic exchanges might take place between amoeba-resistant bacteria. These amoebal pathogens are able to escape the phagocytic behaviour of their host. They belong to different bacterial phyla and often show a larger genome size than human-infecting pathogens. This characteristic is proposed to be the result of frequent gene exchanges with other bacteria that share a sympatric lifestyle and contrasts with the genome reduction observed among strict human pathogens.Results: We sequenced the genome of a new amoebal pathogen, Legionella drancourtii, and compared its gene content to that of a Chlamydia-related bacterium, Parachlamydia acanthamoebae. Phylogenetic reconstructions identified seven potential horizontal gene transfers (HGTs) between the two amoeba-resistant bacteria, including a complete operon of four genes that encodes an ABC-type transporter. These comparisons pinpointed potential cases of gene exchange between P. acanthamoebae and Legionella pneumophila, as well as gene exchanges between other members of the Legionellales and Chlamydiales orders. Moreover, nine cases represent possible HGTs between representatives from the Legionellales or Chlamydiales and members of the Rickettsiales order.Conclusions: This study identifies numerous gene exchanges between intracellular Legionellales and Chlamydiales bacteria, which could preferentially occur within common inclusions in their amoebal hosts. Therefore it contributes to improve our knowledge on the intra-amoebal gene properties associated to their specific lifestyle

Salmonella Strains Isolated from Galápagos Iguanas Show Spatial Structuring of Serovar and Genomic Diversity

It is thought that dispersal limitation primarily structures host-associated bacterial populations because host distributions inherently limit transmission opportunities. However, enteric bacteria may disperse great distances during food-borne outbreaks. It is unclear if such rapid long-distance dispersal events happen regularly in natural systems or if these events represent an anthropogenic exception. We characterized Salmonella enterica isolates from the feces of free-living Galápagos land and marine iguanas from five sites on four islands using serotyping and genomic fingerprinting. Each site hosted unique and nearly exclusive serovar assemblages. Genomic fingerprint analysis offered a more complex model of S. enterica biogeography, with evidence of both unique strain pools and of spatial population structuring along a geographic gradient. These findings suggest that even relatively generalist enteric bacteria may be strongly dispersal limited in a natural system with strong barriers, such as oceanic divides. Yet, these differing results seen on two typing methods also suggests that genomic variation is less dispersal limited, allowing for different ecological processes to shape biogeographical patterns of the core and flexible portions of this bacterial species' genome