The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and compared them with each other and with the model strain PAO1. Phenotypic analysis of CF isolates showed significant variability in colonization and virulence-related traits suggesting different strategies for adaptation to the CF lung. Genomic analysis indicated these strains shared a large set of core genes with the standard laboratory strain PAO1, and identified the genetic basis for some of the observed phenotypic differences. Proteomics revealed that in a conventional laboratory medium PAO1 expressed 827 proteins that were absent in the CF isolates while the CF isolates shared a distinctive signature set of 703 proteins not detected in PAO1. PAO1 expressed many transporters for the uptake of organic nutrients and relatively few biosynthetic pathways. Conversely, the CF isolates expressed a narrower range of transporters and a broader set of metabolic pathways for the biosynthesis of amino acids, carbohydrates, nucleotides and polyamines. The proteomic data suggests that in a common laboratory medium PAO1 may transport a diverse set of “ready-made” nutrients from the rich medium, whereas the CF isolates may only utilize a limited number of nutrients from the medium relying mainly on their own metabolism for synthesis of essential nutrients. These variations indicate significant differences between the metabolism and physiology of P. aeruginosa CF isolates and PAO1 that cannot be detected at the genome level alone. The widening gap between the increasing genomic data and the lack of phenotypic data means that researchers are increasingly reliant on extrapolating from genomic comparisons using experimentally characterized model organisms such as PAO1. While comparative genomics can provide valuable information, our data suggests that such extrapolations may be fraught with peril.

Immunity and nutrition are two essential modulators of individual fitness. However, while the implications of immune function and nutrition on an individual's lifespan and reproduction are well established, the interplay between feeding behaviour, infection and immune function remains poorly understood. Asking how ecological and physiological factors affect immune responses and resistance to infections is a central theme of eco‐immunology. In this study, we used the fruit fly, Drosophila melanogaster, to investigate how infection through septic injury modulates nutritional intake and how macronutrient balance affects survival to infection by the pathogenic Gram‐positive bacterium Micrococcus luteus. Our results show that infected flies maintain carbohydrate intake, but reduce protein intake, thereby shifting from a protein‐to‐carbohydrate (P:C) ratio of ~1:4 to ~1:10 relative to non‐infected and sham‐infected flies. Strikingly, the proportion of flies dying after M. luteus infection was significantly lower when flies were fed a low‐P high‐C diet, revealing that flies shift their macronutrient intake as means of nutritional self‐medication against bacterial infection. These results are likely due to the effects of the macronutrient balance on the regulation of the constitutive expression of innate immune genes, as a low‐P high‐C diet was linked to an upregulation in the expression of key antimicrobial peptides. Together, our results reveal the intricate relationship between macronutrient intake and resistance to infection and integrate the molecular cross‐talk between metabolic and immune pathways into the framework of nutritional immunology.

The filamentous fungus Scedosporium aurantiacum and the bacterium Pseudomonas aeruginosa are opportunistic pathogens isolated from lungs of the cystic fibrosis (CF) patients. P. aeruginosa has been known to suppress the growth of a number of CF related fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. However, the interactions between P. aeruginosa and S. aurantiacum have not been investigated in depth. Hence we assessed the effect of P. aeruginosa reference strain PAO1 and two clinical isolates PASS1 and PASS2 on the growth of two clinical S. aurantiacum isolates WM 06.482 and WM 08.202 using solid plate assays and liquid cultures, in a synthetic medium mimicking the nutrient condition in the CF sputum. Solid plate assays showed a clear inhibition of growth of both S. aurantiacum strains when cultured with P. aeruginosa strains PASS1 and PAO1. The inhibitory effect was confirmed by confocal microscopy. In addition to using chemical fluorescent stains, strains tagged with yfp (P. aeruginosa PASS1) and mCherry (S. aurantiacum WM 06.482) were created to facilitate detailed microscopic observations on strain interaction. To our knowledge, this is the first study describing successful genetic transformation of S. aurantiacum. Inhibition of growth was observed only in co-cultures of P. aeruginosa and S. aurantiacum; the cell fractions obtained from independent bacterial monocultures failed to initiate a response against the fungus. In the liquid co-cultures, biofilm forming P. aeruginosa strains PASS1 and PAO1 displayed higher inhibition of fungal growth when compared to PASS2. No change was observed in the inhibition pattern when direct cell contact between the bacterial and fungal strains was prevented using a separation membrane suggesting the involvement of extracellular metabolites in the fungal inhibition. However, one of the most commonly described bacterial virulence factors, pyocyanin, had no effect against either of the S. aurantiacum strains. This study shows that P. aeruginosa has a substantial inhibitory effect on the growth of the recently described CF fungal pathogen S. aurantiacum. The findings also highlighted that P. aeruginosa biofilm formation is important but not crucial for inhibiting the growth of S. aurantiacum in a lung- mimicking environment.

Cystic fibrosis (CF) is a congenital disease that results in great morbidity and mortality mainly in the Caucasian population. Although CF is a monogenic disease caused by mutation in the CF conductance transmembrane regulator (CFTR) gene, most of the related mortality can be attributed to infection mediated by opportunistic bacterial and fungal pathogens. Over the past decade, advancements in the field of proteomics have helped to gain insight into the repertoire of host and pathogen proteins involved in CF pathophysiology. This review provides an overview of the contributions of proteomic studies in advancing our knowledge of the biology of CF and disease progression associated with pathogen infection and host defense responses.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.

Galleria mellonella has risen to fame as an invertebrate model organism given its ethical advantages, low maintenance costs, rapid reproduction time, short life cycle, high number of progeny, tolerance for human body temperatures, innate immune system and similarities to mammalian host models. It is increasingly being utilised to evaluate in vivo toxicity and efficacy of chemical compounds and antimicrobials, modelling microbial (bacterial, fungal, viral) pathogenicity and assessing host-pathogen interaction during infection. During this molecular age of genomic, transcriptomic, proteomic, and genetic manipulation approaches, our understanding of microbial pathogenicity and host-pathogen interactions has deepened from high-throughput molecular studies performed in G. mellonella. In this review, we describe the use of G. mellonella in a broad range of studies involving omics, drug resistance, functional analysis and host-microbial community relationships. The future of G. mellonella in the molecular age is bright, with a multitude of new approaches and uses for this model from clinical to biotechnological on the horizon.

Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). We undertook Biolog Phenotype Microarray testing of P. aeruginosa CF isolates to investigate their catabolic capabilities compared to P. aeruginosa laboratory strains PAO1 and PA14. One strain, PASS4, displayed an unusual phenotype, only showing strong respiration on adenosine and inosine. Further testing indicated that PASS4 could grow on DNA as a sole carbon source, with a higher biomass production than PAO1. This suggested that PASS4 was specifically adapted to metabolize extracellular DNA, a substrate present at high concentrations in the CF lung. Transcriptomic and proteomic profiling of PASS4 and PAO1 when grown with DNA as a sole carbon source identified a set of upregulated genes, including virulence and host-adaptation genes. PASS4 was unable to utilize N -Acetyl- D -glucosamine, and when we selected PASS4 mutants able to grow on this carbon source, they also displayed a gain in ability to catabolize a broad range of other carbon sources. Genome sequencing of the mutants revealed they all contained mutations within the purK gene, encoding a key protein in the de novo purine biosynthesis pathway. This suggested that PASS4 was a purine auxotroph. Growth assays in the presence of 2 mM adenosine and the complementation of PASS4 with an intact purK gene confirmed this conclusion. Purine auxotrophy may represent a viable microbial strategy for adaptation to DNA-rich environments such as the CF lung.

21 Immunity and nutrition are two essential modulators of individual fitness 22  However, while the implications of immune function and nutrition on an individual's 23 lifespan and reproduction are known, the interplay between feeding behaviour, infection, 24 and immune function, remains poorly understood. 25  In this study, we used the fruit fly, Drosophila melanogaster, to investigate how infection 26 through septic injury modulates nutritional intake, and how macronutrient balance affects 27 survival to infection by the pathogenic Gram-positive bacterium Micrococcus luteus. 28 Our results show that infected flies maintain carbohydrate intake, but reduce protein intake, 29 thereby shifting from a protein-to-carbohydrate (P:C) ratio of ~1:4 to ~1:10 relative to non-30 infected and sham-infected flies. 31 Strikingly, we found that the proportion of flies dying after M. luteus infection was 32 significantly lower when flies were fed a low-P high-C diet, revealing that flies shift their 33 macronutrient intake as means of nutritional self-medication against bacterial infection. 34 This is likely due to the effects of macronutrient balance on the regulation of the 35 constitutive expression of innate immune genes, as a low-P high-C diet was linked to an 36 up-regulation in the expression of key antimicrobial peptides. 37 Together, our results reveal the intricate relationship between macronutrient intake and 38 resistance to infection, and integrate the molecular cross-talk between metabolic and 39 immune pathways into the framework of nutritional immunology. 40 41 42 83 a detailed investigation of the dietary modulation of constitutive innate immune gene expression 84 in an age-dependent manner. The effects of nutrition were measured through a geometric 85 manipulation of the dietary protein and carbohydrate balance. Our observations unveiled 86 nutritional regulations of innate immune gene expression and resistance to bacterial infections, and 87 link these findings to nutritional self-medication.88 5 89 91 We first hypothesized that infection through septic injury with the pathogen Micrococcus luteus 92 would modulate the nutritional selection of Drosophila melanogaster. Adult flies were offered a 93 choice between two capillaries filled with either a sucrose or a yeast solution, and food intake was 94 measured every two days for six days (Ja et al., 2007) (see Methods). While non-, sham-and M. 95 luteus-infected flies ingested similar quantities of carbohydrate (cumulative consumption of 96 carbohydrate over six days, One-way ANOVA, F2,36=1.775, p=0.185, Supplementary Table 1), the 97 consumption of protein was significantly different between the treatments (i.e., cumulative 98 consumption of protein for six days, One way ANOVA, F2,36=5.853, p=0.007, Supplementary 99 Table 1). Protein consumption was the lowest for flies infected with M. luteus and was the greatest 100 for sham-infected and non-infected flies (Fig. 1). This reduction in protein intake by infected flies 101 resulted in a marked ch...