Differential Exoproteome and Biochemical Characterisation of Neoparamoeba perurans

Infection with the protozoan ectoparasite Neoparamoeba perurans, the causative agent of AGD, remains a global threat to salmonid farming. This study aimed to analyse the exoproteome of both an attenuated and virulent N. perurans isolate using proteomics and cytotoxicity testing. A disproportionate presence of proteins from the co-cultured microbiota of N. perurans was revealed on searching an amalgamated database of bacterial, N. perurans and Amoebozoa proteins. LC‑MS/MS identified 33 differentially expressed proteins, the majority of which were upregulated in the attenuated exoproteome. Proteins of putative interest found in both exoproteomes were maltoporin, ferrichrome-iron receptor, and putative ferric enterobactin receptor. Protease activity remained significantly elevated in the attenuated exoproteome compared with the virulent exoproteome. Similarly, the attenuated exoproteome had a significantly higher cytotoxic effect on rainbow trout gill cell line (RTgill W1) cells compared with the virulent exoproteome. The presence of a phosphatase and serine protease in the virulent exoproteome may facilitate AGD infection but do not appear to be key players in causing cytotoxicity. Altogether, this study reveals prolonged culture of N. perurans affects the exoproteome composition in favour of nutritional acquisition, and that the current culturing protocol for virulent N. perurans does not facilitate the secretion of virulence factors.

Phytotransferrin endocytosis mediates a direct cell surface-to-chloroplast iron trafficking axis in marine diatoms

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, respiration, nitrate assimilation, nitrogen fixation and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is internalized via endocytosis, however proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically and biotechnologically important microalgae.

2606. A Divergent Ferrichrome Receptor Associated with an Insertional Element (IS3) Identified on Novel Locus in Clinical Strain of Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is an aerobic Gram-negative bacterium that causes life-threatening acute and chronic infections in humans. Genotypic mutations and phenotypic variations are key features of its antimicrobial resistance and adaptation to the host environment. Pyoverdine associated genes and divergent receptors play a key role in acute Pseudomonas infections. This study seeks to address the heterogeneity of ferrichrome-iron receptor (fpvA) expression, its effect on pathogenicity and its propensity to cause acute infections clinically. Genetic and phenotypic variation of a clinical isolates of P. aeruginosa (PA097 and PA115) were identified by complete genome sequencing method. Methods An IRB-approved prospective study collected 38 P. aeruginosa clinical isolates and stored at Carilion Medical Center. Two genetically unrelated clinical strains were selected from tracheal aspirates: PA097 and PA115. These isolates were characterized by pyoverdine (pvd) quantification in planktonic culture filtrate at OD405 nm. Multiplex PCR was carried out using primers for fpvA receptors. Quantification of iron acquisition was done on chrome azurol S (CAS) agar. Genomes for PA115 and PA097 were sequenced by Illumina Next-generation DNA sequencing. Results Genome assembly shows a 6.3 Mb genome size in PA115 with G+C content of 66.4%. Seven insertion sequence elements were located. We found a 101 kb locus for pvD and a highly diversified fpvA associated with an insertional element (IS3). PA115, exhibits rich green pigment of pvd followed by PAO1 and PA097 in LB media (Figure 1A) and also in Planktonic culture filtrate (Fig 1B) for quantitative estimation of pvd (Figure 1C). On CAS agar, PA115 showed high uptake of iron by orange pigment compared with lower pigmentation in PAO1 and PA097 (Fig 1D). We confirmed the ferrichrome-iron receptor as fpvAIIb in PA115 by Multiplex PCR seen in sequencing of PA115 (Figure 2). Conclusion We found high genetic and phenotypic variation in clinical isolate of P. aeruginosa (PA115) from an acute pneumonia patient. The novel IS element found in its receptor gene locus suggests an increased role in pvd expression and iron uptake from the host. Increased pvd expression and diversified fpvAIIb association with an IS3 element may indicate higher virulence in the PA115 strain. Disclosures All authors: No reported disclosures.

Interspecies Variations in Bordetella Catecholamine Receptor Gene Regulation and Function

Bordetella bronchisepticacan use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and althoughBordetella pertussishas thebfrDandbfrEgenes, the role of these genes in iron uptake has not been demonstrated. In this study, thebfrDandbfrEgenes ofB. pertussiswere shown to be functional inB. bronchiseptica, but neitherB. bronchiseptica bfrDnorbfrEimparted catecholamine utilization toB. pertussis. Gene fusion analyses found that expression ofB. bronchiseptica bfrAwas increased during iron starvation, as is common for iron receptor genes, but that expression of thebfrDandbfrEgenes of both species was decreased during iron limitation. As shown previously forB. pertussis,bfrDexpression inB. bronchisepticawas also dependent on the BvgAS virulence regulatory system; however, in contrast to the case inB. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression ofbfrDinB. bronchiseptica. Further studies using aB. bronchisepticabvgASmutant expressing theB. pertussisbvgASgenes revealed that the interspecies differences inbfrDmodulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to thein vivofitness ofB. bronchisepticaandB. pertussis. Additional evidence of thein vivoimportance of theB. pertussisreceptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with theB. pertussisBfrD and BfrE proteins.

Immunization with the Yersiniabactin Receptor, FyuA, Protects against Pyelonephritis in a Murine Model of Urinary Tract Infection

ABSTRACTUrinary tract infections (UTI) are common and represent a substantial economic and public health burden. Roughly 80% of these infections are caused by a heterogeneous group of uropathogenicEscherichia coli(UPEC) strains. Antibiotics are standard therapy for UTI, but a rise in antibiotic resistance has complicated treatment, making the development of a UTI vaccine more urgent. Iron receptors are a promising new class of vaccine targets for UTI, as UPEC require iron to colonize the iron-limited host urinary tract and genes encoding iron acquisition systems are highly expressed during infection. Previously, three of six UPEC siderophore and heme receptors were identified as vaccine candidates by intranasal immunization in a murine model of ascending UTI. To complete the assessment of iron receptors as vaccine candidates, an additional six UPEC iron receptors were evaluated. Of the six vaccine candidates tested in this study (FyuA, FitA, IroN, the gene product of the CFT073 locusc0294, and two truncated derivatives of ChuA), only FyuA provided significant protection (P= 0.0018) against UPEC colonization. Intranasal immunization induced a robust and long-lived humoral immune response. In addition, the levels of FyuA-specific serum IgG correlated with bacterial loads in the kidneys [Spearman's rank correlation coefficient ρ(14) = −0.72,P= 0.0018], providing a surrogate of protection. FyuA is the fourth UPEC iron receptor to be identified from our screens, in addition to IutA, Hma, and IreA, which were previously demonstrated to elicit protection against UPEC challenge. Together, these iron receptor antigens will facilitate the development of a broadly protective, multivalent UTI vaccine to effectively target diverse strains of UPEC.

Involvement of Fe Uptake Systems and AmpC β-Lactamase in Susceptibility to the Siderophore Monosulfactam BAL30072 in Pseudomonas aeruginosa

ABSTRACTBAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistantPseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 inP. aeruginosaPAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression ofampCand the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of thefecIpromoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. AfecImutant and afecAmutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. ThepiuCgene, coding for a Fe(II)-dependent dioxygenase located next to thepiuAiron receptor gene, was also downregulated. The MICs of BAL30072 forpiuCandpiuAtransposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility ofP. aeruginosaPAO1 to BAL30072.