An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 with 7-hydroxytropolone is pivotal for its antibacterial activity.

Pseudomonas donghuensis P482 exhibits broad antimicrobial activity against phytopathogens, including the soft rot bacteria of the Dickeya genus. Here, we report that under limited nutrient availability, the antibacterial activity of P. donghuensis P482 against Dickeya solani requires the reciprocal action of two iron scavengers: 7-hydroxytropolone (7-HT) and a newly characterized pyoverdine (PVDP482 ) and is quenched in the iron-augmented environment. Further, we show that the biosynthesis of pyoverdine and 7-HT is metabolically coordinated, and the functional BV82_4709 gene involved in 7-HT synthesis is pivotal for expressing the BV82_3755 gene, essential for pyoverdine biosynthesis and vice versa. The synthesis of both scavengers is under the control of Gac/Rsm, but only PVD is controlled by Fur. The isoelectric focusing profile of the P482 siderophore differs from that of the other Pseudomonas spp. tested. This finding led to the unveiling of the chemical structure of the new pyoverdine PVDP482 . To summarize, the antibacterial activity of P. donghuensis P482 is attributed to 7-HT and PVDP482 varies depending on the nutrient and iron availability, highlighting the importance of these factors in the competition between P482 and D. solani.

Substitutions at position 263 within the von Willebrand factor type A domain determine the functionality of complement C2 protein.

The complement system is one of the first defense lines protecting from invading pathogens. However, it may turn offensive to the body's own cells and tissues when deregulated by the presence of rare genetic variants that impair physiological regulation and/or provoke abnormal activity of key enzymatic components. Factor B and complement C2 are examples of paralogs engaged in the alternative and classical/lectin complement pathway, respectively. Pathogenic mutations in the von Willebrand factor A domain (vWA) of FB have been known for years. Despite substantial homology between two proteins and the demonstration that certain substitutions in FB translated to C2 result in analogous phenotype, there was a limited number of reports on pathogenic C2 variants in patients. Recently, we studied a cohort of patients suffering from rare kidney diseases and confirmed the existence of two gain-of-function and three loss-of-function mutations within the C2 gene sequences coding for the vWA domain (amino acids 254-452) or nearly located unstructured region (243-253) of C2 protein. Herein, we report the functional consequences of amino acid substitution of glutamine at position 263. The p.Q263G variant resulted in the gain-of-function phenotype, similarly to a homologous mutation p.D279G in FB. Conversely, the p.Q263P variant found in a patient with C3 glomerulopathy resulted in the loss of C2 function. Our results confirm that the N-terminal part of the vWA domain is a hot spot crucial for the complement C2 function.

Gain-of-Function Mutations R249C and S250C in Complement C2 Protein Increase C3 Deposition in the Presence of C-Reactive Protein.

The impairment of the alternative complement pathway contributes to rare kidney diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). We recently described an aHUS patient carrying an exceptional gain-of-function (GoF) mutation (S250C) in the classical complement pathway component C2 leading to the formation of hyperactive classical convertases. We now report the identification of the same mutation and another C2 GoF mutation R249C in two other patients with a glomerulopathy of uncertain etiology. Both mutations stabilize the classical C3 convertases by a similar mechanism. The presence of R249C and S250C variants in serum increases complement-dependent cytotoxicity (CDC) in antibody-sensitized human cells and elevates deposition of C3 on ELISA plates coated with C-reactive protein (CRP), as well as on the surface of glomerular endothelial cells. Our data justify the inclusion of classical pathway genes in the genetic analysis of patients suspected of complement-driven renal disorders. Also, we point out CRP as a potential antibody-independent trigger capable of driving excessive complement activation in carriers of the GoF mutations in complement C2.

Compatibility of Distinct Label-Free Proteomic Workflows in Absolute Quantification of Proteins Linked to the Oocyte Quality in Human Follicular Fluid.

We present two separate label-free quantitative workflows based on different high-resolution mass spectrometers and LC setups, which are termed after the utilized instrument: Quad-Orbitrap (nano-LC) and Triple Quad-TOF (micro-LC) and their directed adaptation toward the analysis of human follicular fluid proteome. We identified about 1000 proteins in each distinct workflow using various sample preparation methods. With assistance of the Total Protein Approach, we were able to obtain absolute protein concentrations for each workflow. In a pilot study of twenty samples linked to diverse oocyte quality status from four donors, 455 and 215 proteins were quantified by the Quad-Orbitrap and Triple Quad-TOF workflows, respectively. The concentration values obtained from both workflows correlated to a significant degree. We found reasonable agreement of both workflows in protein fold changes between tested groups, resulting in unified lists of 20 and 22 proteins linked to oocyte maturity and blastocyst development, respectively. The Quad-Orbitrap workflow was best suited for an in-depth analysis without the need of extensive fractionation, especially of low abundant proteome, whereas the Triple Quad-TOF workflow allowed a more robust approach with a greater potential to increase in effectiveness with the growing number of analyzed samples after the initial effort of building a comprehensive spectral library.

Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions.

In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC). While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterized. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes, and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1) and downstream (oriC2) of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5'-TTCAC-3' (4-8 nt), which, together with the significant changes in the DNA-binding motif of corresponding DnaAs, determines the unique molecular mechanism of DnaA-DNA interaction. Our results will facilitate identification of oriCs and subsequent identification of factors which regulate chromosome replication in other Epsilonproteobacteria. Since replication is controlled at the initiation step, it will help to better characterize life cycles of these species, many of which are considered as emerging pathogens.