Virulence Induction in Pseudomonas aeruginosa under Inorganic Phosphate Limitation: a Proteomics Perspective.

Inorganic phosphate (Pi) is a central nutrient and signal molecule for bacteria. Pi limitation was shown to increase the virulence of several phylogenetically diverse pathogenic bacteria with different lifestyles. Hypophosphatemia enhances the risk of death in patients due to general bacteremia and was observed after surgical injury in humans. Phosphate therapy, or the reduction of bacterial virulence by the administration of Pi or phosphate-containing compounds, is a promising anti-infective therapy approach that will not cause cytotoxicity or the emergence of antibiotic-resistant strains. The proof of concept of phosphate therapy has been obtained using primarily Pseudomonas aeruginosa (PA). However, a detailed understanding of Pi-induced changes at protein levels is missing. Using pyocyanin production as proxy, we show that the Pi-mediated induction of virulence is a highly cooperative process that occurs between 0.2 to 0.6 mM Pi. We present a proteomics study of PA grown in minimal medium supplemented with either 0.2 mM or 1 mM Pi and rich medium. About half of the predicted PA proteins could be quantified. Among the 1,471 dysregulated proteins comparing growth in 0.2 mM to 1 mM Pi, 1,100 were depleted under Pi-deficient conditions. Most of these proteins are involved in general and energy metabolism, different biosynthetic and catabolic routes, or transport. Pi depletion caused accumulation of proteins that belong to all major families of virulence factors, including pyocyanin synthesis, secretion systems, quorum sensing, chemosensory signaling, and the secretion of proteases, phospholipases, and phosphatases, which correlated with an increase in exoenzyme production and antibacterial activity. IMPORTANCE Antibiotics are our main weapons to fight pathogenic bacteria, but the increase in antibiotic-resistant strains and their consequences represents a major global health challenge, revealing the necessity to develop alternative antimicrobial strategies that do not involve the bacterial killing or growth inhibition. P. aeruginosa has been placed second on the global priority list to guide research on the development of new antibiotics. One of the most promising alternative strategies is the phosphate therapy for which the proof of concept has been obtained for P. aeruginosa. This article reports the detailed changes at the protein levels comparing P. aeruginosa grown under Pi-abundant and Pi-depleted conditions. These data describe in detail the molecular mechanisms underlying phosphate therapy. Apart from Pi, several other phosphate-containing compounds have been used for phosphate therapy and this study will serve as a reference for comparative studies aimed at evaluating the effect of alternative compounds.

Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria.

Mobile genomic islands distribute functional traits between microbes and habitats, yet it remains unclear how their proteins adapt to new environments. Here we used a comparative phylogenomic and proteomic approach to show that the marine bacterium Pseudoalteromonas haloplanktis ANT/505 acquired a genomic island with a functional pathway for pectin catabolism. Bioinformatics and biochemical experiments revealed that this pathway encodes a series of carbohydrate-active enzymes including two multi-modular pectate lyases, PelA and PelB. PelA is a large enzyme with a polysaccharide lyase family 1 (PL1) domain and a carbohydrate esterase family 8 domain, and PelB contains a PL1 domain and two carbohydrate-binding domains of family 13. Comparative phylogenomic analyses indicate that the pathway was most likely acquired from terrestrial microbes, yet we observed multi-modular orthologues only in marine bacteria. Proteomic experiments showed that P. haloplanktis ANT/505 secretes both pectate lyases into the environment in the presence of pectin. These multi-modular enzymes may therefore represent a marine innovation that enhances physical interaction with pectins to reduce loss of substrate and enzymes by diffusion. Our results revealed that marine bacteria can catabolize pectin, and highlight enzyme fusion as a potential adaptation that may facilitate microbial consumption of polymeric substrates in aquatic environments.

Global relative quantification with liquid chromatography-matrix-assisted laser desorption ionization time-of-flight (LC-MALDI-TOF)--cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences.

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Oxidative stress triggers thiol oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus aureus.

The high-resolution two-dimensional protein gel electrophoresis technique combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to analyse the oxidative stress response in Staphylococcus aureus COL. Exponentially growing cells were supplemented with 100 mM H2O2 leading to a growth arrest lasting 30 min. The comparison of the two-dimensional pattern of cytoplasmic protein extracts of stressed and unstressed cells revealed only a few changes in the protein synthesis profile. However, the isoelectric points of Gap (glyceraldehyde-3-phosphate dehydrogenase), AhpC (alkylhydroperoxide reductase) and MvaS (HMG-CoA-synthase) changed strikingly. For analysis of the modification of Gap, tandem hybrid mass spectrometry (Q-Star) was used. The observed pI shift resulted from the oxidation to sulphonic acid of cysteine 151, which is crucial for catalytic activity. A drop in ATP and a complete inactivation of Gap was accompanied by the growth arrest. About 30 min after the addition of H2O2, the damaged Gap was still present, but a new protein spot at the original location became visible, representing the newly synthesized enzyme that is active again. This is accompanied by the restoration of Gap enzyme activity, ATP levels and recovery of growth. There is a strong correlation between growth, ATP level and Gap activity under oxidative stress conditions, indicating that the H2O2-triggered Gap inactivation might be one reason for growth arrest under these conditions. Our data indicate that the damaged Gap protein was not repaired.