l-Serine Catabolism via an Oxygen-Labile l-Serine Dehydratase Is Essential for Colonization of the Avian Gut by Campylobacter jejuni

Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of l-serine from a defined medium containing a mixture of amino acids led to a dramatic decrease in cell yields. As C. jejuni does not have a biosynthetic serine requirement, this supports earlier suggestions that l-serine is a preferentially catabolized amino acid. Serine transport was found to be mediated by at least two systems in strain 11168; a high-capacity, low-affinity l-serine-specific system encoded by Cj1625c (sdaC) and a higher-affinity l-serine/l-threonine system responsible for residual l-serine transport in an sdaC mutant. Catabolism of l-serine to pyruvate and ammonia is carried out by SdaA (encoded by Cj1624c), which was overexpressed, purified, and shown to be an oxygen-labile iron-sulfur enzyme. l-Serine dehydratase activity in an sdaA mutant was reduced 10-fold compared to that in the wild type, but the residual activity (due to the anabolic l-threonine dehydratase) could not support either growth on or utilization of l-serine in defined media. However, although sdaA mutants showed no obvious growth defect in complex media, they completely failed to colonize 3-week-old chickens as assayed both by cloacal swabs taken over a 6-week period and by cecal colony counts postmortem. In contrast, the isogenic parent strain colonized chickens to high levels within 1 week of inoculation. The results show that an active SdaA is essential for colonization of the avian gut by C. jejuni and imply that catabolism of l-serine is crucially important for the growth of this bacterium in vivo

Fosfomycin Enhances the Active Transport of Tobramycin in Pseudomonas aeruginosa

Elevated levels of mucins present in bronchiectatic airways predispose patients to bacterial infections and reduce the effectiveness of antibiotic therapies by directly inactivating antibiotics. Consequently, new antibiotics that are not inhibited by mucins are needed to treat chronic respiratory infections caused by Pseudomonas aeruginosa and Staphylococcus aureus. In these studies, we demonstrate that fosfomycin synergistically enhances the activity of tobramycin in the presence of mucin. The bactericidal killing of a novel 4:1 (wt/wt) combination of fosfomycin-tobramycin (FTI) is superior (>9 log10 CFU/ml) relative to its individual components fosfomycin and tobramycin. Additionally, FTI has a mutation frequency resulting in an antibiotic resistance >3 log10 lower than for fosfomycin and 4 log10 lower than for tobramycin for P. aeruginosa. Mechanistic studies revealed that chemical adducts are not formed, suggesting that the beneficial effects of the combination are not due to molecular modification of the components. FTI displayed time-kill kinetics similar to tobramycin and killed in a concentration-dependent fashion. The bactericidal effect resulted from inhibition of protein biosynthesis rather than cell wall biosynthesis. Studies using radiolabeled antibiotics demonstrated that tobramycin uptake was energy dependent and that fosfomycin enhanced the uptake of tobramycin in P. aeruginosa in a dose-dependent manner. Lastly, mutants resistant to fosfomycin and tobramycin were auxotrophic for specific carbohydrates and amino acids, suggesting that the resistance arises from mutations in specific active transport mechanisms. Overall, these data demonstrate that fosfomycin enhances the uptake of tobramycin, resulting in increased inhibition of protein synthesis and ultimately bacterial killing

Essential Role of Ferritin Pfr in Helicobacter pylori Iron Metabolism and Gastric Colonization

The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the Helicobacter pylori ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled H. pylori to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the pfr gene restricted growth of H. pylori under these conditions. The lowered total iron content in the pfr mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an H. pylori feoB mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of pfr transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant H. pylori Fur to the pfr promoter region. The functions of H. pylori Pfr in iron metabolism are essential for survival in the gastric mucosa, as the pfr mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the pfr phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for H. pylori to survive in its hostile natural environment

Proteomic analysis of purified protein derivative of mycobacterium tuberculosis

BACKGROUND: Purified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as “purified,” in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterization is likely to help in short-listing the relevant antigens required to prepare a less complex and more potent reagent for diagnostic purposes. RESULTS: Proteomic analysis of Connaught Tuberculin 68 (PPD-CT68), a tuberculin preparation generated from M. tuberculosis, was carried out in this study. PPD-CT68 is the protein component of a commercially available tuberculin preparation, Tubersol, which is used for tuberculin skin testing. Using a high resolution LTQ-Orbitrap Velos mass spectrometer, we identified 265 different proteins. The identified proteins were compared with those identified from PPD M. bovis, PPD M. avium and PPD-S2 from previous mass spectrometry-based studies. In all, 142 proteins were found to be shared between PPD-CT68 and PPD-S2 preparations. Out of the 354 proteins from M. tuberculosis–derived PPDs (i.e. proteins in either PPD-CT68 or PPD-S2), 37 proteins were found to be shared with M. avium PPD and 80 were shared with M. bovis PPD. Alignment of PPD-CT68 proteins with proteins encoded by 24 lung infecting bacteria revealed a number of similar proteins (206 bacterial proteins shared epitopes with 47 PPD-CT68 proteins), which could potentially be involved in causing cross-reactivity. The data have been deposited to the ProteomeXchange with identifier PXD000377. CONCLUSIONS: Proteomic and bioinformatics analysis of different PPD preparations revealed commonly and differentially represented proteins. This information could help in delineating the relevant antigens represented in various PPDs, which could further lead to development of a lesser complex and better defined skin test antigen with a higher specificity and sensitivity