Lysine Trimethylation of EF-Tu Mimics Platelet-Activating Factor To Initiate Pseudomonas aeruginosa Pneumonia

Barbier, Mariette; Owings, Joshua P.; Martínez-Ramos, Inmaculada; Damron, F. Heath; Gomila, Rosa M.; Blázquez, Jesús; Goldberg, Joanna B.; Albertí, Sebastián

doi:10.1128/mbio.00207-13

Cited by 48 publications

(83 citation statements)

References 30 publications

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“…Rather, as discussed below, carnitine is typically generated by metabolizing appropriate trimethylated precursors. While evidence for true de novo synthesis is lacking, N 1 -trimethyllysine is the starting point for mammalian carnitine synthesis (Vaz & Wanders, 2002) and bacteria are known to synthesize N 1 -trimethyllysine (Barbier et al, 2013;Klagsbrun & Furano, 1975). Therefore, it remains a formal possibility that bacteria can use this N 1 -trimethyllysine to synthesize carnitine in a manner similar to mammals.…”

Section: Multiple Paths For Carnitine Acquisitionmentioning

confidence: 99%

Carnitine in bacterial physiology and metabolism

2015

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Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant, and can also enhance thermotolerance, cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field. Received 5 January 2015 Accepted 17 March 2015Introduction Carnitine (c-trimethylamino-b-hydroxybutyric acid) ( Fig. 1) is a quaternary amine compound that can be produced by all domains of life, and was discovered in muscle extract in 1905 by Gulewitsch & Krimberg (1905) and Kutscher (1905). It was shown to be essential for larval development of the mealworm Tenebrio molitor and was originally designated vitamin B T based on this requirement. Later, it was discovered that carnitine can be synthesized in mammals and is now considered to be a quasi-nutrient or conditionally essential nutrient (Flanagan et al., 2010), as neonates have reduced biosynthesis and rely on placental transfer of carnitine in utero and exogenous sources after birth (Combs, 2012). Fifty years after the discovery of carnitine, it was demonstrated that assorted Gram-positive and Gram-negative bacteria could use carnitine in either aerobic or anaerobic environments for a variety of cellular functions, including as an electron acceptor, as a compatible solute to survive environmental insults or as a sole carbon, nitrogen and energy source. Bacterial carnitine metabolism was most recently reviewed in 1998 (Bieber, 1988;Bremer, 1983;Kleber, 1997;Rebouche & Seim, 1998) and the field has seen important advances. This review summarizes what we knew at the time of the previous reviews and emphasizes what we have learned since, including: (i) how anaerobic bacteria synthesize and utilize crotonobetaine and carnitine as final electron acceptors, (ii) the impact of carnitine degradation by the intestinal microbiota and the genes responsible for this anaerobic conversion, (iii) the genes involved in aerobic degradation of carnitine, and (iv) how carnitine as a compatible solute impacts survival within and outside of the host. Carnitine in the environmentRecent work makes it clear that while animals represent the most readily accessible source of carnitine, carnitine is often present and sometimes abundant in soil and natural waters. Quaternary ammonium compounds are abundant in a number of soil ecosystems, including comprising a quarter of the most abundant organic nitrogen compounds in the soil water of a subalpine gras...

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Section: Multiple Paths For Carnitine Acquisitionmentioning

confidence: 99%

Carnitine in bacterial physiology and metabolism

2015

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“…We recently recognized that Pseudomonas aeruginosa EF-Tu is trimethylated at lysine 5 in a temperature-dependent manner (13). Through screening of a P. aeruginosa transposon mutant library at 22°C, a strain lacking methylated EF-Tu was identified, and subsequent analysis revealed a transposon insertion in eftM.…”

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Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Owings

Kuiper²,

Prezioso

et al. 2016

Journal of Biological Chemistry

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Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25°C) but not at higher temperatures (37°C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-L-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37°C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25°C. Irreversible protein unfolding at 37°C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.Protein post-translational modification adds an additional level of complexity that can influence protein function as well as change the protein charge and tertiary structure. The protein post-translational modification landscape is vast; more than half of the natural amino acids are substrates for chemical modification, and lysine, for example, can be modified with at least 10 different post-translational modifications, including methylation (1).Although first discovered on the bacterial flagellum (2), the study of lysine methylation in prokaryotes has lagged behind that of eukaryotes. In eukaryotes, the most well studied effect of lysine methylation is within the field of epigenetics, where patterns of methylation form the "histone code," and serve as another level of DNA transcriptional control (3). In bacteria, methylated lysines have been found on flagella, specific outer membrane proteins, and the ribosome translational machinery; however, for the most part, the functional consequences of these modifications are not known (2, 4 -8).Post-translational modification of proteins involved in protein synthesis has the potential to exert a significant effect on bacterial gene expression. Lysine methylation of components of the translational machinery, including essential translation factors such as elongation factor-thermo-unstable (EF-Tu), 6 which binds to and delivers aminoacyla...

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“…Along with the pneumococcus, H. influenzae, Neisseria meningitidis, and Neisseria gonorrheae carry ChoP as surface modifications, and most of these pathogens use ChoP to bind PAFR directly, reflecting a common strategy of passive, structural mimicry of PAF (73)(74)(75). Our work implies that active, enzymatic mechanisms exploiting ChoP mimicry may underlie the welldescribed resistance of these organisms to neutrophils and may expose a broadly applicable target for therapeutic intervention.…”

Section: Subversion Of Paf Signaling Is Conserved In Haemophilus Inflmentioning

confidence: 99%