Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus.

BACKGROUND: Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation. RESULTS: In order to elucidate how global metabolic processes are affected by the absence of formylated proteins the exometabolome of an S. aureus fmt mutant was compared with that of the parental strain and the transcription of corresponding enzymes was analyzed to identify possible regulatory changes. The mutant consumed glucose and other carbon sources slower than the wild type. While the turnover of several metabolites remained unaltered fmt inactivation led to increases pyruvate release and, concomitantly, reduced pyruvate dehydrogenase activity. In parallel, the release of the pyruvate-derived metabolites lactate, acetoin, and alanine was reduced. The anaerobic degradation of arginine was also reduced in the fmt mutant compared to the wild-type strain. Moreover, the lack of formylated proteins caused increased susceptibility to the antibiotics trimethoprim and sulamethoxazole suggesting that folic acid-dependant pathways were perturbed in the mutant. CONCLUSIONS: These data indicate that formylated proteins are crucial for specific bacterial metabolic processes and they may help to understand why it has remained important during bacterial evolution to initiate protein biosynthesis with a formylated tRNAMet.

Pyruvate formate lyase acts as a formate supplier for metabolic processes during anaerobiosis in Staphylococcus aureus.

Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (Δfdh and Δpfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δpfl mutant. In compensation reactions, serine and threonine were consumed better by the Δpfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δpfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the Δpfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H(+), whose regeneration requires respiration.

Formyl peptide receptor-mediated proinflammatory consequences of peptide deformylase inhibition in Staphylococcus aureus.

The biosynthesis of proteins with N-terminal formylated methionine residues and subsequent protein deformylation are unique and invariant bacterial processes. They are exploited by the capacity of the human innate immune system to sense formylated peptides (FPs) and targeted by the deformylation-blocking antibiotic actinonin. We show that human polymorphonuclear leukocytes respond via the formyl peptide receptor (FPR) with increased calcium ion fluxes, chemotactic migration, IL-8 release, and CD11b upregulation to the human pathogen Staphylococcus aureus upon actinonin treatment. These data underscore the crucial role of bacterial FPs in innate immunity and indicate that deformylase inhibition may have considerable proinflammatory consequences.

A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.