Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus.IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.KEYWORDS cyclophilin, Nuc, PI-PLC, PPIase, parvulin, PpiB, PrsA, Staphylococcus aureus, nuclease, protease T he proline peptide bond is unique in nature in that both the cis and trans forms can occur in vivo, with the cis conformation existing approximately 6.5% of the time (1). In contrast, for all other naturally occurring amino acids, steric hindrance between side chains precludes the cis form and overwhelmingly favors the trans form (2). The presence of both the cis and trans forms of proline peptide bonds has important consequences for protein tertiary structure. In certain cases, the isomerization state of
Bacterial toxin-antitoxin systems trigger the onset of a persister state by inhibiting essential cellular processes. The TacT toxin of Salmonella enterica is known to induce a persister state in macrophages through the acetylation of aminoacyl-tRNAs. Here, we show that the TacT toxin and the TacA antitoxin work as a complex that modulates TacT activity via the acetylation state of TacA. TacT acetylates TacA at residue K44, a modification that is removed by the NAD+-dependent CobB sirtuin deacetylase. TacA acetylation increases the activity of TacT, downregulating protein synthesis. TacA acetylation altered binding to its own promoter, although this did not change tacAT expression levels. These claims are supported by results from in vitro protein synthesis experiments used to monitor TacT activity, in vivo growth analyses, electrophoretic mobility shift assays, and quantitative reverse transcription-PCR (RT-qPCR) analysis. TacT is the first example of a Gcn5-related N-acetyltransferase that modifies nonprotein and protein substrates.
N-terminal (Nt)-acylation is the irreversible addition of an acyl moiety to the terminal alpha amino group of a peptide chain. This type of modification alters the nature of the N terminus, which can interfere with the function of the modified protein by disrupting protein interactions, function, localization, degradation, hydrophobicity, or charge. Nt acylation is found in all domains of life and is a highly common occurrence in eukaryotic cells. However, in prokaryotes very few cases of Nt acylation have been reported. It was once thought that Nt acylation of proteins, other than ribosomal proteins, was uncommon in prokaryotes, but recent evidence suggests that this modification may be more common than once realized. In this review, we discuss what is known about prokaryotic Nt acetylation and the acetyltransferases that are responsible, as well as recent advancements in this field and currently used methods to study Nt acetylation.
The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low phosphate low magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, a known activator of SPI-2. Here we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC ( i.e., tktD, tktE) of this bacterium. Transketolases contribute to the non-redox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR binding sites between stm2344 and pagR.
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