Lipoyl-E2-PDH Gets a Second Job
Authors: Seth W. Dickey, Michael Otto
Journal: Cell Host & Microbe (2017)

Pyruvate dehydrogenase (PDH) plays a well-known metabolic role inside cells. In this issue of Cell Host & Microbe, Grayczyk et al., 2017Grayczyk J.P. Harvey C.J. Laczkovich I. Alonzo 3rd, F. Cell Host Microbe. 2017; 22 (this issue): 678-687Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar show that the bacterial pathogen Staphylococcus aureus unexpectedly secretes and repurposes the lipoylated E2 subunit of PDH to suppress TLR-mediated activation of host macrophages by bacterial lipoproteins. Pyruvate dehydrogenase (PDH) plays a well-known metabolic role inside cells. In this issue of Cell Host & Microbe, Grayczyk et al., 2017Grayczyk J.P. Harvey C.J. Laczkovich I. Alonzo 3rd, F. Cell Host Microbe. 2017; 22 (this issue): 678-687Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar show that the bacterial pathogen Staphylococcus aureus unexpectedly secretes and repurposes the lipoylated E2 subunit of PDH to suppress TLR-mediated activation of host macrophages by bacterial lipoproteins. Staphylococcus aureus and its methicillin-resistant strains (better known as MRSA) are the infectious jack-of-all trades in evading, subverting, and killing host immune cells, especially the innate immune arm comprised of the phagocytic macrophages and neutrophils. S. aureus achieves this by producing a diverse array of virulence factors that make it one of the deadliest pathogens in the developed world. Virulence factors that directly interact with host cell components typically have well-characterized secretion systems and are localized primarily outside of bacterial cells. Thus, we categorize these factors as "secreted," as opposed to the "cytoplasmic" proteins that are thought to reside normally only inside bacterial cells. However, a growing body of evidence is challenging the latter tenet. Several studies have found "cytoplasmic" proteins—especially those involved in metabolism—outside the cell, either bound to the bacterial surface or secreted into the extracellular space (Henderson and Martin, 2011Henderson B. Martin A. Infect. Immun. 2011; 79: 3476-3491Crossref PubMed Scopus (338) Google Scholar). Although these findings could be dismissed as an artifact of non-physiological cell lysis, one touted reason for their significance is that these cytoplasmic proteins moonlight outside: they serve additional and unrelated functions outside of cells than they do inside. Based on this "outside moonlighting" hypothesis, pathogenic bacteria would not only benefit from reducing their genome size and minimizing protein production, but would also greatly increase their capacity to promote virulence and evolve new virulence traits by deploying cytoplasmic proteins to interact directly with host cell factors. Several outside-moonlighting proteins show such virulence behaviors, such as adhesion to host tissue or antiphagocytic effects, in vitro. (Henderson and Martin, 2011Henderson B. Martin A. Infect. Immun. 2011; 79: 3476-3491Crossref PubMed Scopus (338) Google Scholar). Moreover, the outside moonlighting hypothesis is supported by the fact that secretion of bacterial cytoplasmic proteins may be regulated by either controlled cell lysis (Lewis, 2000Lewis K. Microbiol. Mol. Biol. Rev. 2000; 64: 503-514Crossref PubMed Scopus (485) Google Scholar) or some other as yet poorly defined mechanism (Ebner et al., 2016Ebner P. Rinker J. Götz F. Curr. Genet. 2016; 62: 19-23Crossref PubMed Scopus (44) Google Scholar). Indeed, cell-wall degrading enzymes (autolysins) (Pasztor et al., 2010Pasztor L. Ziebandt A.K. Nega M. Schlag M. Haase S. Franz-Wachtel M. Madlung J. Nordheim A. Heinrichs D.E. Götz F. J. Biol. Chem. 2010; 285: 36794-36803Crossref PubMed Scopus (92) Google Scholar) and surfactant-like amphipathic peptides (Ebner et al., 2017Ebner P. Luqman A. Reichert S. Hauf K. Popella P. Forchhammer K. Otto M. Götz F. Cell Rep. 2017; 20: 1278-1286Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) increase the abundance of cytoplasmic proteins outside of cells. In this issue, Grayczyk et al., 2017Grayczyk J.P. Harvey C.J. Laczkovich I. Alonzo 3rd, F. Cell Host Microbe. 2017; 22 (this issue): 678-687Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar report a case of an S. aureus cytoplasmic protein moonlighting outside, with evidence that connects a detailed mechanism to physiological relevance in animal models of infection. The authors began by screening a widely used transposon bank of S. aureus mutants (Fey et al., 2013Fey P.D. Endres J.L. Yajjala V.K. Widhelm T.J. Boissy R.J. Bose J.L. Bayles K.W. MBio. 2013; 4: e00537-12Crossref PubMed Scopus (503) Google Scholar) and discovered that culture supernatants of ΔlipA, a gene encoding a lipoic-acid biosynthetic enzyme, led to increased ex vivo activation of murine bone-marrow derived macrophages (BMMs). Notably, this effect depended on macrophages expressing TLR2, a classic pathogen-associated molecular pattern (PAMP) receptor that detects bacterial lipoproteins or lipopeptides. However, as there were no detected alterations, including lipoprotein shedding, in the ΔlipA secretome, the authors focused on known lipoylated proteins (note the distinction compared to lipoproteins) and discovered that the E2 subunit of pyruvate dehydrogenase (lipoyl-E2-PDH), which in the cytosol helps convert pyruvate to acetyl-CoA, is secreted (Figure 1). Intriguingly, its secretion was dependent on the S. aureus autolysin Atl, suggesting lipoyl-E2-PDH secretion is regulated. Using synthetic lipopeptides known to activate TLR2, the authors confirmed that purified lipoyl-E2-PDH mediated the BMM immune suppression. This line of investigation revealed additional biochemical insights. TLR2 can dimerize with TLR1 or TLR6 to detect tri- or di-acylated lipoproteins, respectively (Gay and Gangloff, 2007Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (508) Google Scholar). Therefore, because lipoyl-E2-PDH only suppressed activation by the tri-acylated, but not the di-acylated, lipopeptide, the authors concluded that lipoyl-E2-PDH specifically inhibits activation of the TLR1/2 heterodimer. Additionally, a lipoylated peptide was necessary because a lipoylated tripeptide DKLA—but neither the unmodified DKA nor free lipoic acid—replicated the TLR1/2 mediated BMM suppression at a similar concentration to that in which lipoyl-E2-PDH was required. Thus, the authors have proposed a model in which S. aureus secretes lipoylated E2-PDH to directly inhibit tri-acylated lipoprotein activation of TLR1/2, thereby achieving macrophage immunosuppression (Figure 1). The pièce de résistance of this report is the evidence that strongly supports the physiological relevance of the proposed model. First, the authors demonstrate that lipoyl-E2-PDH inhibits macrophage activation at a concentration of 10 nM. This is potent and within the range of the measured level of secreted lipoyl-E2-PDH that S. aureus produces throughout its growth cycle in vitro. Then, using a mouse intraperitoneal model of infection, the authors show that among macrophages that infiltrate the infection site, more were activated in the presence of the ΔlipA mutant than in the presence of WT S. aureus and that this increased activation leads to greater control of the bacterial infection. Finally, the authors excluded a metabolic deficiency in explaining the reduced bacterial load of the ΔlipA mutant by showing that, in a kidney abscess model in which macrophages cannot infiltrate the walled-off abscess to reach the site of infection, bacterial numbers were similar between ΔlipA and WT S. aureus. There are clear avenues for follow-up studies. From a biochemical viewpoint, the potent inhibition of TLR1/2 by DKLA opens new opportunities for structure and function analyses that may uncover key aspects of TLR signaling. The requirement for lipoic acid suggests that DKLA binds to a lipid-binding pocket on TLR1/2, which in turn would suggest a competitive mechanism that presumably prevents dimerization and subsequent downstream activation. In addition, this study could have far-reaching consequences because in an age of antibiotic resistance, alternative targets of bacterial pathogens are being sought to treat or prevent disease (Dickey et al., 2017Dickey S.W. Cheung G.Y.C. Otto M. Nat. Rev. Drug Discov. 2017; 16: 457-471Crossref PubMed Scopus (416) Google Scholar). Not only does this study uncover lipoyl-E2-PDH as a candidate factor to target, but by boosting support for the outside moonlighting hypothesis in general, Grayczyk et al., 2017Grayczyk J.P. Harvey C.J. Laczkovich I. Alonzo 3rd, F. Cell Host Microbe. 2017; 22 (this issue): 678-687Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar may refocus attention to target a cohort of "cytoplasmic" proteins that mischievously moonlight outside of cells. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (1ZIA-AI000904-17) and the Postdoctoral Research Associate Program of the National Institute of General Medical Sciences (1FI2GM11999101) in the U.S. National Institutes of Health. A Lipoylated Metabolic Protein Released by Staphylococcus aureus Suppresses Macrophage ActivationGrayczyk et al.Cell Host & MicrobeOctober 19, 2017In BriefMacrophages are crucial to the control of bacterial infection. Grayczyk et al. show that the lipoylated E2 subunit of pyruvate dehydrogenase from Staphylococcus aureus moonlights as an immunosuppressant and blocks macrophage activation by triacylated lipopeptides. The synthesis of lipoyl-proteins by S. aureus suppresses macrophage responses during infection and promotes virulence. Full-Text PDF Open Archive