Characterization of MroQ-Dependent Maturation and Export of the Staphylococcus aureus Accessory Gene Regulatory System Autoinducing Peptide.

Gram-positive bacteria produce small autoinducing peptides (AIPs), which act to regulate expression of genes that promote adaptive traits, including virulence. The Gram-positive pathogen Staphylococcus aureus generates a cyclic AIP that controls expression of virulence factors via the accessory gene regulatory (Agr) system. S. aureus strains belong to one of four Agr groups (Agr-I, -II, -III, and -IV); each group harbors allelic variants of AgrD, the precursor of AIP. In a prior screen for S. aureus virulence factors, we identified MroQ, a putative peptidase. A ΔmroQ mutant closely resembled a Δagr mutant and had significant defects in AIP production in an Agr-I strain. Here, we show that expression of AgrD-I in a ΔmroQ mutant leads to accumulation of an AIP processing intermediate at the membrane that coincides with a loss of secreted mature AIP, indicating that MroQ promotes maturation of AgrD-I. MroQ is conserved in all Agr sequence variants, suggesting either identical function among all Agr types or activity specific to Agr-I strains. Our data indicate that MroQ is required for AIP maturation and activity in Agr-I, -II, and -IV strains irrespective of background. However, MroQ is not required for Agr-III activity despite an identifiable role in peptide maturation. Isogenic Δagr and Δagr ΔmroQ strains complemented with Agr-I to -IV validated the critical role of MroQ in the generation of active AIP-I, -II, and -IV but not AIP-III. These findings were reinforced by skin infection studies with mice. Our data substantiate the prevailing model that MroQ is a mediator of cyclic peptide maturation.

Branched chain fatty acid synthesis drives tissue-specific innate immune response and infection dynamics of Staphylococcus aureus.

Fatty acid-derived acyl chains of phospholipids and lipoproteins are central to bacterial membrane fluidity and lipoprotein function. Though it can incorporate exogenous unsaturated fatty acids (UFA), Staphylococcus aureus synthesizes branched chain fatty acids (BCFA), not UFA, to modulate or increase membrane fluidity. However, both endogenous BCFA and exogenous UFA can be attached to bacterial lipoproteins. Furthermore, S. aureus membrane lipid content varies based upon the amount of exogenous lipid in the environment. Thus far, the relevance of acyl chain diversity within the S. aureus cell envelope is limited to the observation that attachment of UFA to lipoproteins enhances cytokine secretion by cell lines in a TLR2-dependent manner. Here, we leveraged a BCFA auxotroph of S. aureus and determined that driving UFA incorporation disrupted infection dynamics and increased cytokine production in the liver during systemic infection of mice. In contrast, infection of TLR2-deficient mice restored inflammatory cytokines and bacterial burden to wildtype levels, linking the shift in acyl chain composition toward UFA to detrimental immune activation in vivo. In in vitro studies, bacterial lipoproteins isolated from UFA-supplemented cultures were resistant to lipase-mediated ester hydrolysis and exhibited heightened TLR2-dependent innate cell activation, whereas lipoproteins with BCFA esters were completely inactivated after lipase treatment. These results suggest that de novo synthesis of BCFA reduces lipoprotein-mediated TLR2 activation and improves lipase-mediated hydrolysis making it an important determinant of innate immunity. Overall, this study highlights the potential relevance of cell envelope acyl chain repertoire in infection dynamics of bacterial pathogens.

Staphylococcus aureus adapts to the host nutritional landscape to overcome tissue-specific branched-chain fatty acid requirement.

During infection, pathogenic microbes adapt to the nutritional milieu of the host through metabolic reprogramming and nutrient scavenging. For the bacterial pathogen Staphylococcus aureus, virulence in diverse infection sites is driven by the ability to scavenge myriad host nutrients, including lipoic acid, a cofactor required for the function of several critical metabolic enzyme complexes. S. aureus shuttles lipoic acid between these enzyme complexes via the amidotransferase, LipL. Here, we find that acquisition of lipoic acid, or its attachment via LipL to enzyme complexes required for the generation of acetyl-CoA and branched-chain fatty acids, is essential for bacteremia, yet dispensable for skin infection in mice. A lipL mutant is auxotrophic for carboxylic acid precursors required for synthesis of branched-chain fatty acids, an essential component of staphylococcal membrane lipids and the agent of membrane fluidity. However, the skin is devoid of branched-chain fatty acids. We showed that S. aureus instead scavenges host-derived unsaturated fatty acids from the skin using the secreted lipase, Geh, and the unsaturated fatty acid-binding protein, FakB2. Moreover, murine infections demonstrated the relevance of host lipid assimilation to staphylococcal survival. Altogether, these studies provide insight into an adaptive trait that bypasses de novo lipid synthesis to facilitate S. aureus persistence during superficial infection. The findings also reinforce the inherent challenges associated with targeting bacterial lipogenesis as an antibacterial strategy and support simultaneous inhibition of host fatty acid salvage during treatment.

Dynamic Relay of Protein-Bound Lipoic Acid in Staphylococcus aureus.

Staphylococcus aureus competes for myriad essential nutrients during host infection. One of these nutrients is the organosulfur compound lipoic acid, a cofactor required for the activity of several metabolic enzyme complexes. In S. aureus, these include the E2 subunits of three α-ketoacid dehydrogenases and two H proteins, GcvH of the glycine cleavage system and its paralog, GcvH-L. We previously determined that the S. aureus amidotransferase LipL is required for lipoylation of the E2 subunits of pyruvate dehydrogenase (PDH) and branched-chain 2-oxoacid dehydrogenase (BCODH) complexes. The results from this study, coupled with those from Bacillus subtilis, suggested that LipL catalyzes lipoyl transfer from H proteins to E2 subunits. However, to date, the range of LipL targets, the extent of LipL-dependent lipoic acid shuttling between lipoyl domain-containing proteins, and the importance of lipoyl relay in pathogenesis remain unknown. Here, we demonstrate that LipL uses both lipoyl-H proteins as the substrates for lipoyl transfer to all E2 subunits. Moreover, LipL facilitates lipoyl relay between E2 subunits and between H proteins, a property that potentially constitutes an adaptive response to nutrient scarcity in the host, as LipL is required for virulence during infection. Together, these observations support a role for LipL in facilitating flexible lipoyl relay between proteins and highlight the complexity of protein lipoylation in S. aureusIMPORTANCE Protein lipoylation is a posttranslational modification that is evolutionarily conserved from bacteria to humans. Lipoic acid modifications are found on five proteins in S. aureus, four of which are components of major metabolic enzymes. In some bacteria, the amidotransferase LipL is critical for the attachment of lipoic acid to these proteins, and yet it is unclear to what extent LipL facilitates the transfer of this cofactor. We find that S. aureus LipL flexibly shuttles lipoic acid among metabolic enzyme subunits, alluding to a dynamic redistribution mechanism within the bacterial cell. This discovery exemplifies a potential means by which bacteria optimize the use of scarce nutrients when resources are limited.

Control of Staphylococcus aureus Quorum Sensing by a Membrane-Embedded Peptidase.

Gram-positive bacteria process and release small peptides, or pheromones, that act as signals for the induction of adaptive traits, including those involved in pathogenesis. One class of small signaling pheromones is the cyclic autoinducing peptides (AIPs), which regulate expression of genes that orchestrate virulence and persistence in a range of microbes, including staphylococci, listeriae, clostridia, and enterococci. In a genetic screen for Staphylococcus aureus secreted virulence factors, we identified an S. aureus mutant containing an insertion in the gene SAUSA300_1984 (mroQ), which encodes a putative membrane-embedded metalloprotease. A ΔmroQ mutant exhibited impaired induction of Toll-like receptor 2-dependent inflammatory responses from macrophages but elicited greater production of the inflammatory cytokine interleukin-1ß and was attenuated in a murine skin and soft tissue infection model. The ΔmroQ mutant phenocopies an S. aureus mutant containing a deletion of the accessory gene regulatory system (Agr), wherein both strains have significantly reduced production of secreted toxins and virulence factors but increased surface protein A abundance. The Agr system controls virulence factor gene expression in S. aureus by sensing the accumulation of AIP via the histidine kinase AgrC and the response regulator AgrA. We provide evidence to suggest that MroQ acts within the Agr pathway to facilitate the optimal processing or export of AIP for signal amplification through AgrC/A and induction of virulence factor gene expression. Mutation of MroQ active-site residues significantly reduces AIP signaling and attenuates virulence. Altogether, this work identifies a new component of the Agr quorum-sensing circuit that is critical for the production of S. aureus virulence factors.

Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus.

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH-L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α-ketoacid dehydrogenase E2 subunits, while the function of GcvH-L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain-containing proteins; LplA1 targets H proteins and LplA2 targets α-ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH-L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation.

Activation of Bacteroides fragilis toxin by a novel bacterial protease contributes to anaerobic sepsis in mice.

Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis. Enterotoxigenic strains that produce B. fragilis toxin (BFT, fragilysin) contribute to colitis and intestinal malignancy, yet are also isolated in bloodstream infection. It is not known whether these strains harbor unique genetic determinants that confer virulence in extra-intestinal disease. We demonstrate that BFT contributes to sepsis in mice, and we identify a B. fragilis protease called fragipain (Fpn) that is required for the endogenous activation of BFT through the removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad that is characteristic of C11-family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp. and Clostridium spp. Fpn-deficient, enterotoxigenic B. fragilis has an attenuated ability to induce sepsis in mice; however, Fpn is dispensable in B. fragilis colitis, wherein host proteases mediate BFT activation. Our findings define a role for B. fragilis enterotoxin and its activating protease in the pathogenesis of bloodstream infection, which indicates a greater complexity of cellular targeting and activity of BFT than previously recognized. The expression of fpn by both toxigenic and nontoxigenic strains suggests that this protease may contribute to anaerobic sepsis in ways that extend beyond its role in toxin activation. It could thus potentially serve as a target for disease modification.

Regulated intramembrane proteolysis of the virulence activator TcpP in Vibrio cholerae is initiated by the tail-specific protease (Tsp).

Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin-co-regulated pilus. Two proteins in this cascade are ToxR and TcpP - unusual membrane-localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane-localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two-step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V. cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C-terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full-length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP and requires residues TcpPA172 and TcpPI174 for wild-type activity.

Decreased transport restores growth of a Salmonella enterica apbC mutant on tricarballylate.

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in iron-sulfur ([Fe-S]) cluster metabolism. An apbC mutant is unable to grow on tricarballylate as a carbon source. Based on the ability of ApbC to transfer an [Fe-S] cluster to an apoprotein, this defect was attributed to poor loading of the [Fe-S] cluster-containing TcuB enzyme. Consistent with these observations, a previous study showed that overexpression of iscU, which encodes an [Fe-S] cluster molecular scaffold, suppressed the tricarballylate growth defect of an apbC mutant (J. M. Boyd, J. A. Lewis, J. C. Escalante-Semerena, and D. M. Downs, J. Bacteriol. 190:4596-4602, 2008). In this study, tcuC mutations that suppress the growth defect of an apbC mutant by decreasing the intracellular concentration of tricarballylate are described. Collectively, the suppressor analyses support a model in which reduced TcuB activity prevents growth on tricarballylate by (i) decreasing catabolism and (ii) allowing levels of tricarballylate that are toxic to the cell to accumulate. The apbC tcuC mutant strains described here reveal that the balance of the metabolic network can be altered by the accumulation of deleterious metabolites.