IFI207, a young and fast-evolving protein, controls retroviral replication via the STING pathway.

Mammalian AIM-2-like receptor (ALR) proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. In mice, the Alr locus is highly polymorphic at the sequence and copy number level, and we show here that it is one of the most dynamic regions of the genome. One rapidly evolving gene within this region, Ifi207, was introduced to the Mus genome by gene conversion or an unequal recombination event a few million years ago. Ifi207 has a large, distinctive repeat region that differs in sequence and length among Mus species and even closely related inbred Mus musculus strains. We show that IFI207 controls murine leukemia virus (MLV) infection in vivo and that it plays a role in the STING-mediated response to cGAMP, dsDNA, DMXXA, and MLV. IFI207 binds to STING, and inclusion of its repeat region appears to stabilize STING protein. The Alr locus and Ifi207 provide a clear example of the evolutionary innovation of gene function, possibly as a result of host-pathogen co-evolution.IMPORTANCEThe Red Queen hypothesis predicts that the arms race between pathogens and the host may accelerate evolution of both sides, and therefore causes higher diversity in virulence factors and immune-related proteins, respectively . The Alr gene family in mice has undergone rapid evolution in the last few million years and includes the creation of two novel members, MndaL and Ifi207. Ifi207, in particular, became highly divergent, with significant genetic changes between highly related inbred mice. IFI207 protein acts in the STING pathway and contributes to anti-retroviral resistance via a novel mechanism. The data show that under the pressure of host-pathogen coevolution in a dynamic locus, gene conversion and recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.

The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during Listeria monocytogenes infection.

Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. IMPORTANCE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

IMPORTANCE: Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

The Intersection between Bacterial Metabolism and Innate Immunity.

BACKGROUND: The innate immune system is the first line of defense against microbial pathogens and is essential for maintaining good health. If pathogens breach innate barriers, the likelihood of infection is significantly increased. Many bacterial pathogens pose a threat to human health on account of their ability to evade innate immunity and survive in growth-restricted environments. These pathogens have evolved sophisticated strategies to obtain nutrients as well as manipulate innate immune responses, resulting in disease or chronic infection. SUMMARY: The relationship between bacterial metabolism and innate immunity is complex. Although aspects of bacterial metabolism can be beneficial to the host, particularly those related to the microbiota and barrier integrity, others can be harmful. Several bacterial pathogens harness metabolism to evade immune responses and persist during infection. The study of these adaptive traits provides insight into the roles of microbial metabolism in pathogenesis that extend beyond energy balance. This review considers recent studies on bacterial metabolic pathways that promote infection by circumventing several facets of the innate immune system. We also discuss relationships between innate immunity and antibiotics and highlight future directions for research in this field. KEY MESSAGES: Pathogenic bacteria have a remarkable capacity to harness metabolism to manipulate immune responses and promote pathogenesis. While we are beginning to understand the multifaceted and complex metabolic adaptations that occur during infection, there is still much to uncover with future research.

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus.

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials.

A Staphylococcal Glucosaminidase Drives Inflammatory Responses by Processing Peptidoglycan Chains to Physiological Lengths.

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1ß (IL-1ß). However, how bacterial processing of peptidoglycan drives IL-1ß production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1ß production. A ΔsagB mutant fails to promote IL-1ß production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1ß production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a ΔsagB mutant leads to reduced caspase-independent cleavage of pro-IL-1ß, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1ß production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1ß production by innate immune cells through a previously undescribed mechanism related to IL-1ß maturation.

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.

Listeria monocytogenes two component system PieRS regulates secretion chaperones PrsA1 and PrsA2 and enhances bacterial translocation across the intestine.

Listeria monocytogenes (Lm) is a widespread environmental Gram-positive bacterium that can transition into a pathogen following ingestion by a susceptible host. To cross host barriers and establish infection, Lm is dependent upon the regulated secretion and activity of many proteins including PrsA2, a peptidyl-prolyl cis-trans isomerase with foldase activity. PrsA2 contributes to the stability and activity of a number of secreted virulence factors that are required for Lm invasion, replication, and cell-to-cell spread within the infected host. In contrast, a second related secretion chaperone, PrsA1, has thus far no identified contributions to Lm pathogenesis. Here we describe the characterization of a two-component signal transduction system PieRS that regulates the expression of a regulon that includes the secretion chaperones PrsA1 and PrsA2. PieRS regulated gene products are required for bacterial resistance to ethanol exposure and are important for bacterial survival during transit through the gastrointestinal tract. PrsA1 was also found to make a unique contribution to Lm survival in the GI tract, revealing for the first time a non-overlapping requirement for both secretion chaperones PrsA1 and PrsA2 during the process of intra-gastric infection.

Toward Uncovering the Complexities of Bacterial Interspecies Communication and Competition on the Skin.

The skin is an inhospitable environment for microbial growth and survival. Hallmarks of the skin microenvironment include low moisture, high acidity, high lipid content, and paucity of essential nutrients, which together establish an antimicrobial barrier that defends against pathogens. Yet, commensal microbes and some opportunistic pathogens call this harsh environment home. The coagulase-negative staphylococci (CoNS) comprise a major constituent of the commensal skin microbiome. Of the CoNS, Staphylococcus epidermidis and Staphylococcus hominis are two common colonizers of human skin. Although comparatively less studied than S. epidermidis, there is a growing appreciation for S. hominis as a beneficial commensal, prompting interest in understanding the mechanisms by which S. hominis interacts with other skin microbes, including those with pathogenic potential. In their recent work, M. M. Severn, M. R. Williams, A. Shahbandi, Z. L. Bunch, et al. [mBio 13(3):e00930-22, 2022, https://doi.org/10.1128/mbio.00930-22] explore quorum sensing as a mediator of S. hominis interbacterial communication that can reduce the virulence of pathogens.

Antibiotic treatment ignites a fire that lasts.

Tissue damage and persistent inflammation are distinctive features of antibiotic-resistant chronic infections. In this issue of Cell Host & Microbe, Tang et al. demonstrate that anti-folate antibiotics trigger the synthesis of a bacterial second messenger, which induces an exuberant immune response and establishes a paradigm for chronic infection.

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.

Fecal transplantation and butyrate improve neuropathic pain, modify immune cell profile, and gene expression in the PNS of obese mice.

Obesity affects over 2 billion people worldwide and is accompanied by peripheral neuropathy (PN) and an associated poorer quality of life. Despite high prevalence, the molecular mechanisms underlying the painful manifestations of PN are poorly understood, and therapies are restricted to use of painkillers or other drugs that do not address the underlying disease. Studies have demonstrated that the gut microbiome is linked to metabolic health and its alteration is associated with many diseases, including obesity. Pathologic changes to the gut microbiome have recently been linked to somatosensory pain, but any relationships between gut microbiome and PN in obesity have yet to be explored. Our data show that mice fed a Western diet developed indices of PN that were attenuated by concurrent fecal microbiome transplantation (FMT). In addition, we observed changes in expression of genes involved in lipid metabolism and calcium handling in cells of the peripheral nerve system (PNS). FMT also induced changes in the immune cell populations of the PNS. There was a correlation between an increase in the circulating short-chain fatty acid butyrate and pain improvement following FMT. Additionally, butyrate modulated gene expression and immune cells in the PNS. Circulating butyrate was also negatively correlated with distal pain in 29 participants with varied body mass index. Our data suggest that the metabolite butyrate, secreted by the gut microbiome, underlies some of the effects of FMT. Targeting the gut microbiome, butyrate, and its consequences may represent novel viable approaches to prevent or relieve obesity-associated neuropathies.

Lipidomic and Ultrastructural Characterization of the Cell Envelope of Staphylococcus aureus Grown in the Presence of Human Serum.

Staphylococcus aureus can incorporate exogenous straight-chain unsaturated and saturated fatty acids (SCUFAs and SCFAs, respectively) to replace some of the normally biosynthesized branched-chain fatty acids and SCFAs. In this study, the impact of human serum on the S. aureus lipidome and cell envelope structure was comprehensively characterized. When S. aureus was grown in the presence of 20% human serum, typical human serum lipids, such as cholesterol, sphingomyelin, phosphatidylethanolamines, and phosphatidylcholines, were present in the total lipid extracts. Mass spectrometry showed that SCUFAs were incorporated into all major S. aureus lipid classes, i.e., phosphatidylglycerols, lysyl-phosphatidylglycerols, cardiolipins, and diglucosyldiacylglycerols. Heat-killed S. aureus retained fewer serum lipids and failed to incorporate SCUFAs, suggesting that association and incorporation of serum lipids with S. aureus require a living or nondenatured cell. Cytoplasmic membranes isolated from lysostaphin-produced protoplasts of serum-grown cells retained serum lipids, but washing cells with Triton X-100 removed most of them. Furthermore, electron microscopy studies showed that serum-grown cells had thicker cell envelopes and associated material on the surface, which was partially removed by Triton X-100 washing. To investigate which serum lipids were preferentially hydrolyzed by S. aureus lipases for incorporation, we incubated individual serum lipid classes with S. aureus and found that cholesteryl esters (CEs) and triglycerides (TGs) are the major donors of the incorporated fatty acids. Further experiments using purified Geh lipase confirmed that CEs and TGs were the substrates of this enzyme. Thus, growth in the presence of serum altered the nature of the cell surface with implications for interactions with the host.IMPORTANCE Comprehensive lipidomics of S. aureus grown in the presence of human serum suggests that human serum lipids can associate with the cell envelope without being truly integrated into the lipid membrane. However, fatty acids derived from human serum lipids, including unsaturated fatty acids, can be incorporated into lipid classes that can be biosynthesized by S. aureus itself. Cholesteryl esters and triglycerides are found to be the major source of incorporated fatty acids upon hydrolysis by lipases. These findings have significant implications for the nature of the S. aureus cell surface when grown in vivo Changes in phospholipid and glycolipid abundances and fatty acid composition could affect membrane biophysics and function and the activity of membrane-targeting antimicrobials. Finally, the association of serum lipids with the cell envelope has implications for the physicochemical nature of the cell surface and its interaction with host defense systems.

Group 1 CD1-restricted T cells contribute to control of systemic Staphylococcus aureus infection.

Staphylococcus aureus (SA) is the causative agent of both skin/soft tissue infections as well as invasive bloodstream infections. Though vaccines have been developed to target both humoral and T cell-mediated immune responses against SA, they have largely failed due to lack of protective efficacy. Group 1 CD1-restricted T cells recognize lipid rather than peptide antigens. Previously found to recognize lipids derived from cell wall of Mycobacterium tuberculosis (Mtb), these cells were associated with protection against Mtb infection in humans. Using a transgenic mouse model expressing human group 1 CD1 molecules (hCD1Tg), we demonstrate that group 1 CD1-restricted T cells can recognize SA-derived lipids in both immunization and infection settings. Systemic infection of hCD1Tg mice showed that SA-specific group 1 CD1-restricted T cell response peaked at 10 days post-infection, and hCD1Tg mice displayed significantly decreased kidney pathology at this time point compared with WT control mice. Immunodominant SA lipid antigens recognized by group 1 CD1-restricted T cells were comprised mainly of cardiolipin and phosphatidyl glycerol, with little contribution from lysyl-phosphatidyl glycerol which is a unique bacterial lipid not present in mammals. Group 1 CD1-restricted T cell lines specific for SA lipids also conferred protection against SA infection in the kidney after adoptive transfer. They were further able to effectively control SA replication in vitro through direct antigen presentation by group 1 CD1-expressing BMDCs. Together, our data demonstrate a previously unknown role for group 1 CD1-restricted SA lipid-specific T cells in the control of systemic MRSA infection.

Suppression of Staphylococcus aureus Superantigen-Independent Interferon Gamma Response by a Probiotic Polysaccharide.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes a variety of diseases. Bloodstream infection is the most severe, with mortality rates reaching 20 to 50%. Exopolysaccharide (EPS) from the probiotic Bacillus subtilis reduces bacterial burden and inflammation during S. aureus bloodstream infection in mice. Protection is due, in part, to hybrid macrophages that restrict S. aureus growth through reactive oxygen species and to limiting superantigen-induced T cell activation and interferon gamma (IFN-γ) production during infection. A decrease in IFN-γ production was observed within 24 h after infection, and here, we investigated how EPS abrogates its production. We discovered that S. aureus uses a rapid, superantigen-independent mechanism to induce host IFN-γ and that this is mediated by interleukin-12 (IL-12) activation of NK cells. Furthermore, we found that EPS limits IFN-γ production by modulating host immunity in a Toll-like receptor 4 (TLR4)-dependent manner, a signaling pathway that is required for EPS-mediated protection from S. aureus infection in vivo We conclude that EPS protects hosts from acute bloodstream S. aureus infection not only by inducing macrophages that restrict S. aureus growth and inhibit superantigen-activated T cells but also by limiting NK cell production of IFN-γ after S. aureus infection in a TLR4-dependent manner.

A Caspase-1 Biosensor to Monitor the Progression of Inflammation In Vivo.

Inflammasomes are multiprotein complexes that coordinate cellular inflammatory responses and mediate host defense. Following recognition of pathogens and danger signals, inflammasomes assemble and recruit and activate caspase-1, the cysteine protease that cleaves numerous downstream targets, including pro-IL-1ß and pro-IL-18 into their biologically active form. In this study, we sought to develop a biosensor that would allow us to monitor the initiation, progression, and resolution of inflammation in living animals. To this end, we inserted a known caspase-1 target sequence into a circularly permuted luciferase construct that becomes bioluminescent upon protease cleavage. This biosensor was activated in response to various inflammatory stimuli in human monocytic cell lines and murine bone marrow-derived macrophages. Next, we generated C57BL/6 transgenic mice constitutively expressing the caspase-1 biosensor. We were able to monitor the spatiotemporal dynamics of caspase-1 activation and onset of inflammation in individual animals in the context of a systemic bacterial infection, colitis, and acute graft-versus-host disease. These data established a model whereby the development and progression of inflammatory responses can be monitored in the context of these and other mouse models of disease.

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.

Staphylococcus aureus Lipoic Acid Synthesis Limits Macrophage Reactive Oxygen and Nitrogen Species Production To Promote Survival during Infection.

Macrophages are critical mediators of innate immunity and must be overcome for bacterial pathogens to cause disease. The Gram-positive bacterium Staphylococcus aureus produces virulence factors that impede macrophages and other immune cells. We previously determined that production of the metabolic cofactor lipoic acid by the lipoic acid synthetase, LipA, blunts macrophage activation. A ΔlipA mutant was attenuated during infection and was more readily cleared from the host. We hypothesized that bacterial lipoic acid synthesis perturbs macrophage antimicrobial functions and therefore hinders the clearance of S. aureus Here, we found that enhanced innate immune cell activation after infection with a ΔlipA mutant was central to attenuation in vivo, whereas a growth defect imparted by the lipA mutation made a negligible contribution to overall clearance. Macrophages recruited to the site of infection with the ΔlipA mutant produced larger amounts of bactericidal reactive oxygen species (ROS) and reactive nitrogen species (RNS) than those recruited to the site of infection with the wild-type strain or the mutant strain complemented with lipA ROS derived from the NADPH phagocyte oxidase complex and RNS derived from the inducible nitric oxide synthetase, but not mitochondrial ROS, were critical for the restriction of bacterial growth under these conditions. Despite enhanced antimicrobial immunity upon primary infection with the ΔlipA mutant, we found that the host failed to mount an improved recall response to secondary infection. Our data suggest that lipoic acid synthesis in S. aureus promotes bacterial persistence during infection through limitation of ROS and RNS generation by macrophages. Broadly, this work furthers our understanding of the intersections between bacterial metabolism and immune responses to infection.

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.