Bacteroides fragilis is the leading cause of anaerobic bacteremia and sepsis 1. Enterotoxigenic strains producing B. fragilis toxin (BFT, fragilysin) contribute to colitis 2 and intestinal malignancy 3, yet are also isolated in bloodstream infection 4,5. 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 and identify a B. fragilis protease, fragipain (Fpn), which is required for endogenous activation of BFT through removal of its auto-inhibitory prodomain. Structural analysis of Fpn reveals a His-Cys catalytic dyad characteristic of C11 family cysteine proteases that are conserved in multiple pathogenic Bacteroides spp and Clostridium spp. Fpn-deficient enterotoxigenic B. fragilis is attenuated in its ability to induce sepsis, 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, indicating a greater complexity of cellular targeting and action of BFT than previously appreciated. The expression of fpn by both toxigenic and non-toxigenic strains suggests this protease may contribute to anaerobic sepsis beyond its role in toxin activation, potentially serving as a target for disease modification.

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-1␤ 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.

SummaryVibrio 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 TcpPunusual 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 wildtype activity.

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.

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.

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.

26Gram-positive bacteria process and release small peptides or "pheromones" that act as signals 27 for the induction of adaptive traits including those involved in pathogenesis. One class of small 28 signaling pheromones is the cyclic auto-inducing peptides (AIPs), which regulate expression of 29 genes that orchestrate virulence and persistence in a range of microbes including 30Staphylococci, Listeria, Clostridia, and Enterococci. In a genetic screen for Staphylococcus 31 aureus secreted virulence factors, we identified a S. aureus mutant containing an insertion in 32 gene SAUSA300_1984 (mroQ), which encodes a putative membrane-embedded 33 metalloprotease. A ΔmroQ mutant exhibits impaired induction of TLR2-dependent 34 inflammatory responses from macrophages, but elicits greater production of the inflammatory 35 cytokine IL-1β and is attenuated in a murine skin and soft tissue infection model. The ΔmroQ 36 mutant phenocopies a S. aureus mutant containing a deletion of the accessory gene regulatory 37 system (Agr), wherein both strains have significantly reduced production of secreted toxins and 38 virulence factors, but increased surface Protein A abundance. The Agr system controls 39 virulence factor gene expression in S. aureus through sensing accumulation of AIP via the 40 histidine kinase AgrC and response regulator AgrA. We provide evidence to suggest that MroQ 41 acts within the Agr pathway to facilitate optimal processing or export of AIP for signal 42 amplification through AgrC/A and induction of virulence factor gene expression. Mutation of 43MroQ active site residues significantly reduces AIP signaling and attenuates virulence. 44Altogether, this work identifies a new component of the Agr quorum sensing circuit that is 45 critical for the production of S. aureus virulence factors. 46 47 48 from the bacterial cell. While many signaling molecules of Gram-negative bacteria are freely 56 diffusible, the peptides of Gram-positives generally must transit the membrane via a dedicated 57 transporter (4, 5). After processing and transport, the peptide is either imported back into the 58 bacterial cell or transmits signal from the extracellular environment by binding to membrane-59 embedded sensor histidine kinases (6). Peptide signaling culminates in a change in gene 60 expression mediated by transcription factors that respond to the peptide. Many Gram-positive 61 bacterial pathogens use these "quorum-sensing" peptides to induce gene expression programs 62 that promote virulence adaptations such as competence, toxin production, biofilm formation, 63 and establishment of persistence traits. Because of its importance in activating virulence 64 programs in S. aureus, quorum-sensing inhibition has been the focus of many therapeutic 65 initiatives (7)(8)(9)(10)(11)(12)(13)(14). 66The ability of S. aureus to infect host tissues and cause acute and chronic disease is 67 partially due to its use of complex gene regulatory systems that control virulence factor gene 68 expression (15-17). S. aureus employs 16 two-compone...

Mutants of Iron and sulfur are essential to almost all bacteria, and cofactors containing these elements ([Fe-S] clusters) are nearly ubiquitous. Evolution has harnessed the flexible biochemical and biophysical properties of these cofactors for a variety of cellular functions, including enzymatic reactions, electron transfer, DNA metabolism, and environmental sensing (reviewed in references 8 and 27).Three systems for [Fe-S] cluster biosynthesis have been described in bacteria. Work with Azotobacter vinelandii led to the discovery of the nif (nitrogen fixation) (55) and isc (iron-sulfur cluster) (54) [Fe-S] cluster biosynthetic operons. A third system, encoded by the suf (sulfur utilization factor) operon, was discovered in Escherichia coli (47). The genome of Salmonella enterica serovar Typhimurium encodes both the iscSUA-hscAB-fdx-orf3 and sufABCDSE operons (36,47).Both bioinformatics and in vivo genetic approaches have identified several loci outside the isc, suf, and nif operons that impact [Fe-S] cluster metabolism. The apbC, rseC, and apbE loci were discovered in Salmonella enterica by using sensitive genetic screens that exploited phenotypes described for strains lacking isc operon functions (6,7,38,(44)(45)(46). ApbC is a 40-kDa cytoplasmic protein that can bind and rapidly transfer [Fe-S] clusters to a target protein (15) and hydrolyze ATP (16,44). Both of these activities are necessary for in vivo function (16). ApbC is a representative member of an ancient family of [Fe-S] cluster biosynthetic proteins that evolved before the divergence of the Archaea and Eucarya (13). To our knowledge, all organisms that have sequenced genomes and are known to metabolize iron encode a copy of an apbC homologue. In contrast, the functions of the rseC and apbE gene products remain unknown.The erpA, nfuA, cyaY, and ygfZ genes were identified in bacteria by using bioinformatic approaches, and subsequent physiological studies found that these gene products function in 4,35,37,49). The NfuA and ErpA proteins show sequence similarity to the proposed [Fe-S] cluster-trafficking pro-