Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

The Orphan Nuclear Receptors Steroidogenic Factor-1 and Liver Receptor Homolog-1: Structure, Regulation, and Essential Roles in Mammalian Reproduction.

Nuclear receptors are intracellular proteins that act as transcription factors. Proteins with classic nuclear receptor domain structure lacking identified signaling ligands are designated orphan nuclear receptors. Two of these, steroidogenic factor-1 (NR5A1, also known as SF-1) and liver receptor homolog-1 (NR5A2, also known as LRH-1), bind to the same DNA sequences, with different and nonoverlapping effects on targets. Endogenous regulation of both is achieved predominantly by cofactor interactions. SF-1 is expressed primarily in steroidogenic tissues, LRH-1 in tissues of endodermal origin and the gonads. Both receptors modulate cholesterol homeostasis, steroidogenesis, tissue-specific cell proliferation, and stem cell pluripotency. LRH-1 is essential for development beyond gastrulation and SF-1 for genesis of the adrenal, sexual differentiation, and Leydig cell function. Ovary-specific depletion of SF-1 disrupts follicle development, while LRH-1 depletion prevents ovulation, cumulus expansion, and luteinization. Uterine depletion of LRH-1 compromises decidualization and pregnancy. In humans, SF-1 is present in endometriotic tissue, where it regulates estrogen synthesis. SF-1 is underexpressed in ovarian cancer cells and overexpressed in Leydig cell tumors. In breast cancer cells, proliferation, migration and invasion, and chemotherapy resistance are regulated by LRH-1. In conclusion, the NR5A orphan nuclear receptors are nonredundant factors that are crucial regulators of a panoply of biological processes, across multiple reproductive tissues.

Steroidogenic factor 1 (SF-1; Nr5a1) regulates the formation of the ovarian reserve.

The ovarian follicle reserve, formed pre- or perinatally, comprises all oocytes for lifetime reproduction. Depletion of this reserve results in infertility. Steroidogenic factor 1 (SF-1; Nr5a1) and liver receptor homolog 1 (LRH-1; Nr5a2) are two orphan nuclear receptors that regulate adult endocrine function, but their role in follicle formation is unknown. We developed models of conditional depletion of SF-1 or LRH-1 from prenatal ovaries. Depletion of SF-1, but not LRH-1, resulted in dramatically smaller ovaries and fewer primordial follicles. This was mediated by increased oocyte death, resulting from increased ovarian inflammation and increased Notch signaling. Major dysregulated genes were Iroquois homeobox 3 and 5 and their downstream targets involved in the establishment of the ovarian laminin matrix and oocyte-granulosa cell gap junctions. Disruptions of these pathways resulted in follicles with impaired basement membrane formation and compromised oocyte-granulosa communication networks, believed to render them more prone to atresia. This study identifies SF-1 as a key regulator of the formation of the ovarian reserve.

Skin-resident dendritic cells mediate postoperative pain via CCR4 on sensory neurons.

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.

The role of steroidogenic factor 1 (SF-1) in steroidogenic cell function of the testes and ovaries of mature mice.

In brief: The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility. Abstract: Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Landscape context affects the sustainability of organic farming systems.

Organic agriculture promotes sustainability compared to conventional agriculture. However, the multifunctional sustainability benefits of organic farms might be mediated by landscape context. Assessing how landscape context affects sustainability may aid in targeting organic production to landscapes that promote high biodiversity, crop yields, and profitability. We addressed this using a meta-analysis spanning 60 crop types on six continents that assessed whether landscape context affected biodiversity, yield, and profitability of organic vs. conventional agroecosystems. We considered landscape metrics reflecting landscape composition (percent cropland), compositional heterogeneity (number and diversity of cover types), and configurational heterogeneity (spatial arrangement of cover types) across our study systems. Organic sites had greater biodiversity (34%) and profits (50%) than conventional sites, despite lower yields (18%). Biodiversity gains increased as average crop field size in the landscape increased, suggesting organic farms provide a "refuge" in intensive landscapes. In contrast, as crop field size increased, yield gaps between organic and conventional farms increased and profitability benefits of organic farming decreased. Profitability of organic systems, which we were only able to measure for studies conducted in the United States, varied across landscapes in conjunction with production costs and price premiums, suggesting socioeconomic factors mediated profitability. Our results show biodiversity benefits of organic farming respond differently to landscape context compared to yield and profitability benefits, suggesting these sustainability metrics are decoupled. More broadly, our results show that the ecological, but not the economic, sustainability benefits of organic agriculture are most pronounced in more intensive agricultural landscapes.

Dominant-negative pathogenic variant BRIP1 c.1045G>C is a high-risk allele for non-mucinous epithelial ovarian cancer: A case-control study.

BRIP1 is a moderate susceptibility epithelial ovarian cancer (EOC) gene. Having identified the BRIP1 c.1045G>C missense variant in a number of families with EOC, we aimed to investigate the frequency of this and BRIP1.2392C>T pathogenic variant in patients with breast cancer (BC) and/or EOC. A case-control study of 3767 cases and 2043 controls was undertaken investigating the presence of these variants using Sanger sequencing and gene panel data. Individuals with BC and/or EOC were grouped by family history. BRIP1 c.1045G>C was associated with increased risk of BC/EOC (OR = 37.7; 95% CI 5.3-444.2; P = 0.0001). The risk was highest for women with EOC (OR = 140.8; 95% CI 23.5-1723.0; P < 0.0001) and lower for BC (OR = 11.1; 95% CI 1.2-106.5; P = 0.1588). BRIP1 c.2392C>T was associated with smaller risks for BC/EOC (OR = 5.4; 95%CI 2.4-12.7; P = 0.0003), EOC (OR = 5.9; 95% CI 1.3-23.0; p = 0.0550) and BC (OR = 5.3; 95%CI 2.3-12.9; P = 0.0009). Our study highlights the importance of BRIP1 as an EOC susceptibility gene, especially in familial EOC. The variant BRIP1 c.1045G>C, rs149364097, is of particular interest as its dominant-negative effect may confer a higher risk of EOC than that of the previously reported BRIP1 c.2392C>T nonsense variant. Dominant-negative missense variants may confer higher risks than their loss-of-function counterparts.

A trait-based framework for predicting foodborne pathogen risk from wild birds.

Recent foodborne illness outbreaks have heightened pressures on growers to deter wildlife from farms, jeopardizing conservation efforts. However, it remains unclear which species, particularly birds, pose the greatest risk to food safety. Using >11,000 pathogen tests and 1565 bird surveys covering 139 bird species from across the western United States, we examined the importance of 11 traits in mediating wild bird risk to food safety. We tested whether traits associated with pathogen exposure (e.g., habitat associations, movement, and foraging strategy) and pace-of-life (clutch size and generation length) mediated foodborne pathogen prevalence and proclivities to enter farm fields and defecate on crops. Campylobacter spp. were the most prevalent enteric pathogen (8.0%), while Salmonella and Shiga-toxin producing Escherichia coli (STEC) were rare (0.46% and 0.22% prevalence, respectively). We found that several traits related to pathogen exposure predicted pathogen prevalence. Specifically, Campylobacter and STEC-associated virulence genes were more often detected in species associated with cattle feedlots and bird feeders, respectively. Campylobacter was also more prevalent in species that consumed plants and had longer generation lengths. We found that species associated with feedlots were more likely to enter fields and defecate on crops. Our results indicated that canopy-foraging insectivores were less likely to deposit foodborne pathogens on crops, suggesting growers may be able to promote pest-eating birds and birds of conservation concern (e.g., via nest boxes) without necessarily compromising food safety. As such, promoting insectivorous birds may represent a win-win-win for bird conservation, crop production, and food safety. Collectively, our results suggest that separating crop production from livestock farming may be the best way to lower food safety risks from birds. More broadly, our trait-based framework suggests a path forward for co-managing wildlife conservation and food safety risks in farmlands by providing a strategy for holistically evaluating the food safety risks of wild animals, including under-studied species.

Hepatic proteomic assessment of oral ingestion of titanium dioxide nano fiber (TDNF) in Sprague Dawley rats.

Titanium dioxide nanofibers (TDNF) have been widely employed in pigments, sunscreens, paints, ointments, toothpaste and photocatalytic splitting of water. However, their potential toxicity has not been thoroughly examined. The goal of the present study is to examine hepatic effects associated with the ingestion of TDNF. TDNF was fabricated via electrospinning method and characterized. Six to seven weeks old male Sprague Dawley rats ingested (oral gavage) a total of 0 ppm, 40, 60 ppm TDNF for two weeks. After sacrifice, the liver was assessed for cellular effects using proteomic approach. The fibers diameter ranged from 0.18 - 0.29 µm, forming clusters and majority of the fibers were in the rutile phase. Proteomics assessment revealed more that more than 400 hundred proteins in the liver may be affected. These proteins are involved in such processes as catalysis of fatty acids by CoA, homocysteine metabolism, beta oxidation and the condensation of carbamoyl phosphate in the urea cycle among others. Further analysis of the protein associations showed that 325 biological processes, 140 molecular functions and 70 cellular components appear to be affected from the ingestion of TNDF. Quantitative analysis of specific mRNA transcripts indicated CMBL, GSTM1 and SDS were differentially expressed.

Complex life histories predispose aphids to recent abundance declines.

Many animals change feeding habits as they progress through life stages, exploiting resources that vary in space and time. However, complex life histories may bring new risks if rapid environmental change disrupts the timing of these switches. Here, we use abundance times series for a diverse group of herbivorous insects, aphids, to search for trait and environmental characteristics associated with declines. Our meta dataset spanned three world regions and >300 aphid species, tracked at 75 individual sites for 10-50 years. Abundances were generally falling, with median changes of -8.3%, -5.6%, and -0.1% per year in the central USA, northwestern USA, and United Kingdom, respectively. Aphids that obligately alternated between host plants annually and those that were agricultural pests exhibited the steepest declines, relative to species able to persist on the same host plant year-round or those in natural areas. This suggests that host alternation might expose aphids to climate-induced phenology mismatches with one or more of their host plant species, with additional risks from exposure to insecticides and other management efforts. Warming temperatures through time were associated with milder aphid declines or even abundance increases, particularly at higher latitudes. Altogether, while a warming world appeared to benefit some aphid species in some places, most aphid species that had time-sensitive movements among multiple host plants seemed to face greater risk of decline. More generally, this suggests that recent human-induced rapid environmental change is rebalancing the risks and rewards associated with complex life histories.

Recent climate change is creating hotspots of butterfly increase and decline across North America.

Some insect populations are experiencing dramatic declines, endangering the crucial ecosystem services they provide. Yet, other populations appear robust, highlighting the need to better define patterns and underlying drivers of recent change in insect numbers. We examined abundance and biodiversity trends for North American butterflies using a unique citizen-science dataset that has recorded observations of over 8 million butterflies across 456 species, 503 sites, nine ecoregions, and 26 years. Butterflies are a biodiverse group of pollinators, herbivores, and prey, making them useful bellwethers of environmental change. We found great heterogeneity in butterfly species' abundance trends, aggregating near zero, but with a tendency toward decline. There was strong spatial clustering, however, into regions of increase, decrease, or relative stasis. Recent precipitation and temperature appeared to largely drive these patterns, with butterflies generally declining at increasingly dry and hot sites but increasing at relatively wet or cool sites. In contrast, landscape and butterfly trait predictors had little influence, though abundance trends were slightly more positive around urban areas. Consistent with varying responses by different species, no overall directional change in butterfly species richness or evenness was detected. Overall, a mosaic of butterfly decay and rebound hotspots appeared to largely reflect geographic variability in climate drivers. Ongoing controversy about insect declines might dissipate with a shift in focus to the causes of heterogeneous responses among taxa and sites, with climate change emerging as a key suspect when pollinator communities are broadly impacted.

Guidelines for the diagnosis and treatment of neuroangiostrongyliasis: updated recommendations.

A subcommittee of the Hawaii Governor's Joint Task Force on Rat Lungworm Disease developed preliminary guidelines for the diagnosis and treatment of neuroangiostrongyliasis (NAS) in 2018 (Guidelines, 2018). This paper reviews the main points of those guidelines and provides updates in areas where our understanding of the disease has increased. The diagnosis of NAS is described, including confirmation of infection by real-time polymerase chain reaction (RTi-PCR) to detect parasite DNA in the central nervous system (CNS). The treatment literature is reviewed with recommendations for the use of corticosteroids and the anthelminthic drug albendazole. Long-term sequelae of NAS are discussed and recommendations for future research are proposed.

Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†.

Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph.

Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.

Highly diversified crop-livestock farming systems reshape wild bird communities.

Agricultural intensification is a leading threat to bird conservation. Highly diversified farming systems that integrate livestock and crop production might promote a diversity of habitats useful to native birds foraging across otherwise-simplified landscapes. At the same time, these features might be attractive to nonnative birds linked to a broad range of disservices to both crop and livestock production. We evaluated the influence of crop-livestock integration on wild bird richness and density along a north-south transect spanning the U.S. West Coast. We surveyed birds on 52 farms that grew primarily mixed vegetables and fruits alone or integrated livestock into production. Crop-livestock systems harbored higher native bird density and richness relative to crop-only farms, a benefit more pronounced on farms embedded in nonnatural landscapes. Crop-livestock systems bolstered native insectivores linked to the suppression of agricultural pest insects but did not bolster native granivores that may be more likely to damage crops. Crop-livestock systems also significantly increased the density of nonnative birds, primarily European Starlings (Sturnus vulgaris) and House Sparrows (Passer domesticus) that may compete with native birds for resources. Models supported a small, positive correlation between nonnative density and overall native bird density as well as between nonnative density and native granivore density. Relative to crop-only farms, on average, crop-livestock systems exhibited 1.5 times higher patch richness, 2.4 times higher density of farm structures, 7.3 times smaller field sizes, 2.4 times greater integration of woody crops, and 5.3 times greater integration of pasture/hay habitat on farm. Wild birds may have responded to this habitat diversity and/or associated food resources. Individual farm factors had significantly lower predictive power than farming system alone (change in C statistic information criterion (ΔCIC) = 80.2), suggesting crop-livestock systems may impact wild birds through a suite of factors that change with system conversion. Collectively, our findings suggest that farms that integrate livestock and crop production can attract robust native bird communities, especially within landscapes devoted to intensified food production. However, additional work is needed to demonstrate persistent farm bird communities through time, ecophysiological benefits to birds foraging on these farms, and net effects of both native and nonnative wild birds in agroecosystems.

Using Contrast Motion to Generate Patient-Specific Blood Flow Simulations During Invasive Coronary Angiography.

Virtual fractional flow reserve (vFFR) is an emerging technology employing patient-specific computational fluid dynamics (CFD) simulations to infer the hemodynamic significance of a coronary stenosis. Patient-specific boundary conditions are an important aspect of this approach and while most efforts make use of lumped parameter models to capture key phenomena, they lack the ability to specify the associated parameters on a patient-specific basis. When applying vFFR in a catheter laboratory setting using X-ray angiograms as the basis for creating the simulations, there is some indirect functional information available through the observation of the radio-opaque contrast agent motion. In this work, we present a novel method for tuning the lumped parameter arterial resistances (commonly incorporated in such simulations), based on simulating the physics of the contrast motion and comparing the observed and simulated arrival times of the contrast front at key points within a coronary tree. We present proof of principle results on a synthetically generated coronary tree comprised of multiple segments, demonstrating that the method can successfully optimize the arterial resistances to reconstruct the underlying velocity and pressure fields, providing a potential new means to improve the patient specificity of simulation-based technologies in this area.

Sperm-borne glutathione-S-transferase omega 2 accelerates the nuclear decondensation of spermatozoa during fertilization in mice†.

The postacrosomal sheath (PAS) of the perinuclear theca (PT) is the first compartment of the sperm head to solubilize into the ooplasm upon sperm-oocyte fusion, implicating its constituents in zygotic development. This study investigates the role of one such constituent, glutathione-S-transferase omega 2 (GSTO2), an oxidative-reductive enzyme found in the PAS and perforatorial regions of the PT. GSTO2 uses the conjugation of reduced glutathione, an electron donor shown to be compulsory in sperm disassembly within the ooplasm. The proximity of GSTO2 to the condensed sperm nucleus led us to hypothesize that this enzyme may facilitate nuclear decondensation by reducing disulfide bonds before the recruitment of GSTO enzymes from within the ooplasm. To test this hypothesis, we utilized a cell permeable isozyme-specific inhibitor, which fluoresces when bound to the active site of GSTO2, to functionally inhibit spermatozoa before performing intracytoplasmic sperm injections (ICSI) in mice. The technique allowed for targeted inhibition of solely PT-residing GSTO2, as all that is required for complete zygotic development is the injection of the mouse spermatozoon head. ICSI showed that inhibition of PT-anchored GSTO2 caused a delay in sperm nuclear decondensation, and further resulted in untimely embryo cleavage, and an increase in fragmentation beginning at the morula stage. The confounding effects of these developmental delays ultimately resulted in decreased blastocyst formation. This study implicates PT-anchored GSTO2 as an important facilitator of nuclear decondensation and reinforces the notion that the PAS-PT is a critical sperm compartment harboring molecules that facilitate zygotic development.

Basal levels of (p)ppGpp differentially affect the pathogenesis of infective endocarditis in Enterococcus faecalis.

The alarmone (p)ppGpp mediates the stringent response and has a recognized role in bacterial virulence. We previously reported a stringent response-like state in Enterococcus faecalis isolated from a rabbit foreign body abscess model and showed that E. faecalis mutants with varying levels of cellular (p)ppGpp [Δrel, ΔrelQ and the (p)ppGpp0 ΔrelΔrelQ] had differential abilities to persist within abscesses. In this study, we investigated whether (p)ppGpp contributes to the pathogenesis of E. faecalis infective endocarditis (IE), a biofilm infection of the heart valves. While the stringent response was not activated in heart valve-associated E. faecalis, deletion of the gene encoding the bifunctional (p)ppGpp synthetase/hydrolase Rel significantly impaired valve colonization. These results indicate that the presence of (p)ppGpp is dispensable for E. faecalis to cause IE, whereas the ability to regulate (p)ppGpp levels is critical for valve colonization. Next, we characterized how basal (p)ppGpp levels affect processes associated with IE pathogenesis. Despite being defective in binding to BSA-coated polystyrene surfaces, the Δrel strain bound to collagen- and fibronectin-coated surfaces and ex vivo porcine heart valves as well as the parent and ΔrelΔrelQ strains, ruling out the possibility that the impaired IE phenotype was due to an attachment defect. Moreover, differences in cellular (p)ppGpp levels did not affect extracellular gelatinase activity but significantly impaired enterococcal invasion of human coronary artery endothelial cells. Taken together, this study uncovers for the first time the fact that differences in basal (p)ppGpp levels, rather than the stringent response, differentially affect processes that contribute to the pathogenesis of IE.