Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

Neutrophil "plucking" on megakaryocytes drives platelet production and boosts cardiovascular disease.

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and 'instruct' them with pattern-recognition and motility programs.

Coordinated navigation within tissues is essential for cells of the innate immune system to reach the sites of inflammatory processes, but the signals involved are incompletely understood. Here we demonstrate that NG2(+) pericytes controlled the pattern and efficacy of the interstitial migration of leukocytes in vivo. In response to inflammatory mediators, pericytes upregulated expression of the adhesion molecule ICAM-1 and released the chemoattractant MIF. Arteriolar and capillary pericytes attracted and interacted with myeloid leukocytes after extravasating from postcapillary venules, 'instructing' them with pattern-recognition and motility programs. Inhibition of MIF neutralized the migratory cues provided to myeloid leukocytes by NG2(+) pericytes. Hence, our results identify a previously unknown role for NG2(+) pericytes as an active component of innate immune responses, which supports the immunosurveillance and effector function of extravasated neutrophils and macrophages.

Mechanosensing via a GpIIb/Src/14-3-3ζ axis critically regulates platelet migration in vascular inflammation.

Platelets are not only the first responders in thrombosis and hemostasis but also central players in inflammation. Compared with platelets recruited to thrombi, immune-responsive platelets use distinct effector functions including actin-related protein complex 2/3-dependent migration along adhesive substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cellular level is incompletely understood. Here, we use time-resolved morphodynamic profiling of individual platelets to show that migration, in contrast to clot retraction, requires anisotropic myosin IIa-activity at the platelet rear which is preceded by polarized actin polymerization at the front to initiate and maintain migration. Integrin GPIIb-dependent outside-in signaling via Gα13 coordinates polarization of migrating platelets to trigger tyrosine kinase c-Src/14-3-3ζ-dependent lamellipodium formation and functions independent of soluble agonists or chemotactic signals. Inhibitors of this signaling cascade, including the clinically used ABL/c-Src inhibitor dasatinib, interfere predominantly with the migratory capacity of platelets, without major impairment of classical platelet functions. In murine inflammation models, this translates to reduced migration of platelets visualized by 4D intravital microscopy, resulting in increased inflammation-associated hemorrhage in acute lung injury. Finally, platelets isolated from patients with leukemia treated with dasatinib who are prone to clinically relevant hemorrhage exhibit prominent migration defects, whereas other platelet functions are only partially affected. In summary, we define a distinct signaling pathway essential for migration and provide novel mechanistic insights explaining dasatinib-related platelet dysfunction and bleeding.

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI.

Impairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbß3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

International Care programs for Pediatric Post-COVID Condition (Long COVID) and the way forward.

BACKGROUND: Pediatric Post-COVID-Condition (PPCC) clinics treat children despite limited scientific substantiation. By exploring real-life management of children diagnosed with PPCC, the International Post-COVID-Condition in Children Collaboration (IP4C) aimed to provide guidance for future PPCC care. METHODS: We performed a cross-sectional international, multicenter study on used PPCC definitions; the organization of PPCC care programs and patients characteristics. We compared aggregated data from PPCC cohorts and identified priorities to improve PPCC care. RESULTS: Ten PPCC care programs and six COVID-19 follow-up research cohorts participated. Aggregated data from 584 PPCC patients was analyzed. The most common symptoms included fatigue (71%), headache (55%), concentration difficulties (53%), and brain fog (48%). Severe limitations in daily life were reported in 31% of patients. Most PPCC care programs organized in-person visits with multidisciplinary teams. Diagnostic testing for respiratory and cardiac morbidity was most frequently performed and seldom abnormal. Treatment was often limited to physical therapy and psychological support. CONCLUSIONS: We found substantial heterogeneity in both the diagnostics and management of PPCC, possibly explained by scarce scientific evidence and lack of standardized care. We present a list of components which future guidelines should address, and outline priorities concerning PPCC care pathways, research and international collaboration. IMPACT: Pediatric Post-COVID Condition (PPCC) Care programs have been initiated in many countries. Children with PPCC in different countries are affected by similar symptoms, limiting many to participate in daily life. There is substantial heterogeneity in diagnostic testing. Access to specific diagnostic tests is required to identify some long-term COVID-19 sequelae. Treatments provided were limited to physical therapy and psychological support. This study emphasizes the need for evidence-based diagnostics and treatment of PPCC. The International Post-COVID Collaboration for Children (IP4C) provides guidance for guideline development and introduces a framework of priorities for PPCC care and research, to improve PPCC outcomes.

Candida albicans Hyphal Morphogenesis within Macrophages Does Not Require Carbon Dioxide or pH-Sensing Pathways.

The opportunistic fungal pathogen Candida albicans has evolved a variety of mechanisms for surviving inside and escaping macrophages, including the initiation of filamentous growth. Although several distinct models have been proposed to explain this process at the molecular level, the signals driving hyphal morphogenesis in this context have yet to be clarified. Here, we evaluate the following three molecular signals as potential hyphal inducers within macrophage phagosomes: CO2, intracellular pH, and extracellular pH. Additionally, we revisit previous work suggesting that the intracellular pH of C. albicans fluctuates in tandem with morphological changes in vitro. Using time-lapse microscopy, we observed that C. albicans mutants lacking components of the CO2-sensing pathway were able to undergo hyphal morphogenesis within macrophages. Similarly, a rim101Δ strain was competent in hyphal induction, suggesting that neutral/alkaline pH sensing is not necessary for the initiation of morphogenesis within phagosomes either. Contrary to previous findings, single-cell pH-tracking experiments revealed that the cytosolic pH of C. albicans remains tightly regulated both within macrophage phagosomes and under a variety of in vitro conditions throughout the process of morphogenesis. This finding suggests that intracellular pH is not a signal contributing to morphological changes.

Candida auris-macrophage cellular interactions and transcriptional response.

The pathogenic yeast Candida auris represents a global threat of the utmost clinical relevance. This emerging fungal species is remarkable in its resistance to commonly used antifungal agents and its persistence in the nosocomial settings. The innate immune system is one the first lines of defense preventing the dissemination of pathogens in the host. C. auris is susceptible to circulating phagocytes, and understanding the molecular details of these interactions may suggest routes to improved therapies. In this work, we examined the interactions of this yeast with macrophages. We found that macrophages avidly phagocytose C. auris; however, intracellular replication is not inhibited, indicating that C. auris resists the killing mechanisms imposed by the phagocyte. Unlike Candida albicans, phagocytosis of C. auris does not induce macrophage lysis. The transcriptional response of C. auris to macrophage phagocytosis is very similar to other members of the CUG clade (C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae), i.e., downregulation of transcription/translation and upregulation of alternative carbon metabolism pathways, transporters, and induction of oxidative stress response and proteolysis. Gene family expansions are common in this yeast, and we found that many of these genes are induced in response to macrophage co-incubation. Among these, amino acid and oligopeptide transporters, as well as lipases and proteases, are upregulated. Thus, C. auris shares key transcriptional signatures shared with other fungal pathogens and capitalizes on the expansion of gene families coding for potential virulence attributes that allow its survival, persistence, and evasion of the innate immune system.

Direct 16S/18S rRNA Gene PCR Followed by Sanger Sequencing as a Clinical Diagnostic Tool for Detection of Bacterial and Fungal Infections: a Systematic Review and Meta-Analysis.

rRNA gene Sanger sequencing is being used for the identification of cultured pathogens. A new diagnostic approach is sequencing of uncultured samples by using the commercial DNA extraction and sequencing platform SepsiTest (ST). The goal was to analyze the clinical performance of ST with a focus on nongrowing pathogens and the impact on antibiotic therapy. A literature search used PubMed/Medline, Cochrane, Science Direct, and Google Scholar. Eligibility followed PRISMA-P criteria. Quality and risk of bias were assessed drawing on QUADAS-2 (quality assessment of diagnostic accuracy studies, revised) criteria. Meta-analyses were performed regarding accuracy metrics compared to standard references and the added value of ST in terms of extra found pathogens. We identified 25 studies on sepsis, infectious endocarditis, bacterial meningitis, joint infections, pyomyositis, and various diseases from routine diagnosis. Patients with suspected infections of purportedly sterile body sites originated from various hospital wards. The overall sensitivity (79%; 95% confidence interval [CI], 73 to 84%) and specificity (83%; 95% CI, 72 to 90%) were accompanied by large effect sizes. ST-related positivity was 32% (95% CI, 30 to 34%), which was significantly higher than the culture positivity (20%; 95% CI, 18 to 22%). The overall added value of ST was 14% (95% CI, 10 to 20%) for all samples. With 130 relevant taxa, ST uncovered high microbial richness. Four studies demonstrated changes of antibiotic treatment at 12% (95% CI, 9 to 15%) of all patients upon availability of ST results. ST appears to be an approach for the diagnosis of nongrowing pathogens. The potential clinical role of this agnostic molecular diagnostic tool is discussed regarding changes of antibiotic treatment in cases where culture stays negative.

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo.

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

Abdominal Symptoms Assessed With the CFAbd-Score are Associated With Intestinal Inflammation in Patients With Cystic Fibrosis.

OBJECTIVES: This prospective study evaluated the relationship between fecal markers of intestinal inflammation and cystic fibrosis (CF)-associated abdominal symptoms. These were assessed using the CFAbd-Score, a CF-specific patient-related outcome measure developed and validated, following FDA guidelines. METHODS: In feces from patients with CF (n = 41) and healthy volunteers (n = 27), concentrations of fecal calprotectin (FC), M2-pyruvate kinase (M2-PK), interleukins IL-1ß, IL-6, IL-8, and neutrophilic elastase (NE) were measured. Abdominal symptoms during the 2 preceding weeks were recorded using the CFAbd-Score. This patient-reported outcome measure (PROM) for assessment of the multi-organic abdominal involvement in CF includes 28 items in five domains. RESULTS: Inflammatory parameters FC, IL-1ß, M2-PK, and NE in feces, as well as CFAbd-Scores resulted significantly higher in CF patients than in healthy controls (all P < 0.01). Furthermore, significant differences between both groups were found for pain-symptoms, disorders of bowel movement, impaired quality of life, as well as disorders of eating and appetite. With 83% sensitivity and 74% specificity, FC was the most reliable measure for CF-related intestinal inflammation, which, in the CFAbd-Score, was associated to significantly higher rates of abdominal pain, as well as to general quality of life items such as gastrointestinal-related impaired sleep and frustration. CONCLUSION: Using the CFAbd-Score as a CF-specific PROM for identification and quantification of abdominal symptoms revealed that abdominal pain and impaired quality of life are associated with intestinal inflammation in CF.

One year monitoring of SARS-CoV-2 prevalence in a German cohort of patients with cystic fibrosis.

BACKGROUND: In Germany, the first case of coronavirus disease 2019 (COVID-19) was registered on 28 January 2020. By February 2021, the third wave of the pandemic began. So far, only few data are available on the SARS-CoV-2 prevalence and the clinical impact of an infection in patients with cystic fibrosis (CF). METHODS: From February 2020 until March 2021, we screened 156 CF patients for anti-SARS-CoV-2 IgG antibodies (serology) and the presence of SARS-CoV-2 in deep throat saliva or nasopharyngeal swabs (molecular testing). From patients with confirmed SARS-CoV-2 infection, we recorded symptoms and collected clinical data. RESULTS: In total, 13 patients (8.3%) were tested positive for SARS-CoV-2 infection, most of them during the second and the beginning third wave of the pandemic. Ten positive tested patients described symptoms linked to COVID-19. The most common symptom was cough followed by fatigue and headache. SARS-CoV-2 infection did not impair lung function. No positive tested patient needed to be hospitalized. CONCLUSIONS: SARS-CoV-2 infections in patients with CF are not as rare as initially anticipated, as frequent testing revealed. However, infected patients did not experience more severe clinical courses or worse clinical outcome. Our observation is in line with published reports indicating that individuals with CF are not at higher risk for severe COVID-19.

Eosinophil-platelet interactions promote atherosclerosis and stabilize thrombosis with eosinophil extracellular traps.

Clinical observations implicate a role of eosinophils in cardiovascular diseases because markers of eosinophil activation are elevated in atherosclerosis and thrombosis. However, their contribution to atherosclerotic plaque formation and arterial thrombosis remains unclear. In these settings, we investigated how eosinophils are recruited and activated through an interplay with platelets. Here, we provide evidence for a central importance of eosinophil-platelet interactions in atherosclerosis and thrombosis. We show that eosinophils support atherosclerotic plaque formation involving enhanced von Willebrand factor exposure on endothelial cells and augmented platelet adhesion. During arterial thrombosis, eosinophils are quickly recruited in an integrin-dependent manner and engage in interactions with platelets leading to eosinophil activation as we show by intravital calcium imaging. These direct interactions induce the formation of eosinophil extracellular traps (EETs), which are present in human thrombi and constitute a substantial part of extracellular traps in murine thrombi. EETs are decorated with the granule protein major basic protein, which causes platelet activation by eosinophils. Consequently, targeting of EETs diminished thrombus formation in vivo, which identifies this approach as a novel antithrombotic concept. Finally, in our clinical analysis of coronary artery thrombi, we identified female patients with stent thrombosis as the population that might derive the greatest benefit from an eosinophil-inhibiting strategy. In summary, eosinophils contribute to atherosclerotic plaque formation and thrombosis through an interplay with platelets, resulting in mutual activation. Therefore, eosinophils are a promising new target in the prevention and therapy of atherosclerosis and thrombosis.

Development and evaluation of an undergraduate curriculum on non-communicable disease research in Guam: The Pacific Islands Cohort of College Students (PICCS).

BACKGROUND: The non-communicable disease (NCD) epidemic among Pacific Islanders prompted the declaration of a regional state of NCD emergency throughout the United States-Affiliated Pacific Islands (USAPIs) in 2010. Subsequently, the University of Guam Health Science Program launched a pilot study on NCD research in its undergraduate curriculum modeled after the Pacific Data for Decision Making (DDM) framework - a field epidemiology training program employed in the USAPIs. The primary objective of the research is to conduct annual assessments of student health indicators with plans for longitudinal follow-up. Here, development and evaluation of the undergraduate research curriculum are described. METHODS: The Pacific DDM framework covering knowledge and skills in resourcing, types of data and indicators, data sources, data management, information products, and data dissemination and use were incorporated in undergraduate core courses of the Health Science Program. During the data collection pilot years, 2013 and 2014, a survey containing questions predominantly on NCD risk factors was launched at the university. The survey was administered by upperclassmen in the Health Science Program and evolved into the Pacific Islands Cohort of College Students (PICCS) research study. The initial years were spent developing the infrastructure. Program outputs were tracked annually to measure program success. RESULTS: Students in the Health Science Program obtained research knowledge and skills through various courses while enrolled in the program. The PICCS data collection continued annually as a cross-sectional survey from 2015 to current. Numerous successes have resulted including student abstracts and publications, acceptances to summer programs and fellowships, a sustained annual health fair for college students, a grant award, and other program-related impacts. CONCLUSION: The PICCS framework provided the organizational structure and documented tools, protocols, roles, and responsibilities to enhance consistency and reproducibility. Undergraduate students applied their knowledge and skills to an ongoing study focused on NCD risk factor surveillance of college students. Additionally, multiple research successes have been achieved through the PICCS curriculum. Plans are underway to begin the longitudinal design of the PICCS research study and sustain it through the curriculum, with room for adaptation as courses are updated over time.

N95 respirator reuse, decontamination methods, and microbial burden: A randomized controlled trial.

PURPOSE: To evaluate the effectiveness and ease of N95 respirator decontamination methods in a clinic setting and to identify the extent of microbial colonization on respirators associated with reuse. METHODS: In a prospective fashion, N95 respirators (n = 15) were randomized to a decontamination process (time, dry heat, or ultraviolet C light [UVC]) in outpatient clinics. Each respirator was re-used up to 5 separate clinic sessions. Swabs on each respirator for SARS-CoV-2, bacteria, and fungi were obtained before clinic, after clinic and post-treatment. Mask integrity was checked after each treatment (n = 68). Statistical analyses were performed to determine factors for positive samples. RESULTS: All three decontamination processes reduced bacteria counts similarly. On multivariate mixed model analysis, there were an additional 8.1 colonies of bacteria (95% CI 5.7 to 10.5; p < 0.01) on the inside compared to the outside surface of the respirators. Treatment resulted in a decrease of bacterial load by 8.6 colonies (95% CI -11.6 to -5.5; p < 0.01). Although no decontamination treatment affected the respirator filtration efficiency, heat treatments were associated with the breakdown of thermoplastic elastomer straps. Contamination with fungal and SARS-CoV-2 viral particles were minimal to non-existent. CONCLUSIONS: Time, heat and UVC all reduced bacterial load on reused N95 respirators. Fungal contamination was minimal. Heat could permanently damage some elastic straps making the respirators nonfunctional. Given its effectiveness against microbes, lack of damage to re-treated respirators and logistical ease, UVC represents an optimal decontamination method for individual N95 respirators when reuse is necessary.

The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species also are among the most important and problematic, opportunistic nosocomial pathogens. Surprisingly, these two species antagonize each other's virulence in both nematode infection and in vitro biofilm models. We report here the identification of the E. faecalis bacteriocin, EntV, produced from the entV (ef1097) locus, as both necessary and sufficient for the reduction of C. albicans virulence and biofilm formation through the inhibition of hyphal formation, a critical virulence trait. A synthetic version of the mature 68-aa peptide potently blocks biofilm development on solid substrates in multiple media conditions and disrupts preformed biofilms, which are resistant to current antifungal agents. EntV68 is protective in three fungal infection models at nanomolar or lower concentrations. First, nematodes treated with the peptide at 0.1 nM are completely resistant to killing by C. albicans The peptide also protects macrophages and augments their antifungal activity. Finally, EntV68 reduces epithelial invasion, inflammation, and fungal burden in a murine model of oropharyngeal candidiasis. In all three models, the peptide greatly reduces the number of fungal cells present in the hyphal form. Despite these profound effects, EntV68 has no effect on C. albicans viability, even in the presence of significant host-mimicking stresses. These findings demonstrate that EntV has potential as an antifungal agent that targets virulence rather than viability.

Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo.

OBJECTIVE: Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT. APPROACH AND RESULTS: Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis. CONCLUSIONS: Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.