Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Physiological Responses and Adaptations to Lower Load Resistance Training: Implications for Health and Performance.

Resistance training is a method of enhancing strength, gait speed, mobility, and health. However, the external load required to induce these benefits is a contentious issue. A growing body of evidence suggests that when lower load resistance training [i.e., loads < 50% of one-repetition maximum (1RM)] is completed within close proximity to concentric failure, it can serve as an effective alternative to traditional higher load (i.e., loads > 70% of 1RM) training and in many cases can promote similar or even superior physiological adaptations. Such findings are important given that confidence with external loads and access to training facilities and equipment are commonly cited barriers to regular resistance training. Here, we review some of the mechanisms and physiological changes in response to lower load resistance training. We also consider the evidence for applying lower loads for those at risk of cardiovascular and metabolic diseases and those with reduced mobility. Finally, we provide practical recommendations, specifically that to maximize the benefits of lower load resistance training, high levels of effort and training in close proximity to concentric failure are required. Additionally, using lower loads 2-3 times per week with 3-4 sets per exercise, and loads no lower than 30% of 1RM can enhance muscle hypertrophy and strength adaptations. Consequently, implementing lower load resistance training can be a beneficial and viable resistance training method for a wide range of fitness- and health-related goals.

The relationship between adrenocortical candidate gene expression and clinical response to hydrocortisone in patients with septic shock.

PURPOSE: To determine if adrenocortical gene expression is associated with clinical outcomes or response to corticosteroid treatment in septic shock. METHODS: A pre-specified nested cohort study of a randomised controlled trial of hydrocortisone compared to placebo in septic shock. Blood was collected for RNAseq analysis prior to treatment with hydrocortisone or placebo. The expression of adrenocortical candidate genes related to pituitary releasing hormones, mineralocorticoid and glucocorticoid receptors, intracellular glucocorticoid metabolism and transport proteins was measured. RESULTS: From May 2014 to April 2017, 671 patients were enrolled in the nested cohort study, from which 494 samples were available for analysis. We found no evidence of an association between candidate gene expression levels and either 90-day mortality, 28-day mortality or time to shock reversal. We observed evidence of a significant interaction between expression and treatment group for time to shock reversal in two genes; GLCCI1 (HR 3.81, 95%CI 0.57-25.47 vs. HR 0.64, 95%CI 0.13-3.07 for hydrocortisone and placebo respectively, p for interaction 0.008) and BHSD1 (HR 0.55, 95%CI 0.28-1.09 vs. HR 1.32 95%CI 0.67-2.60, p for interaction 0.01). CONCLUSIONS: In patients with septic shock, there is no association between adrenocortical candidate gene expression and mortality. Patients with higher expression of GLCCI1 who received hydrocortisone achieved shock resolution faster than those receiving placebo; conversely, patients who had higher expression of BHSD1 who received hydrocortisone achieved shock resolution slower than those who received placebo. Variation in gene expression may be a mechanism for heterogeneity of treatment response to corticosteroids in septic shock.

Citrullination of extracellular histone H3.1 reduces antibacterial activity and exacerbates its proteolytic degradation.

BACKGROUND: Cystic fibrosis (CF), involves excessive airway accumulation of neutrophils, often in parallel with severe infection caused by Pseudomonas aeruginosa. Free histones are known to possess bactericidal properties, but the degree of antibacterial activity exerted on specific lung-based pathogens is largely unknown. Neutrophils have a high content of peptidyl deiminase 4 (PADI4), which citrullinate cationic peptidyl-arginines. In histone H3.1, several positions in the NH2-terminal tail are subject to citrullination. METHODS: Full-length and segmented histone subunit H3.1 was investigated for bactericidal activity towards P. aeruginosa (strain PAO1). PADI4-induced citrullination of histone H3.1 was assessed for antibacterial activity towards P. aeruginosa. Next, the effect of neutrophil elastase (NE)-mediated proteolysis of histone H3.1 was investigated. Finally, PADI4, H3.1, and citrullinated H3.1 were examined in healthy control and CF patient lung tissues. RESULTS: Full-length histone H3.1 and sections of the histone H3.1 tail, displayed bactericidal activity towards P. aeruginosa. These antibacterial effects were reduced following citrullination by PADI4 or proteolysis by NE. Interestingly, citrullination of histone H3.1 exacerbated NE-mediated degradation. In CF lung tissue, citrullinated histone H3.1 and PADI4 immunoreactivity was abundant. Degraded histone H3.1 was detected in the sputum of CF patients but was absent in the sputum of healthy controls. CONCLUSIONS: Citrullination impairs the antibacterial activity of histone H3.1 and exacerbates its proteolytic degradation by NE. Citrullination is likely to play an important role during resolution of acute inflammation. However, in chronic inflammation akin to CF, citrullination may dampen host defense and promote pathogen survival, as exemplified by P. aeruginosa.

BPI-ANCA is expressed in the airways of cystic fibrosis patients and correlates to platelet numbers and Pseudomonas aeruginosa colonization.

BACKGROUND: Autoantibodies to bactericidal/permeability-increasing protein (BPI), BPI-ANCA, are often present in serum of patients with cystic fibrosis (CF), and correlate to airway colonization with Pseudomonas aeruginosa. The aim of the study was to investigate if BPI-ANCA IgA is also present in the airways of CF patients, and if its presence correlates with neutrophil counts, platelets, and P. aeruginosa DNA in sputum. METHODS: BPI-ANCA IgA was quantified in serum and sputum samples from adult CF patients (n = 45) by ELISA. Sputum neutrophil counts, platelets, and platelet-neutrophil complexes were assessed by flow cytometry, and P. aeruginosa DNA was analysed with RT-PCR. RESULTS: Serum BPI-ANCA IgA was present in 44% of the study participants, and this group also had significantly enhanced BPI-ANCA levels in sputum compared to serum negative patients. Sputum levels of BPI-ANCA IgA correlated with P. aeruginosa DNA (r = 0.63, p = 0.0003) and platelet counts in sputum (r = 0.60, p = 0.0002). CONCLUSIONS: BPI-ANCA is expressed in the airways of CF patients and correlates with P. aeruginosa load and platelet counts, suggesting a link to airway inflammation and mucosal immunity.

Functions of the WNT Signaling Network in Shaping Host Responses to Infection.

It is well-established that aberrant WNT expression and signaling is associated with developmental defects, malignant transformation and carcinogenesis. More recently, WNT ligands have emerged as integral components of host responses to infection but their functions in the context of immune responses are incompletely understood. Roles in the modulation of inflammatory cytokine production, host cell intrinsic innate defense mechanisms, as well as the bridging of innate and adaptive immunity have been described. To what degree WNT responses are defined by the nature of the invading pathogen or are specific for subsets of host cells is currently not well-understood. Here we provide an overview of WNT responses during infection with phylogenetically diverse pathogens and highlight functions of WNT ligands in the host defense against infection. Detailed understanding of how the WNT network orchestrates immune cell functions will not only improve our understanding of the fundamental principles underlying complex immune response, but also help identify therapeutic opportunities or potential risks associated with the pharmacological targeting of the WNT network, as currently pursued for novel therapeutics in cancer and bone disorders.