Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.British Society for Antimicrobial Chemotherapy BSAC-2018-0095NC3Rs NC/V001582/1Biological Sciences Research Council BB/V007823/1Academy of Medical Sciences SBF006\104

Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo.

The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Type VI Secretion System: a bacterial killing machine and biocontrol weapon

The T6SS is a bacterial nanomachine used to inject effectors (toxins) into target cells. The system is found exclusively in Gram negative bacteria and frequently targets other prokaryotic cells; thus being considered a potent antimicrobial weapon.Our model organism, P. putida, encodes three T6SS systems (K1, K2 and K3). Each one of those contains the complete set of genes to encode the core components necessary to assemble a functional machinery (Tss components), accessory components (Tag proteins) and over eleven effectors including nucleases and pore-forming colicins together with their cognate immunity pairs (Tke1-Tki1, Tke2-Tki2, Tke3-Tki3, …). Among these systems, the K1-T6SS is a potent antibacterial device considered a biocontrol mechanism used by P. putida to kill a broad range of bacteria, including resilient phytopathogens such as Agrobacterium tumefaciens, Pseudomonas syringae, Pectobacterium caratovorum and Xanthomonas campestris.The structure of the T6SS resembles an inverted bacteriophage with a tube (Hcp proteins) surrounded by a contractile sheath (TssBC proteins) and capped with a puncturing tip (VgrG trimer). The cytosolic part of the T6SS docks onto a membrane complex (TssJLM) by interacting with a phage baseplate-like structure (TssAEFGK). Upon contraction of the sheath, the tube-tip complex, loaded with the effectors, is ejected and penetrates the target cell. The system is then disassembled and partially recycled for the next round of firing.Although the structure of the T6SS is very well conserved, some systems contain variations of core components that can be coupled to accessory proteins allowing to fine-tune different mechanisms including the assembly and/or the firing of the system. In this seminar, I will talk about TssA and its associate proteins (TagA, TagB, TagJ), an example of a core component that shows variability at domain level and that in combination with accessory proteins is involved in different aspects of the system assembly and thus is implicated in the system functionality

A novel stabilization mechanism for the type VI secretion system sheath

The type VI secretion system (T6SS) is a phage-derived contractile nanomachine primarily involved in interbacterial competition. Its pivotal component, TssA, is indispensable for the assembly of the T6SS sheath structure, the contraction of which propels a payload of effector proteins into neighboring cells. Despite their key function, TssA proteins exhibit unexpected diversity and exist in two major forms, a short form (TssAS) and a long form (TssAL). While TssAL proteins interact with a partner, called TagA, to anchor the distal end of the extended sheath, the mechanism for the stabilization of TssAS-containing T6SSs remains unknown. Here we discover a class of structural components that interact with short TssA proteins and contribute to T6SS assembly by stabilizing the polymerizing sheath from the baseplate. We demonstrate that the presence of these components is important for full sheath extension and optimal firing. Moreover, we show that the pairing of each form of TssA with a different class of sheath stabilization proteins results in T6SS apparatuses that either reside in the cell for some time or fire immediately after sheath extension. We propose that this diversity in firing dynamics could contribute to the specialization of the T6SS to suit bacterial lifestyles in diverse environmental niches

SpvD induces an intra-nuclear accumulation of importins.

<p>(A) HeLa cells were cotransfected with FLAG-KPNA1 or FLAG-KPNA3 and pRK5myc-SpvD or pRK5myc-SpvC. Samples were fixed, permeabilised in Triton X-100, labelled with anti-FLAG (red), anti-myc (blue) and anti-lamin (green) antibodies and analysed by confocal microscopy. Scale bar, 10 μm. White arrows indicate nuclear lamina-associated KPNAs. (B) Quantification of cells with nuclear lamina-associated KPNA1 or KPNA3 after transfection with pRK5myc-SpvD or pRK5myc-SpvC. Values are expressed as mean ± SEM of at least 4 independent experiments (*p < 0.05; *** p < 0.005). (C) HeLa cells were cotransfected with FLAG-KPNA1 and pRK5myc-SpvD. Samples were fixed, permeabilised in Triton X-100 or Saponin and labelled as in A. Scale bar, 8 um. (D) HeLa cells were cotransfected with FLAG-KPNA1 plasmids then infected for 14 h with <i>Salmonella</i> strains. Samples were fixed, permeabilised in Triton X-100 and labelled with anti-FLAG (red) and anti-<i>Salmonella</i> (blue) antibodies. Cell nuclei were stained with DRAQ5 (green). Scale bar, 10 μm. (E) Quantification of cells with nuclear lamina-associated KPNA1 after infection with <i>Salmonella</i> strains. Values are expressed as mean ± SEM of at least 4 independent experiments (** p < 0.01; *** p < 0.005). (F) HeLa cells were infected for 14 h with <i>Salmonella</i> strains. Nuclear and total cell extracts were analysed by SDS-PAGE and immunoblotting with anti-KPNA1, anti-histone H3 and anti-GAPDH antibodies. (G) Ratio of KPNA1 in the nucleus normalised to wild-type -infected cells. Values are expressed as mean ± SEM of 3 independent experiments. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05).</p

Effect of SpvD on virulence and cytokine expression <i>in vivo</i>.

<p>(A) Lack of SpvD causes virulence attenuation of <i>Salmonella</i> in the mouse model of systemic infection. C57BL/6 mice were inoculated by intraperitoneal injection (i.p.) with equal numbers (2.5 x 10⁴ CFU) of indicated bacteria. Bacteria were recovered from infected spleens 3 days post-inoculation. CI values were calculated as described in materials and methods. The scatter plot displays values obtained for individual mice and the mean is indicated (line) (*p < 0.05; *** p < 0.005). (B-D) C57BL/6 mice were inoculated by i.p. with indicated bacteria, RNAs from splenic macrophages were recovered at 2 days post-inoculation and <i>serpinB2</i> (B), <i>tnf</i>-α (C) and <i>il1-</i>β (D) mRNA levels were analysed by qRT-PCR. The transcript levels were normalized to the levels of <i>rsp9</i>, which were constant under all conditions used, and then expressed relative to those of wild-type-infected mice. Results are expressed as mean ± SEM of at least 3 independent experiments. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05). (E) Model depicting pathways targeted by SpvC and SpvD to overcome PAMP-induced defences in host cells. Immunoblots are representative of three independent experiments.</p

SpvD prevents nuclear accumulation of p65 but not degradation of IĸBα.

<p>(A) SpvD does not inhibit activation of an AP-1-regulated promoter. HEK-293 cells were cotransfected with an AP-1-dependent luciferase reporter plasmid, pTK-Renilla luciferase and pRK5myc, pRK5myc-SpvC or pRK5myc-SpvD. Cells were then stimulated with PMA for 6 h. Firefly luciferase activity was normalized against Renilla luciferase activity. Results are expressed as fold induction compared to non-transfected (NT) cells. Values are expressed as mean ± SEM of 3 independent experiments and P-values were obtained using two-tailed unpaired Student's t-test (*** p < 0.005). (B-C) SpvD prevents nuclear translocation of p65. (B) Representative immunofluorescence fields of p65 localisation using anti-p65 (red) in <i>TLR4</i>^-/- BMMs infected for 10 h with indicated <i>Salmonella</i> strains (blue). Cell nuclei were stained with DRAQ5 (green). Scale bar, 8 μm. (C) Quantification above background levels of p65 intensity in the nucleus was analyzed by three-dimensional (3D) confocal microscopy. Background levels were determined using signals measured in uninfected cells. P-values were obtained using two-tailed unpaired Student's t-test (*p < 0.05; ** p < 0.01). (D) SpvD inhibits activation of an NF-ĸB -regulated promoter. HEK-293 cells were co-transfected with an NF-ĸB -dependent luciferase reporter plasmid, pTK-Renilla luciferase and pRK5myc, pRK5myc-SpvC, pRK5myc-SpvD or pRK5myc-SpvD_K185A at the indicated amounts (in ng). The NF-ĸB pathway was activated with TNF-α and luciferase activity was measured after 8 h. Results are expressed as fold activation in relation to unstimulated and non-transfected (NT) cells. Values are expressed as mean ± SEM of at least 3 independent experiments and statistical significances were calculated using ANOVA followed by Bonferonni's multiple comparison test against NT cells (*p < 0.05; ** p < 0.01; *** p < 0.005). (E) Degradation of IĸBα is not affected by SpvD. HEK-293 cells transfected by pRK5myc-SpvD or pRK5myc-YopP were prepared at the indicated times after TNF-α stimulation and analysed by SDS-PAGE and immunoblotting with anti- IĸBα and anti-tubulin antibodies. Ratio of IĸBα normalised to unstimulated cells is indicated below immunoblots.</p