A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several priA mutants. The export efficiency can be substantial; in a strain carrying the prUA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all

Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms.

We previously reported that a number of Treponema pallidum membrane proteins appear to reside on the cell surface, since intact treponemes radiolabeled by overnight incubation in medium containing [35S]methionine bind immunoglobulin G (IgG) antibodies directed against these proteins. In the present study, it was found that freshly extracted organisms radiolabeled in vitro for only 2 h inefficiently bound IgG antibodies directed against just two proteins of molecular weights 40,000 and 34,000. An in vitro incubation period of greater than 8 h was required before IgG antibodies present in rabbit syphilitic serum could recognize additional protein antigens on the cell surface. Treatment of aged treponemes, but not freshly extracted organisms, with 0.04% sodium dodecyl sulfate selectively removed a membranous layer from the treponemal surface. Only three treponemal proteins were found associated with this structure, including the same 40,000- and 34,000-molecular-weight proteins mentioned above. These two proteins most likely represent endoflagellar subunits, since they were precipitated with rabbit antisera prepared against purified endoflagellar subunits of the cultivable treponemal strain Treponema phagedenis. Further evidence also was obtained that cells of T. pallidum actively secrete into their extracellular environment a unique class of low-molecular-weight proteins

Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo.

We radiolabeled Leptospira proteins with [35S]methionine. Solubilized extracts of radiolabeled L. interrogans serovar hardjo strain hardjoprajitno were analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. We compared the protein profile obtained in this manner to the protein profiles of various [35S]methionine-labeled Leptospira spp. The profiles of the pathogenic L. interrogans strains were very similar but not identical and exhibited no obvious relationship to those of the two nonpathogenic species. We used solubilized, radiolabeled hardjoprajitno extracts and a sensitive radioimmunoprecipitation procedure to identify protein antigens recognized by immunoglobulin G antibodies present in various rabbit anti-hardjo sera. Homologous hyperimmune rabbit serum efficiently precipitated a large subset of proteins, the majority of which were between 30,000 and 66,500 daltons. Radioimmunoprecipitations with sera prepared against each of four recent hardjo isolates cultured from infected cattle produced similar results. Immunoprecipitations done with various radiolabeled Leptospira extracts and anti-hardjoprajitno serum demonstrated that the pathogenic leptospires possessed a number of cross-reactive major and minor protein antigens. By cell fractionation procedures, we found that most of the major protein antigens were present in the outer envelope. These proteins were exposed on the leptospiral cell surface because intact radiolabeled leptospires bound antibodies directed against them

Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein

Procalcitonin Is Not a Reliable Biomarker of Bacterial Coinfection in People With Coronavirus Disease 2019 Undergoing Microbiological Investigation at the Time of Hospital Admission

Abstract Admission procalcitonin measurements and microbiology results were available for 1040 hospitalized adults with coronavirus disease 2019 (from 48 902 included in the International Severe Acute Respiratory and Emerging Infections Consortium World Health Organization Clinical Characterisation Protocol UK study). Although procalcitonin was higher in bacterial coinfection, this was neither clinically significant (median [IQR], 0.33 [0.11–1.70] ng/mL vs 0.24 [0.10–0.90] ng/mL) nor diagnostically useful (area under the receiver operating characteristic curve, 0.56 [95% confidence interval, .51–.60]).</jats:p

Implementation of corticosteroids in treating COVID-19 in the ISARIC WHO Clinical Characterisation Protocol UK:prospective observational cohort study

BACKGROUND: Dexamethasone was the first intervention proven to reduce mortality in patients with COVID-19 being treated in hospital. We aimed to evaluate the adoption of corticosteroids in the treatment of COVID-19 in the UK after the RECOVERY trial publication on June 16, 2020, and to identify discrepancies in care. METHODS: We did an audit of clinical implementation of corticosteroids in a prospective, observational, cohort study in 237 UK acute care hospitals between March 16, 2020, and April 14, 2021, restricted to patients aged 18 years or older with proven or high likelihood of COVID-19, who received supplementary oxygen. The primary outcome was administration of dexamethasone, prednisolone, hydrocortisone, or methylprednisolone. This study is registered with ISRCTN, ISRCTN66726260. FINDINGS: Between June 17, 2020, and April 14, 2021, 47 795 (75·2%) of 63 525 of patients on supplementary oxygen received corticosteroids, higher among patients requiring critical care than in those who received ward care (11 185 [86·6%] of 12 909 vs 36 415 [72·4%] of 50 278). Patients 50 years or older were significantly less likely to receive corticosteroids than those younger than 50 years (adjusted odds ratio 0·79 [95% CI 0·70–0·89], p=0·0001, for 70–79 years; 0·52 [0·46–0·58], p80 years), independent of patient demographics and illness severity. 84 (54·2%) of 155 pregnant women received corticosteroids. Rates of corticosteroid administration increased from 27·5% in the week before June 16, 2020, to 75–80% in January, 2021. INTERPRETATION: Implementation of corticosteroids into clinical practice in the UK for patients with COVID-19 has been successful, but not universal. Patients older than 70 years, independent of illness severity, chronic neurological disease, and dementia, were less likely to receive corticosteroids than those who were younger, as were pregnant women. This could reflect appropriate clinical decision making, but the possibility of inequitable access to life-saving care should be considered. FUNDING: UK National Institute for Health Research and UK Medical Research Council

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

Vitamin D insufficiency in COVID-19 and influenza A, and critical illness survivors: a cross-sectional study

Objectives: The steroid hormone vitamin D has roles in immunomodulation and bone health. Insufficiency is associated with susceptibility to respiratory infections. We report 25-hydroxy vitamin D (25(OH)D) measurements in hospitalised people with COVID-19 and influenza A and in survivors of critical illness to test the hypotheses that vitamin D insufficiency scales with illness severity and persists in survivors. Design: Cross-sectional study. Setting and participants: Plasma was obtained from 295 hospitalised people with COVID-19 (International Severe Acute Respiratory and emerging Infections Consortium (ISARIC)/WHO Clinical Characterization Protocol for Severe Emerging Infections UK study), 93 with influenza A (Mechanisms of Severe Acute Influenza Consortium (MOSAIC) study, during the 2009–2010 H1N1 pandemic) and 139 survivors of non-selected critical illness (prior to the COVID-19 pandemic). Total 25(OH)D was measured by liquid chromatography-tandem mass spectrometry. Free 25(OH)D was measured by ELISA in COVID-19 samples. Outcome measures: Receipt of invasive mechanical ventilation (IMV) and in-hospital mortality. Results: Vitamin D insufficiency (total 25(OH)D 25–50 nmol/L) and deficiency (<25 nmol/L) were prevalent in COVID-19 (29.3% and 44.4%, respectively), influenza A (47.3% and 37.6%) and critical illness survivors (30.2% and 56.8%). In COVID-19 and influenza A, total 25(OH)D measured early in illness was lower in patients who received IMV (19.6 vs 31.9 nmol/L (p<0.0001) and 22.9 vs 31.1 nmol/L (p=0.0009), respectively). In COVID-19, biologically active free 25(OH)D correlated with total 25(OH)D and was lower in patients who received IMV, but was not associated with selected circulating inflammatory mediators. Conclusions: Vitamin D deficiency/insufficiency was present in majority of hospitalised patients with COVID-19 or influenza A and correlated with severity and persisted in critical illness survivors at concentrations expected to disrupt bone metabolism. These findings support early supplementation trials to determine if insufficiency is causal in progression to severe disease, and investigation of longer-term bone health outcomes

Co-infections, secondary infections, and antimicrobial use in patients hospitalised with COVID-19 during the first pandemic wave from the ISARIC WHO CCP-UK study: a multicentre, prospective cohort study

Background: Microbiological characterisation of co-infections and secondary infections in patients with COVID-19 is lacking, and antimicrobial use is high. We aimed to describe microbiologically confirmed co-infections and secondary infections, and antimicrobial use, in patients admitted to hospital with COVID-19. Methods: The International Severe Acute Respiratory and Emerging Infections Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study is an ongoing, prospective cohort study recruiting inpatients from 260 hospitals in England, Scotland, and Wales, conducted by the ISARIC Coronavirus Clinical Characterisation Consortium. Patients with a confirmed or clinician-defined high likelihood of SARS-CoV-2 infection were eligible for inclusion in the ISARIC WHO CCP-UK study. For this specific study, we excluded patients with a recorded negative SARS-CoV-2 test result and those without a recorded outcome at 28 days after admission. Demographic, clinical, laboratory, therapeutic, and outcome data were collected using a prespecified case report form. Organisms considered clinically insignificant were excluded. Findings: We analysed data from 48 902 patients admitted to hospital between Feb 6 and June 8, 2020. The median patient age was 74 years (IQR 59–84) and 20 786 (42·6%) of 48 765 patients were female. Microbiological investigations were recorded for 8649 (17·7%) of 48 902 patients, with clinically significant COVID-19-related respiratory or bloodstream culture results recorded for 1107 patients. 762 (70·6%) of 1080 infections were secondary, occurring more than 2 days after hospital admission. Staphylococcus aureus and Haemophilus influenzae were the most common pathogens causing respiratory co-infections (diagnosed ≤2 days after admission), with Enterobacteriaceae and S aureus most common in secondary respiratory infections. Bloodstream infections were most frequently caused by Escherichia coli and S aureus. Among patients with available data, 13 390 (37·0%) of 36 145 had received antimicrobials in the community for this illness episode before hospital admission and 39 258 (85·2%) of 46 061 patients with inpatient antimicrobial data received one or more antimicrobials at some point during their admission (highest for patients in critical care). We identified frequent use of broad-spectrum agents and use of carbapenems rather than carbapenem-sparing alternatives. Interpretation: In patients admitted to hospital with COVID-19, microbiologically confirmed bacterial infections are rare, and more likely to be secondary infections. Gram-negative organisms and S aureus are the predominant pathogens. The frequency and nature of antimicrobial use are concerning, but tractable targets for stewardship interventions exist. Funding: National Institute for Health Research (NIHR), UK Medical Research Council, Wellcome Trust, UK Department for International Development, Bill &amp; Melinda Gates Foundation, EU Platform for European Preparedness Against (Re-)emerging Epidemics, NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool, and NIHR HPRU in Respiratory Infections at Imperial College London