The Binary Protein Interactome of Treponema pallidum – The Syphilis Spirochete

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Genetic Dissection of the Francisella novicida Restriction Barrier▿ †

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 106-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes

BAC Library of T. pallidum DNA in E. coli

Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T. pallidum ORFs. Using this minimal set of clones, at least 12 T. pallidum proteins were shown to react with pooled sera from rabbits immunized with T. pallidum, indicating that at least some T. pallidum genes are transcribed and expressed in E. coli

Transcriptome of Treponema pallidum: Gene Expression Profile during Experimental Rabbit Infection

RNA transcript levels in the syphilis spirochete Treponema pallidum subsp. pallidum (Nichols) isolated from experimentally infected rabbits were determined by the use of DNA microarray technology. This characterization of the T. pallidum transcriptome during experimental infection provides further insight into the importance of gene expression levels for the survival and pathogenesis of this bacterium

Systematic Cloning of Treponema pallidum Open Reading Frames for Protein Expression and Antigen Discovery

A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp. pallidum. Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences. A computational prediction of signal sequences identified 248 T. pallidum proteins that are potentially secreted from the cell. These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins. To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T. pallidum. Twelve of the 85 proteins bound significant levels of antibody. Of these 12 proteins, seven had previously been identified as T. pallidum antigens, and the remaining five represent novel antigens. These results demonstrate the potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete

Evaluating the Clinical Utility of Routine Sentinel Lymph Node Biopsy and the Value of Adjuvant Chemotherapy in Elderly Patients Diagnosed With Oestrogen Receptor Positive, Clinically Node Negative Breast Cancer

Background: Sentinel lymph node biopsy (SLNB) provides staging information and guides adjuvant therapy in early breast cancer (EBC). Routine SLNB in oncogeriatricians with low-risk EBC remains controversial. Aims: To evaluate axillary management in elderly patients diagnosed with oestrogen receptor positive (ER+), clinically lymph node negative (cLN−) EBC, and to assess whether SLNB affects further axillary management or adjuvant chemotherapy (ACTX) decision making. Methods: Female patients aged > 65 years, diagnosed with ER+, human epidermal growth factor receptor-2 negative (HER2−), and cLN− breast cancer (BC), who underwent surgery and SLNB were included. Clinicopathological predictors of ACTX and completion axillary lymph node dissection (CALND) were determined. Kaplan-Meier analyses assessed survival outcomes. Results: A total of 253 patients were included (median age: 72 years, range: 66-90), all underwent SLNB; 50 (19.8%) had lymphatic metastasis on SLNB (SLNB+). Of these, 19 proceeded to CALND (38.0%), 10 (52.6%) of whom had further axillary disease (ALND+). 20 of the 50 SLNB+ patients received ACTX (40.0%) as did 31 of the 203 SLNB− patients (15.2%) ( P   25 (OR: 4.37, 95% CI: 1.38-13.80, P  = .012) independently predicted receiving ACTX. Receiving ACTX and proceeding to CALND did not improve disease-free ( P  = .485 and P  = .345) or overall survival ( P  = .981 and P  = .646). Conclusions: Routine SNLB may not be necessary in elderly patients diagnosed with ER+, cLN− EBC. Future oncogeriatric practice is likely to see genomic testing guiding ACTX prescription in this group

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing the Treponema pallidum Proteome

To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at different rates during the time course of infection

Genome Scale Identification of Treponema pallidum Antigens

Antibody responses for 882 of the 1,039 proteins in the proteome of Treponema pallidum were examined. Sera collected from infected rabbits were used to systematically identify 106 antigenic proteins, including 22 previously identified antigens and 84 novel antigens. Additionally, sera collected from rabbits throughout the course of infection demonstrated a progression in the breadth and intensity of humoral immunoreactivity against a representative panel of T. pallidum antigens