Differential Expression of the ToxR Regulon in Classical and E1 Tor Biotypes of Vibrio cholerae is Due to Biotype-Specific Control over toxT Expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74784/1/j.1365-2958.1998.00890.x.pd

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

Functional and genetic analysis of the lambdoid bacteriophage H-19B, a natural vector for the Shiga-like toxin genes.

Mobile genetic elements are associated with horizontal transfer of virulence factors among bacteria. One type of genetic element capable of transferring virulence factors are bacterial viruses, or bacteriophages. This study focuses on H-19B, a member of the lambdoid bacteriophage family, that carries the genes encoding Shiga-like toxin I. Following infection of a bacterial host, H-19B can integrate into the host genome, thereby transferring its virulence property to that host and contributing to the pathogenicity of the bacteria. The primary objective of these studies was to use both comparative analysis and functional studies to determine if the unique composition of genes that make up the H-19B genome has any direct correlation with its selection as a Shiga-like toxin vector. A secondary objective was to gain a better understanding of important transcriptional regulatory mechanisms employed by the phage through the use of functional and mutational analyses. Identification of genes and subsequent comparative studies confirmed that the H-19B genome, like those of other lambdoid phages, is a genetic mosaic of genes found in the lambdoid genus. I confirmed that H-19B has the common genetic organization of lambdoid phages through shared regions of homology and also identified multiple putative open reading frames that are not found in any of the gene data bases. Identification of genes and sequences that suggested major phage regulatory sites by sequence homology and genome location also showed significant differences from other lambdoid sequences suggesting variant mechanistic processes. Sequence analysis located the stx genes in the late operon, and I verified significance of this location by demonstrating that toxin production can be enhanced through expression of the upstream phage promoter. Functional analysis of the transcriptional regulatory region, nutR, downstream of the early promoter confirmed antitermination ability when provided with the phage N regulatory protein. However, mutational analysis of this region suggests a difference in multiple component requirements for the N-mediated antitermination mechanism used by H-19B in comparison to that shown for other lambdoid phages.Ph.D.Biological SciencesGeneticsHealth and Environmental SciencesMicrobiologyPathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131496/2/9909954.pd

SalY of the Streptococcus pyogenes Lantibiotic Locus Is Required for Full Virulence and Intracellular Survival in Macrophages▿

Streptococcus pyogenes utilizes numerous mechanisms for evading the host immune response but has only recently been found to survive in the intracellular environment. In this study, we demonstrate the requirement of a putative ABC transporter permease for intracellular survival in macrophages. The highly attenuated S. pyogenes mutant, SalY, was identified from a transposon mutagenesis screen, with over 200-fold attenuation in virulence in a zebrafish invasive-disease model. Sequencing of the region surrounding the insertion identified a locus that is highly conserved in other S. pyogenes genomes and is homologous to an operon involved in lantibiotic production. In vitro analysis demonstrated that the SalY mutant is deficient in intracellular survival in murine macrophages, a phenotype also observed in zebrafish macrophages in vivo. Macrophage crude cell lysates added to bacterial cultures resulted in the death of the SalY mutant but only growth inhibition of the wild-type strain. Specific depletion of zebrafish macrophages in vivo restored the ability of the SalY mutant to cause disease to wild-type levels. The SalY-infected, macrophage-depleted zebrafish exhibit large lesions and invasive dissemination at a rate and level similar to those of the wild type. In contrast, an M protein mutant with a degree of attenuation similar to that of the SalY mutant did not regain full virulence by in vivo depletion of macrophages. The putative SalY ABC transporter may be an example of the ability of S. pyogenes to adapt and evolve new survival strategies that allow dissemination and growth in previously uninhabitable sites

Large-Scale Screen Highlights the Importance of Capsule for Virulence in the Zoonotic Pathogen Streptococcus iniae

Zoonotic pathogens have the unique ability to cross the species barrier, causing disease in both humans and specific animal hosts. Streptococcus iniae is a zoonotic pathogen of both fish and humans, and the clinical presentations of S. iniae infections in fish and humans are very similar to those caused by various human-specific streptococcal pathogens. Virulence mechanisms required for infection by this pathogen of either host have yet to be determined. Using the previously reported zebrafish infectious disease model, we performed a large-scale screening to determine genes required for systemic infection. Screening 1,128 signature-tagged transposon mutants through the zebrafish model allowed identification of 41 potential mutants that were unable to survive within the host environment. Greater than 50% of the mutants that could be identified through homology searches were highly homologous to genes found in other human-specific streptococcal pathogens, while 32% were found to have no homology to any sequences found in the databases, suggesting as yet unknown gram-positive bacterial virulence factors. A large percentage of the insertions were found to be located in several putative capsule synthesis genes, an important virulence component for other systemic pathogens. Density gradient assays demonstrated that several of these putative capsule mutants have dissimilar buoyant densities, suggesting different levels of capsule synthesis. Putative capsule mutants were also less resistant to phagocytosis in whole-blood assays than wild-type S. iniae. Our initial large-scale characterization of S. iniae virulence highlights the importance of the capsule for successful infection