Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome

Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion

IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion

La técnica de la microgota como alternativa para el recuento de Azospirillum spp. dentro del protocolo de la Red de Control de Calidad de Inoculantes (REDCAI)

La evaluación de la calidad de los inoculantes comerciales es fundamental para garantizar una adecuada respuesta de los cultivos a la inoculación dentro de un marco de bioseguridad. En este sentido, el objetivo de este trabajo fue la estandarización y validación de la técnica de la microgota para la cuantificación de Azospirillum como metodología alternativa a la técnica de siembra en superficie, propuesta actualmente en el protocolo consenso de la Red de Calidad de Inoculantes, REDCAI. Entre 14 y 25 laboratorios, tanto privados como públicos, participaron de tres ensayos independientes. A partir de ellos se obtuvieron resultados reproducibles y robustos que permiten confirmar que ambas técnicas son equivalentes y concluir que la técnica de recuento por la microgota es una alternativa adecuada para ser incluida dentro del mencionado protocolo consenso.Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.Fil: Di Salvo, Luciana Paula. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: García, Julia E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Amigo, Josefina Alejandra. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia. Departamento de Ecología. Cátedra de Microbiología Agrícola; ArgentinaFil: Anriquez, Analia Liliana. Universidad Nacional de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; ArgentinaFil: Barlocco, Claudia. Instituto Nacional de Investigación Agropecuaria; UruguayFil: Benintende, Silvia Mercedes. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias; ArgentinaFil: Bochatay, Tatiana. BASF Agricultural Specialities S.A.; ArgentinaFil: Bortolato, Marta Alejandra. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; ArgentinaFil: Cassan, Fabricio Dario. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Ramirez Castaño, Carolina. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Catafesta, Melina. Bio Nova; ArgentinaFil: Coniglio, Nayla Anahí. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Díaz, Marisa. Rizobacter Argentina; ArgentinaFil: Galian, Liliana Rosa. Universidad Nacional de Lomas de Zamora. Facultad de Ciencias Agrarias; ArgentinaFil: Gallace, María Eugenia. Universidad Nacional de La Pampa. Facultad de Agronomía. Recursos Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Confluencia; ArgentinaFil: Garcia, Patricia Graciela. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Confluencia; ArgentinaFil: García de Salamone, Inés E.. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; ArgentinaFil: Landa, Marianela. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Liernur, Germán. No especifíca;Fil: Maneiro, María Laura. Rizobacter Argentina S.A.; ArgentinaFil: Massa, Rosana. Stoller Biociencias S.R.L.; ArgentinaFil: Malinverni, Julieta. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Marchessi, Nicolas Carlos. Universidad Nacional de Lomas de Zamora. Facultad de Ciencias Agrarias; ArgentinaFil: Monteleone, Emilia. Nitrasoil Argentina S.A.; ArgentinaFil: Oviedo, Silvina. Rizobacter Argentina S.A.; ArgentinaFil: Pobliti, Lucrecia. Barenbrug Argentina; ArgentinaFil: Portela, Gabriela Rut. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Radovancich, Débora. Laformed S.A.; ArgentinaFil: Righes, Silvia. Marketing Agrícola S.R.L.; Argentin

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

Mechanisms of unconventional secretion of IL-1 family cytokines

One of the most poorly understood processes in cell biology is the peculiar ability of specific leaderless proteins to be secreted via ER/Golgi-independent mechanisms (‘unconventional protein secretion’). One such leaderless protein is the major immune-activating cytokine, interleukin-1β (IL-1β). Unusual amongst cytokines, IL-1β is expressed in the cytosol as an inactive precursor protein. It requires maturation by the caspase-1 protease, which itself requires activation upon immune cell sensing of infection or cell stress. Despite 25 years of intensive research into IL-1β secretory mechanisms, how it exits the cell is still not well understood. Here we will review the various mechanisms by which macrophages have been proposed to secrete IL-1 family cytokines, and the potential involvement of caspase-1 therein. Since aberrant IL-1β production drives inherited and acquired human diseases (e.g. autoinflammatory diseases, arthritic diseases, gout, Alzheimer’s disease), elucidation of the IL-1β secretory pathway may offer new therapeutic opportunities for treatment across this wide range of human conditions

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-γ-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics

Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors▿

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems

Plasmin(ogen) acquisition by group A Streptococcus protects against C3b-mediated neutrophil killing

The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection

Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity

Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome-caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses