Rab32 restriction of intracellular bacterial pathogens

Experiments in the authors’ laboratory are supported by the Wellcome Trust (grant no. 109680/Z/15/Z), the Royal Society (grant no. RG150386), and Tenovus Scotland (grant no. G14/19) to SS. VSC is recipient of a European Union’s Horizon 2020 research and innovation programme Marie Sklodowska Curie Fellowship (grant no. 706040).Peer reviewedPublisher PD

The pneumococcal MgaSpn virulence transcriptional regulator generates multimeric complexes on linear double-stranded DNA

Este artículo pertenece a la Tesis presentada por Virtudes Solano Collado con título: "Caracterización molecular del regulador transcripcional MgaSpn de Streptococcus pneumoniae"(https://digital.csic.es/handle/10261/98162)The MgaSpn transcriptional regulator contributes to the virulence of Streptococcus pneumoniae. It is thought to be a member of the Mga/AtxA family of global regulators. MgaSpn was shown to activate in vivo the P1623B promoter, which is divergent from the promoter (Pmga) of its own gene. This activation required a 70-bp region (PB activation region) located between both promoters. In this work, we purified an untagged form of the MgaSpn protein, which formed dimers in solution. By gel retardation and footprinting assays, we analysed the binding of MgaSpn to linear double-stranded DNAs. MgaSpn interacted with the PB activation region when it was placed at internal position on the DNA. However, when it was positioned at one DNA end, MgaSpn recognized preferentially the Pmga promoter placed at internal position. In both cases, and on binding to the primary site, MgaSpn spread along the adjacent DNA regions generating multimeric protein-DNA complexes. When both MgaSpn-binding sites were located at internal positions on longer DNAs, electron microscopy experiments demonstrated that the PB activation region was the preferred target. DNA molecules totally or partially covered by MgaSpn were also visualized. Our results suggest that MgaSpn might recognize particular DNA conformations to achieve DNA-binding specificityBFU2009-11868 to A.B.] and [CSD2008-00013- INTERMODS to M.E.] from the Spanish Ministry of Economy and Competitiveness, and [PIE-201320E028 to A.B.] from the Spanish National Research Council. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2009-11868].Peer Reviewe

VARP and Rab9 are dispensable for the Rab32/BLOC-3 dependent salmonella killing in mouse macrophages

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localisation on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing

A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth

Acknowledgments We are very grateful to Leigh Knodler for her generous gift of P22 phages from a S. Typhimurium glmS::Cm::mCherry strain. We thank the Microscopy and Histology Core Facility, the Centre for Genome-Enabled Biology and Medicine (CGEBM), the Iain Fraser Cytometry Centre and the qPCR Facility (University of Aberdeen) for their support and assistance in this work. We thank members of the Spanò/Baldassarre laboratory for their feedback throughout this project. The content of this manuscript has been posted as a preprint on bioRxiv (Solano-Collado et al., 2020). Funding This work was supported by the European Union’s Horizon 2020 research and innovation program Marie Skłodowska-Curie Fellowship (706040_KILLINGTYPHI) to VS-C, the Wellcome Trust (Seed Award 109680/Z/15/Z), the European Union’s Horizon 2020 ERC consolidator award (2016-726152-TYPHI), the BBSRC (BB/N017854/1) and Tenovus Scotland (G14/19) to SS.Peer reviewedPublisher PD

MgaSpn and H-NS: Two unrelated global regulators with similar DNA-binding properties

Global regulators play an essential role in the adaptation of bacterial cells to specific niches. Bacterial pathogens thriving in the tissues and organs of their eukaryotic hosts are a well-studied example. Some of the proteins that recognize local DNA structures rather than specific nucleotide sequences act as global modulators in many bacteria, both Gram-negative and -positive. To this class of regulators belong the H-NS-like proteins, mainly identified in γ-Proteobacteria, and the MgaSpn-like proteins identified in Firmicutes. H-NS and MgaSpn from Escherichia coli and Streptococcus pneumoniae, respectively, neither have sequence similarity nor share structural domains. Nevertheless, they display common features in their interaction with DNA, namely: (i) they bind to DNA in a non-sequence-specific manner, (ii) they have a preference for intrinsically curved DNA regions, and (iii) they are able to form multimeric complexes on linear DNA. Using DNA fragments from the hemolysin operon regulatory region of the E. coli plasmid pHly152, we show in this work that MgaSpn is able to recognize particular regions on extended H-NS binding sites. Such regions are either located at or flanked by regions of potential bendability. Moreover, we show that the regulatory region of the pneumococcal P1623B promoter, which is recognized by MgaSpn, contains DNA motifs that are recognized by H-NS. These motifs are adjacent to regions of potential bendability. Our results suggest that both regulatory proteins recognize similar structural characteristics of DNA

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

FUNDING This study was financially supported by grants BIO2016-76412- C2-2-R (AEI/FEDER, UE) to AB from the Spanish Ministry of Economy and Competitiveness, and PID2019-104553RB-C21 to AB from the Spanish Ministry of Science and Innovation. ACKNOWLEDGMENTS Thanks are due to F. W. Studier for his gift of the E. coli BL21 (DE3) strain and to L. Rodríguez for her technical help in protein purification.Peer reviewedPublisher PD

PclR is a transcriptional activator of the gene that encodes the pneumococcal collagen-like protein PclA

Acknowledgements This work was supported by grant PID2019-104553RB-C21 to A.B. from the Spanish Ministry of Science and Innovation.Peer reviewedPublisher PD

The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages.

Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex-3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens

Caracterización molecular del regulador transcripcional MgaSpn de Streptococcus pneumoniae

240 p.-7 tab.-44 fig.[EN] Bacteria usually live in habitats of changing conditions. During infection, pathogenic bacteria must be able to survive in different environments encountered as the pathogen progresses through its host. This adaptation requires sensing the relevant extracellular signals and linking them to a coordinate change in the expression of genes, which encode factors appropriate to the given situation. Global transcriptional regulators that respond to specific environmental signals are key elements in such regulatory networks. Bacteria often use classical two-component signal transduction systems (TCSs) to link the environmental signals to adaptive responses (Stock et al., 2000). Moreover, in addition to TCSs, stand-alone response regulators have been implicated in the global regulation of virulence gene expression. The term stand-alone has been used to define global transcriptional regulators that (i) are not associated to a membrane-bound sensor histidine kinase, (ii) their activity and/or intracellular concentration changes in response to specific external stimuli and (iii) their signal transduction components have yet to be fully defined (McIver, 2009). The Gram-positive (G+) bacterium Streptococcus pneumoniae, commonly called the pneumococcus, is a member of the normal human nasopharyngeal flora, where it exists asymptomatically as a commensal. However, when the immune system weakens, it can cause serious diseases such as sinusitis, conjunctivitis, otitis media, meningitis and bacteremia (Kadioglu et al., 2008; van der Poll and Opal, 2009). S. pneumoniae remains as a main cause of morbidity and mortality worldwide as a result of its increasing resistance to antibiotics. Recent data estimate that pneumococcal pneumonia kills annually around 1.2 million children younger than five years, more than AIDS, malaria and tuberculosis combined (www.who.int/mediacentre/factsheets/fs331/en/index.html). Understanding the molecular mechanisms that control the expression of pneumococcal virulence genes in response to environmental stimuli will offer new insights into the pathogenesis of this bacterium. Searching for homologies we found that the genome of the pneumococcal R6 strain (Hoskins et al., 2001), which derives from the D39 clinical isolate (serotype 2), encodes a protein (named MgaSpn by us), which is highly conserved in the pneumococcal strains whose genomes have been totally or partially sequenced. At present, MgaSpn is thought to be a member of the Mga/AtxA family of global response regulators, which includes the Mga and the AtxA virulence regulators encoded by the G+ pathogens S. pyogenes (the Group A Streptococcus, GAS) and Bacillus anthracis, respectively. MgaSpn shares 42.6% of similarity and 21.4% of identity with Mga and 39.9% of similarity and 20.7% of identity with AtxA. Regarding the Mga regulator, it controls the expression of approximately 10% of the GAS genome during the exponential growth phase (Ribardo and McIver, 2006). Mga activates directly the transcription of several virulence genes, which encode factors important for adherence and internalization into non-phagocytic cells, as well as factors that enable the bacterium to evade the host immune responses. Mga also activates the expression of its own gene (McIver et al., 1999; McIver, 2009). In vitro studies using a His-tagged Mga showed that it binds to regions located upstream of the target promoters with low sequence identity.(Full summary in attached document)[ES] Regulación global de la expresión de genes de virulencia en bacterias patógenas La patogenicidad de determinadas bacterias puede entenderse como una respuesta de adaptación rápida a los cambios producidos en las condiciones del entorno que las rodea. La habilidad para detectar y responder a esos cambios es lo que les conferirá cierta ventaja a la hora de colonizar nuevos nichos así como evadir el sistema inmune del organismo infectado. En el caso concreto de las bacterias patógenas, estas respuestas van unidas a cambios en la expresión de determinados genes que codifican factores implicados en virulencia. Normalmente, las bacterias utilizan los sistemas de transducción de señales de dos componentes (TCSs) para conectar los estímulos ambientales con una respuesta adaptativa concreta. Estos sistemas, que están implicados en diversos procesos celulares, se componen de dos proteínas, una histidina quinasa (HK) y un regulador de respuesta (RR). La HK es generalmente una proteína integral de membrana, encargada de captar y responder a un determinado estímulo modificando el estado de fosforilación del RR citosólico. Esta fosforilación provocará cambios conformacionales en el regulador el cual, ahora, podrá actuar como un factor transcripcional activando o reprimiendo la expresión de determinados genes (para una revisión ver Perry et al., 2011). Además de estos sistemas, varios reguladores de respuesta denominados stand-alone han sido implicados en la regulación global de la expresión de genes de virulencia. En general, el término stand-alone hace referencia a i) no están asociados a una HK unida a membrana, ii) su actividad y/o concentración intracelular varía en respuesta a estímulos externos y iii) los mecanismos implicados en la transducción de dichos estímulos no han sido totalmente definidos (McIver, 2009). Dentro de este grupo de reguladores se encuentra la proteína MgaSpn de Streptococcus pneumoniae, cuya caracterización molecular ha sido el objetivo principal de este trabajo. En este momento, y como consecuencia de nuestra investigación, se considera que MgaSpn es un miembro de la familia Mga/AtxA de reguladores de respuesta global, que incluye a los reguladores de virulencia Mga (S. pyogenes) y AtxA (Bacillus anthracis).(Ver resumen completo en documento adjunto)The work was supported by grants from the Spanish Ministry of Education and Science (BFU2006-08487 and fellowship FPI BES-2007-17086), the Community of Madrid/Spanish National Research Council (CCG08 CSIC/SAL-3694) and the Spanish Ministry of Science and Innovation (CSD2008-00013-INTERMODS, BFU2009-11868).Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis.(https://digital.csic.es/handle/10261/42052) Activator role of the pneumococcal Mga-Like virulence transcriptional regulator (https://digital.csic.es/handle/10261/60421) The pneumococcal MgaSpn virulence transcriptional regulator generates multimeric complexes on linear double-stranded DNA (https://digital.csic.es/handle/10261/95584)Peer reviewe

Activator role of the pneumococcal Mga-like virulence transcriptional regulator

11 páginas, 10 figuras, 1 tabla -- PAGS nros. 4197-4207Este artículo pertenece a la Tesis presentada por Virtudes Solano Collado con título: "Caracterización molecular del regulador transcripcional MgaSpn de Streptococcus pneumoniae"(https://digital.csic.es/handle/10261/98162)Global transcriptional regulators that respond to specific environmental signals are crucial in bacterial pathogenesis. In the case of the Gram-positive pathogen Streptococcus pneumoniae (the pneumococcus), the sp1800 gene of the clinical isolate TIGR4 encodes a protein that exhibits homology to the Mga “stand-alone” response regulator of the group A Streptococcus. Such a pneumococcal protein was shown to play a significant role in both nasopharyngeal colonization and development of pneumonia in murine infection models. Moreover, it was shown to repress the expression of several genes located within the rlrA pathogenicity islet. The pneumococcal R6 strain, which derives from the D39 clinical isolate, lacks the rlrA islet but has a gene (here named mgaSpn) equivalent to the sp1800 gene. In this work, and using in vivo approaches, we have identified the promoter of the mgaSpn gene (Pmga) and demonstrated that four neighboring open reading frames of unknown function (spr1623 to spr1626) constitute an operon. Transcription of this operon is under the control of two promoters (P1623A and P1623B) that are divergent from the Pmga promoter. Furthermore, we have shown that the MgaSpn protein activates the P1623B promoter in vivo. This activation requires sequences located around 50 to 120 nucleotides upstream of the P1623B transcription start site. By DNase I footprinting assays, we have also demonstrated that such a region includes an MgaSpn binding site. This is the first report on the activator role of the pneumococcal Mga-like proteinThis work was supported by grants CSD2008-00013-INTERMODS to M.E. and BFU2009-11868 to A.B. from the Spanish Ministry of Science and Innovation and grant PIE-201020E030 to A.B. from the Spanish National Research Council. V.S.-C was the recipient of a fellowship (BES-2007-17086) from the Spanish Ministry of Science and InnovationPeer reviewe