Genomics and bacterial pathogenesis.

Whole-genome sequencing is transforming the study of pathogenic bacteria. Searches for single virulence genes can now be performed on a genomewide scale by a variety of computer and genetic techniques. These techniques are discussed to provide a perspective on the developing field of genomics

Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targetsSpanish Ministerio de Economía, Industria y Competitividad , Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016‐75365‐REuropean Union’s Horizon 2020 e Marie Skłodowska‐Curie grant agreement No 84262

Chemical Reporters for Bacterial Pathogenesis and Beyond

Microbial infection involves complex and dynamic interplay of host defense and pathogen virulence pathways that requires new methods for mechanistic investigation. Towards this goal, I have developed chemical reporters of nucleic acid and protein synthesis and posttranslational modifications to explore their functions in bacterial pathogenesis as well as basic biological pathways. Initially, I developed amino acid chemical reporters that allow the cell-selective labeling of bacterial proteomes to facilitate the analysis of bacterial protein expression during infection of host cells. These reporters are based on unnatural amino acids that are incorporated into the bacterial proteome instead of methionine or phenylalanine when used in combination with mutant aminoacyl-tRNA-synthetases. This technology allows the visualization of bacterial protein synthesis during infection as well as the enrichment, identification and proteomic analysis of bacterial proteins from infected host cells. Additionally, I developed a chemical reporter for the study of protein AMPylation, a key posttranslational modification used by various bacterial pathogens to rewire host-signaling pathways. This reporter is used by all of the known AMPylation enzyme classes and allows the proteomic identification of enzyme-specific substrates in cell lysates. This project also stimulated the development of a chemical reporter for RNA synthesis, which in contrast to previous RNA reporters allows labeling of mRNA polyadenylation tails. In conclusion, I have developed chemical reporters for the cell selective labeling of bacteria in the presence of mammalian cells, a chemical reporter for protein AMPylation, and a chemical reporter for RNA synthesis and mRNA polyadenylation. These reagents should help to identify bacterial proteins that are differentially expressed during infection, new AMPylation substrates and the analysis of mRNA polyadenylation dynamics in basic cell biology and microbial pathogenesis studies in the future

Bacterial pathogenesis as an imperfect symbiosis

Els patogens bacterians adaptats als humans com ara Salmonella enterica serovar Typhi, Helicobacter pylori o Mycobacterium tuberculosis causen infeccions agudes i també infeccions latents i asimptomàtiques. Durant una infecció latent, el patogen atenua la seva virulència i adopta un estil de vida que redueix l'impacte de la infecció sobre el benestar de l'hoste. Estratègies evolutives d'aquest tipus poden dirigir alguns patògens bacterians vers el comensalisme.Human-adapted bacterial pathogens such as Salmonella enterica serovar Typhi, Helicobacter pylori, and Mycobacterium tuberculosis cause acute infections and also latent, asymptomatic infections. During latent infection the pathogen undergoes self-attenuation of virulence, a lifestyle that reduces the impact of infection on host fitness. Evolutionary strategies of this kind may drive certain bacterial pathogens towards commensalism

Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy

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Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis

Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.Fil: Marques Da Silva, Wanderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carvalho, Rodrigo D. De Oliveira. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Dorella, Fernanda A.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Federal da Paraíba. Centro de Biotecnologia; BrasilFil: Souza, Gustavo H. M. F.. Waters Corporation; BrasilFil: Pimenta, Adriano M. C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Silva, Artur. Universidade Federal do Pará; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi