scholarly journals Salmonella typhimurium Virulence Genes Are Induced upon Bacterial Invasion into Phagocytic and Nonphagocytic Cells

1999 ◽  
Vol 67 (11) ◽  
pp. 5690-5698 ◽  
Author(s):  
Cheryl G. Pfeifer ◽  
Sandra L. Marcus ◽  
Olivia Steele-Mortimer ◽  
Leigh A. Knodler ◽  
B. Brett Finlay
Keyword(s):  
Salmonella Typhimurium ◽  
Mammalian Cells ◽  
Early Time ◽  
Gene Cassette ◽  
Bacterial Virulence ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Specific Gene ◽  
Intracellular Bacteria ◽  
Survival And Growth

ABSTRACT Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identifySalmonella typhimurium genes that are induced insideSalmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes inS. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in thessaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene,pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA(induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.

scholarly journals A tractable Drosophila cell system enables rapid identification of Acinetobacter baumannii host factors

2020 ◽  
Author(s):  
Qing-Ming Qin ◽  
Jianwu Pei ◽  
Gabriel Gomez ◽  
Allison Rice-Ficht ◽  
Thomas A. Ficht ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Model System ◽  
Host Factors ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Organelle Biogenesis ◽  
Drosophila Cell

AbstractAcinetobacter baumannii is an important causative agent of nosocomial infections worldwide. The pathogen also readily acquires resistance to antibiotics, and pan-resistant strains have been reported. A. baumannii is widely regarded as an extracellular bacterial pathogen. However, accumulating evidence demonstrates that the pathogen can invade, survive or persist in infected mammalian cells. Unfortunately, the molecular mechanisms controlling these processes remain poorly understood. Here, we show that Drosophila S2 cells provide several attractive advantages as a model system for investigating the intracellular lifestyle of the pathogen, including susceptibility to bacterial intracellular replication and limited infection-induced host cell death. We also show that the Drosophila system can be used to rapidly identify host factors, including MAP kinase proteins, which confer susceptibility to intracellular parasitism. Finally, analysis of the Drosophila system suggested that host proteins that regulate organelle biogenesis and membrane trafficking contribute to regulating the intracellular lifestyle of the pathogen. Taken together, these findings establish a novel model system for elucidating interactions between A. baumannii and host cells, define new factors that regulate bacterial invasion or intracellular persistence, and identify subcellular compartments in host cells that interact with the pathogen.

High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion

2007 ◽  
Vol 292 (2) ◽  
pp. C945-C952 ◽  
Author(s):  
Benjamin E. Steinberg ◽  
Cameron C. Scott ◽  
Sergio Grinstein
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Small Interference Rna ◽  
Phagosomal Maturation ◽  
Large Populations ◽  
Intracellular Proliferation ◽  
Efficient Screening

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fcγ receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response.

scholarly journals Cell-Contact-Stimulated Formation of Filamentous Appendages by Salmonella typhimurium Does Not Depend on the Type III Secretion System Encoded by SalmonellaPathogenicity Island 1

1998 ◽  
Vol 66 (5) ◽  
pp. 2007-2017 ◽  
Author(s):  
Katharine A. Reed ◽  
M. Ann Clark ◽  
Trevor A. Booth ◽  
Christoph J. Hueck ◽  
Samuel I. Miller ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salmonella Typhimurium ◽  
Cell Types ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Cell Contact ◽  
Wild Type ◽  
Absolute Requirement ◽  
Appendage Formation ◽  
Rudimentary Form

ABSTRACT The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717–724, 1994). We have examined the roles of cell contact and Salmonellapathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer’s patch follicle-associated epithelia. Wild-type S. typhimuriumadhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA,invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, anS. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

scholarly journals Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

2003 ◽  
Vol 69 (1) ◽  
pp. 600-606 ◽  
Author(s):  
Hadi Abd ◽  
Thorsten Johansson ◽  
Igor Golovliov ◽  
Gunnar Sandström ◽  
Mats Forsman
Keyword(s):  
Francisella Tularensis ◽  
Fluorescent Protein ◽  
Acanthamoeba Castellanii ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Intracellular Location ◽  
Gfp Fluorescence ◽  
Environmental Reservoir ◽  
Survival And Growth ◽  
Green Fluorescent

ABSTRACT Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.

scholarly journals Identification of Listeria monocytogenes genes contributing to oxidative stress resistance under conditions relevant to host infection

2021 ◽  
Author(s):  
David R. Mains ◽  
Samuel J. Eallonardo ◽  
Nancy E. Freitag
Keyword(s):  
Oxidative Stress ◽  
Listeria Monocytogenes ◽  
Virulence Factors ◽  
Stress Resistance ◽  
Mammalian Cells ◽  
Bacterial Virulence ◽  
Host Cells ◽  
Physiological Adaptation ◽  
Wild Type ◽  
Oxidative Stress Resistance

The Gram-positive bacterium Listeria monocytogenes survives in environments ranging from the soil to the cytosol of infected host cells. Key to L. monocytogenes intracellular survival is the activation of PrfA, a transcriptional regulator that is required for the expression of multiple bacterial virulence factors. Mutations that constitutively activate prfA (prfA* mutations) result in high-level expression of multiple bacterial virulence factors as well as the physiological adaptation of L. monocytogenes for optimal replication within host cells. Here we demonstrate that L. monocytogenes prfA* mutants exhibit significantly enhanced resistance to oxidative stress in comparison to wild type strains. Transposon mutagenesis of L. monocytogenes prfA* strains resulted in the identification of three novel gene targets required for full oxidative stress resistance only in the context of PrfA activation. One gene, lmo0779, predicted to encode an uncharacterized protein, and two additional genes known as cbpA and ygbB, encoding a cyclic-di-AMP binding protein and a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase respectively, contribute to the enhanced oxidative stress resistance of prfA* strains while exhibiting no significant contribution in wild type L. monocytogenes. Transposon inactivation of cbpA and lmo0779 in a prfA* background led to reduced virulence in the liver of infected mice. These results indicate that L. monocytogenes calls upon specific bacterial factors for stress resistance in the context of PrfA activation and thus under conditions favorable for bacterial replication within infected mammalian cells.

Interaction between host cells and Salmonella Typhimurium isolates from septicemic pigs

2009 ◽  
Author(s):  
Nadia Bergeron ◽  
J. Corriveau ◽  
Ann Letellier ◽  
F. Daigle ◽  
L. Lessard ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Host Cells

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals An infection-induced RhoB-Beclin 1-Hsp90 complex enhances clearance of uropathogenic Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chunhui Miao ◽  
Mingyu Yu ◽  
Geng Pei ◽  
Zhenyi Ma ◽  
Lisong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Treatment Strategies ◽  
Uropathogenic Escherichia Coli ◽  
Beclin 1 ◽  
Host Cells ◽  
Infection Model ◽  
Intracellular Bacteria ◽  
Bladder Epithelium ◽  
Binding Residue

AbstractHost cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/− and RhoB−/− mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.

scholarly journals Set1 Targets Genes with Essential Identity and Tumor-Suppressing Functions in Planarian Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1182
Author(s):  
Prince Verma ◽  
Court K. M. Waterbury ◽  
Elizabeth M. Duncan
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mammalian Cells ◽  
Cell Function ◽  
Genotoxic Stress ◽  
Specific Gene ◽  
Cell Identity ◽  
Chromatin Signature ◽  
Lysine Methyltransferase

Tumor suppressor genes (TSGs) are essential for normal cellular function in multicellular organisms, but many TSGs and tumor-suppressing mechanisms remain unknown. Planarian flatworms exhibit particularly robust tumor suppression, yet the specific mechanisms underlying this trait remain unclear. Here, we analyze histone H3 lysine 4 trimethylation (H3K4me3) signal across the planarian genome to determine if the broad H3K4me3 chromatin signature that marks essential cell identity genes and TSGs in mammalian cells is conserved in this valuable model of in vivo stem cell function. We find that this signature is indeed conserved on the planarian genome and that the lysine methyltransferase Set1 is largely responsible for creating it at both cell identity and putative TSG loci. In addition, we show that depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal DNA damage response (DDR). Importantly, this work establishes that Set1 targets specific gene loci in planarian stem cells and marks them with a conserved chromatin signature. Moreover, our data strongly suggest that Set1 activity at these genes has important functional consequences both during normal homeostasis and in response to genotoxic stress.

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