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.

scholarly journals Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of Rickettsia typhi

2008 ◽  
Vol 190 (18) ◽  
pp. 6234-6242 ◽  
Author(s):  
Nicole C. Ammerman ◽  
M. Sayeedur Rahman ◽  
Abdu F. Azad
Keyword(s):  
Host Cell ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Mammalian Host ◽  
Signal Peptides ◽  
Cell Infection ◽  
Rickettsia Typhi ◽  
Sec Translocon

ABSTRACT As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

scholarly journals Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Xuesen Zhao ◽  
Mohit Sehgal ◽  
Zhifei Hou ◽  
Junjun Cheng ◽  
Sainan Shu ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Viral Entry ◽  
Molecular Mechanisms ◽  
Biological Activities ◽  
Viral Envelope ◽  
Host Factors ◽  
Host Cells ◽  
Restriction Factors ◽  
Structural Motifs ◽  
Infectious Entry

ABSTRACTInterferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCEThe differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

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 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 Reconstituting the Mammalian Apoptotic Switch in Yeast

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 145 ◽  
Author(s):  
Peter Polčic ◽  
Marek Mentel
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Model System ◽  
Yeast Cells ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Membranes ◽  
Antiapoptotic Proteins ◽  
The Family ◽  
Induction Of Apoptosis ◽  
Apoptotic Signal

Proteins of the Bcl-2 family regulate the permeabilization of the mitochondrial outer membrane that represents a crucial irreversible step in the process of induction of apoptosis in mammalian cells. The family consists of both proapoptotic proteins that facilitate the membrane permeabilization and antiapoptotic proteins that prevent it in the absence of an apoptotic signal. The molecular mechanisms, by which these proteins interact with each other and with the mitochondrial membranes, however, remain under dispute. Although yeast do not have apparent homologues of these apoptotic regulators, yeast cells expressing mammalian members of the Bcl-2 family have proved to be a valuable model system, in which action of these proteins can be effectively studied. This review focuses on modeling the activity of proapoptotic as well as antiapoptotic proteins of the Bcl-2 family in yeast.

scholarly journals The TIR-domain containing effectors BtpA and BtpB from Brucella abortus block energy metabolism

2019 ◽  
Author(s):  
Julia María Coronas-Serna ◽  
Arthur Louche ◽  
María Rodríguez-Escudero ◽  
Morgane Roussin ◽  
Paul R.C. Imbert ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Interleukin 1 ◽  
Effector Proteins ◽  
Host Cells ◽  
Yeast Cells ◽  
Tir Domain ◽  
Tir Domains

ABSTRACTBrucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

scholarly journals 1School of Life Science and Technology, ShanghaiTech University, Pudong, Shanghai 201210, China; [email protected] (X.Y.); [email protected] (Y.Q.)

2019 ◽  
Vol 20 (8) ◽  
pp. 2028 ◽  
Author(s):  
Yue ◽  
Qian ◽  
Gim ◽  
Lee
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Host Cells ◽  
Scaffolding Protein ◽  
Membrane Domain ◽  
Cellular Functions ◽  
Domain Organization ◽  
Bacterial Proliferation ◽  
Intracellular Organelles

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington’s disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

scholarly journals Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

2004 ◽  
Vol 15 (2) ◽  
pp. 637-648 ◽  
Author(s):  
Seema Dalal ◽  
Meredith F. N. Rosser ◽  
Douglas M. Cyr ◽  
Phyllis I. Hanson
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Atp Hydrolysis ◽  
Brefeldin A ◽  
Transmembrane Conductance Regulator ◽  
Organelle Biogenesis ◽  
Ubiquitinated Proteins ◽  
Organelle Assembly ◽  
Specific Assessment

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.

scholarly journals A Tractable Drosophila Cell System Enables Rapid Identification of Acinetobacter baumannii Host Factors

2020 ◽  
Vol 10 ◽  
Author(s):  
Qing-Ming Qin ◽  
Jianwu Pei ◽  
Gabriel Gomez ◽  
Allison Rice-Ficht ◽  
Thomas A. Ficht ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Rapid Identification ◽  
Cell System ◽  
Host Factors ◽  
Drosophila Cell

scholarly journals Burkholderia thailandensis as a Model System for the Study of the Virulence-Associated Type III Secretion System of Burkholderia pseudomallei

2008 ◽  
Vol 76 (11) ◽  
pp. 5402-5411 ◽  
Author(s):  
Andrea Haraga ◽  
T. Eoin West ◽  
Mitchell J. Brittnacher ◽  
Shawn J. Skerrett ◽  
Samuel I. Miller
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Burkholderia Pseudomallei ◽  
Clinical Symptoms ◽  
Type Iii Secretion System ◽  
Model System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii

ABSTRACT Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of clinical symptoms collectively known as melioidosis. Since it can be acquired by inhalation and is difficult to eradicate due to its resistance to a wide group of antibiotics and capacity for latency, work with B. pseudomallei requires a biosafety level 3 (BSL-3) containment facility. The bsa (Burkholderia secretion apparatus)-encoded type III secretion system (TTSS) has been shown to be required for its full virulence in a number of animal models. TTSSs are export devices found in a variety of gram-negative bacteria that translocate bacterial effector proteins across host cell membranes into the cytoplasm of host cells. Although the Bsa TTSS has been shown to play an important role in the ability of B. pseudomallei to survive and replicate in mammalian cells, escape from the endocytic vacuole, and spread from cell to cell, little is known about its effectors mediating these functions. Using bioinformatics, we identified homologs of several known TTSS effectors from other bacteria in the B. pseudomallei genome. In addition, we show that orthologs of these putative effectors exist in the genome of B. thailandensis, a closely related bacterium that is rarely pathogenic to humans. By generating a Bsa TTSS mutant B. thailandensis strain, we also demonstrated that the Bsa TTSS has similar functions in the two species. Therefore, we propose B. thailandensis as a useful BSL-1 model system to study the role of the Bsa TTSS during Burkholderia infection of mammalian cells and animals.

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