scholarly journals Role for the Src Family Kinase Fyn in Sphingolipid Acquisition by Chlamydiae

2011 ◽  
Vol 79 (11) ◽  
pp. 4559-4568 ◽  
Author(s):  
Jeffrey Mital ◽  
Ted Hackstadt
Keyword(s):  
Host Proteins ◽  
Content Type ◽  
Obligate Intracellular ◽  
Screening Protocol ◽  
Infected Cells ◽  
Protective Environment ◽  
Membrane Bound ◽  
Obligate Intracellular Pathogen ◽  
Interfering Rna ◽  
Chlamydial Inclusion

ABSTRACTThe bacterial obligate intracellular pathogenChlamydia trachomatisreplicates within a membrane-bound vacuole termed the inclusion. From within this protective environment, chlamydiae usurp numerous functions of the host cell to promote chlamydial survival and replication. Here we utilized a small interfering RNA (siRNA)-based screening protocol designed to identify host proteins involved in the trafficking of sphingomyelin to the chlamydial inclusion. Twenty-six host proteins whose deficiency significantly decreased sphingomyelin trafficking to the inclusion and 16 proteins whose deficiency significantly increased sphingomyelin trafficking to the inclusion were identified. The reduced sphingomyelin trafficking caused by downregulation of the Src family tyrosine kinase Fyn was confirmed in more-detailed analyses. Fyn silencing did not alter sphingomyelin synthesis or trafficking in the absence of chlamydial infection but reduced the amount of sphingomyelin trafficked to the inclusion in infected cells, as determined by two independent quantitative assays. Additionally, inhibition of Src family kinases resulted in increased cellular retention of sphingomyelin and significantly decreased incorporation into elementary bodies of bothC. trachomatisandChlamydophila caviae.

scholarly journals Hijacking and Use of Host Kinases by Chlamydiae

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1034
Author(s):  
Prakash Sah ◽  
Erika I. Lutter
Keyword(s):  
Host Cell ◽  
Intracellular Pathogen ◽  
Host Proteins ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Host Cell Apoptosis ◽  
Survival And Development ◽  
Causative Agents ◽  
Cell Processes ◽  
Obligate Intracellular Pathogen

Chlamydia species are causative agents of sexually transmitted infections, blinding trachoma, and animal infections with zoonotic potential. Being an obligate intracellular pathogen, Chlamydia relies on the host cell for its survival and development, subverting various host cell processes throughout the infection cycle. A key subset of host proteins utilized by Chlamydia include an assortment of host kinase signaling networks which are vital for many chlamydial processes including entry, nutrient acquisition, and suppression of host cell apoptosis. In this review, we summarize the recent advancements in our understanding of host kinase subversion by Chlamydia.

scholarly journals Active Escape of Orientia tsutsugamushi from Cellular Autophagy

2012 ◽  
Vol 81 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Youngho Ko ◽  
Ji-Hye Choi ◽  
Na-Young Ha ◽  
Ik-Sang Kim ◽  
Nam-Hyuk Cho ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Cellular Autophagy ◽  
A Cell ◽  
Infected Cells ◽  
Obligate Intracellular Pathogen

ABSTRACTOrientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show thatO. tsutsugamushiinfection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitatedO. tsutsugamushitargeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of theatg3gene does not significantly affect the intracellular growth ofO. tsutsugamushiin vitro. These results suggest thatO. tsutsugamushihas evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

scholarly journals Chlamydia interfere with an interaction between the mannose-6-phosphate receptor and sorting nexins to counteract host restriction

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Cherilyn A Elwell ◽  
Nadine Czudnochowski ◽  
John von Dollen ◽  
Jeffrey R Johnson ◽  
Rachel Nakagawa ◽  
...  
Keyword(s):  
Cell Biology ◽  
Host Bacterium ◽  
Sorting Nexins ◽  
Host Restriction ◽  
Sorting Nexin ◽  
Obligate Intracellular ◽  
Binding Surface ◽  
Membrane Bound ◽  
Mannose 6 Phosphate ◽  
Obligate Intracellular Pathogen

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.

scholarly journals Beclin 1 Is Required for Starvation-Enhanced, but Not Rapamycin-Enhanced, LC3-Associated Phagocytosis of Burkholderia pseudomallei in RAW 264.7 Cells

2012 ◽  
Vol 81 (1) ◽  
pp. 271-277 ◽  
Author(s):  
Xuelei Li ◽  
Mark Prescott ◽  
Ben Adler ◽  
John D. Boyce ◽  
Rodney J. Devenish
Keyword(s):  
Burkholderia Pseudomallei ◽  
Defense Mechanism ◽  
Beclin 1 ◽  
Raw 264.7 Cells ◽  
Early Event ◽  
Raw 264.7 ◽  
Content Type ◽  
Innate Defense ◽  
Infected Cells ◽  
Interfering Rna

LC3-associated phagocytosis (LAP) ofBurkholderia pseudomalleiby murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infectingB. pseudomallei. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP ofB. pseudomalleibut that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent.

scholarly journals Coxiella burnetiiSubverts p62/Sequestosome 1 and Activates Nrf2 Signaling in Human Macrophages

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Caylin G. Winchell ◽  
Amanda L. Dragan ◽  
Katelynn R. Brann ◽  
Frances I. Onyilagha ◽  
Richard C. Kurten ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Content Type ◽  
Sequestosome 1 ◽  
Type Iv ◽  
Host Signaling ◽  
Infected Cells ◽  
Pv Generation ◽  
Interfering Rna

ABSTRACTCoxiella burnetiiis the causative agent of human Q fever, a debilitating flu-like illness that can progress to chronic disease presenting as endocarditis. Following inhalation,C. burnetiiis phagocytosed by alveolar macrophages and generates a lysosome-like replication compartment termed the parasitophorous vacuole (PV). A type IV secretion system (T4SS) is required for PV generation and is one of the pathogen's few known virulence factors. We previously showed thatC. burnetiiactively recruits autophagosomes to the PV using the T4SS but does not alter macroautophagy. In the current study, we confirmed that the cargo receptor p62/sequestosome 1 (SQSTM-1) localizes near the PV in primary human alveolar macrophages infected with virulentC. burnetii. p62 and LC3 typically interact to select cargo for autophagy-mediated degradation, resulting in p62 degradation and LC3 recycling. However, inC. burnetii-infected macrophages, p62 was not degraded when cells were starved, suggesting that the pathogen stabilizes the protein. In addition, phosphorylated p62 levels increased, indicative of activation, during infection. Small interfering RNA experiments indicated that p62 is not absolutely required for intracellular growth, suggesting that the protein serves a signaling role during infection. Indeed, the Nrf2-Keap1 cytoprotective pathway was activated during infection, as evidenced by sustained maintenance of Nrf2 levels and translocation of the protein to the nucleus inC. burnetii-infected cells. Collectively, our studies identify a new p62-regulated host signaling pathway exploited byC. burnetiiduring intramacrophage growth.

scholarly journals EUKARYOTIC SNARE VAMP3 DYNAMICALLY INTERACTS WITH MULTIPLE CHLAMYDIAL INCLUSION MEMBRANE PROTEINS

2020 ◽  
Author(s):  
Duc-Cuong Bui ◽  
Lisa M. Jorgenson ◽  
Scot P. Ouellette ◽  
Elizabeth A. Rucks
Keyword(s):  
De Novo ◽  
Host Protein ◽  
Snare Proteins ◽  
Developmental Cycle ◽  
Inclusion Membrane ◽  
Transient Nature ◽  
Binding Partners ◽  
Infected Cells ◽  
Obligate Intracellular Pathogen ◽  
Chlamydial Inclusion

Chlamydia trachomatis, an obligate intracellular pathogen, undergoes a biphasic developmental cycle within a membrane-bound vacuole called the chlamydial inclusion. To facilitate interactions with the host cell, Chlamydia modifies the inclusion membrane with type III secreted proteins, called Incs. As with all chlamydial proteins, Incs are temporally expressed, modifying the chlamydial inclusion during the early- and mid-developmental cycle. VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion. Their recruitment requires de novo chlamydial protein synthesis during the mid-developmental cycle. Thus, we hypothesize that VAMPs 3 and 4 are recruited by Incs. In chlamydial infected cells, identifying Inc binding partners for SNARE proteins specifically has been elusive. To date, most studies examining chlamydial Inc-eukaryotic proteins have benefitted from stable interacting partners or a robust interaction at a specific time point post-infection. While these types of interactions are the predominant class that have been identified, they are likely the ‘exception’ to chlamydial-host interactions. Therefore, we applied two separate but complementary experimental systems to identify candidate chlamydial Inc binding partners for VAMPs. Based on these results, we created transformed strains of C. trachomatis serovar L2 to inducibly express a candidate Inc-FLAG protein. In chlamydial infected cells, we found that five Incs temporally and transiently interact with VAMP3. Further, loss of incA or ct813 expression altered VAMP3 localization to the inclusion. For the first time, our studies demonstrate the transient nature of certain host protein-Inc interactions that contribute to the chlamydial developmental cycle.

scholarly journals Pathogenic Puppetry: Manipulation of the Host Actin Cytoskeleton by Chlamydia trachomatis

2019 ◽  
Vol 21 (1) ◽  
pp. 90 ◽  
Author(s):  
Liam Caven ◽  
Rey A. Carabeo
Keyword(s):  
Chlamydia Trachomatis ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Host Cells ◽  
Surface Receptors ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Experimental Findings ◽  
Chlamydial Inclusion

The actin cytoskeleton is crucially important to maintenance of the cellular structure, cell motility, and endocytosis. Accordingly, bacterial pathogens often co-opt the actin-restructuring machinery of host cells to access or create a favorable environment for their own replication. The obligate intracellular organism Chlamydia trachomatis and related species exemplify this dynamic: by inducing actin polymerization at the site of pathogen-host attachment, Chlamydiae induce their own uptake by the typically non-phagocytic epithelium they infect. The interaction of chlamydial adhesins with host surface receptors has been implicated in this effect, as has the activity of the chlamydial effector TarP (translocated actin recruitment protein). Following invasion, C. trachomatis dynamically assembles and maintains an actin-rich cage around the pathogen’s membrane-bound replicative niche, known as the chlamydial inclusion. Through further induction of actin polymerization and modulation of the actin-crosslinking protein myosin II, C. trachomatis promotes egress from the host via extrusion of the inclusion. In this review, we present the experimental findings that can inform our understanding of actin-dependent chlamydial pathogenesis, discuss lingering questions, and identify potential avenues of future study.

scholarly journals Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

2016 ◽  
Vol 90 (7) ◽  
pp. 3611-3626 ◽  
Author(s):  
Nikolay Kovalev ◽  
Isabel Fernández de Castro Martín ◽  
Judit Pogany ◽  
Daniel Barajas ◽  
Kunj Pathak ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Electron Microscopic ◽  
Content Type ◽  
Endosomal Sorting ◽  
Infected Cells ◽  
Membrane Bound ◽  
Positive Strand Rna

ABSTRACTPlus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs).Tomato bushy stunt virus(TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae)vps23Δbro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions ofArabidopsishomologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression inNicotiana benthamianaleaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast andin vitro. Electron microscopic imaging ofvps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed inN. benthamianacells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a “measuring string” during VRC assembly and spherule formation.IMPORTANCEPlant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation usingin vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

scholarly journals The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication

2010 ◽  
Vol 84 (17) ◽  
pp. 8571-8583 ◽  
Author(s):  
Linghui Xu ◽  
Siti Khadijah ◽  
Shouguo Fang ◽  
Li Wang ◽  
Felicia P. L. Tay ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Rna Helicase ◽  
Host Proteins ◽  
Bait Protein ◽  
Infectious Bronchitis ◽  
Infected Cells ◽  
Interfering Rna ◽  
Discontinuous Transcription ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.

scholarly journals Gingimaps: Protein Localization in the Oral Pathogen Porphyromonas gingivalis

2020 ◽  
Vol 84 (1) ◽  
Author(s):  
Giorgio Gabarrini ◽  
Stefano Grasso ◽  
Arie Jan van Winkelhoff ◽  
Jan Maarten van Dijl
Keyword(s):  
Rheumatoid Arthritis ◽  
Porphyromonas Gingivalis ◽  
Protein Localization ◽  
Host Proteins ◽  
Comprehensive Overview ◽  
Oral Pathogen ◽  
Content Type ◽  
Autoimmune Antibodies ◽  
Citrullinated Proteins ◽  
Membrane Bound

SUMMARY Porphyromonas gingivalis is an oral pathogen involved in the widespread disease periodontitis. In recent years, however, this bacterium has been implicated in the etiology of another common disorder, the autoimmune disease rheumatoid arthritis. Periodontitis and rheumatoid arthritis were known to correlate for decades, but only recently a possible molecular connection underlying this association has been unveiled. P. gingivalis possesses an enzyme that citrullinates certain host proteins and, potentially, elicits autoimmune antibodies against such citrullinated proteins. These autoantibodies are highly specific for rheumatoid arthritis and have been purported both as a symptom and a potential cause of the disease. The citrullinating enzyme and other major virulence factors of P. gingivalis, including some that were implicated in the etiology of rheumatoid arthritis, are targeted to the host tissue as secreted or outer-membrane-bound proteins. These targeting events play pivotal roles in the interactions between the pathogen and its human host. Accordingly, the overall protein sorting and secretion events in P. gingivalis are of prime relevance for understanding its full disease-causing potential and for developing preventive and therapeutic approaches. The aim of this review is therefore to offer a comprehensive overview of the subcellular and extracellular localization of all proteins in three reference strains and four clinical isolates of P. gingivalis, as well as the mechanisms employed to reach these destinations.

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