scholarly journals Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Kumaraguruparan Ramasamy ◽  
Sowmya Balasubramanian ◽  
Krishnan Manickam ◽  
Lavanya Pandranki ◽  
Alexander B. Taylor ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Mycoplasma Pneumoniae ◽  
Retrograde Transport ◽  
Airway Disease ◽  
The United States ◽  
Community Acquired Pneumonia ◽  
Trauma Patients ◽  
Airway Injury ◽  
Cards Toxin

ABSTRACTMycoplasma pneumoniaeis an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization,M. pneumoniaeexpresses a unique ADP-ribosylating and vacuolating cytotoxin designatedcommunity-acquiredrespiratorydistresssyndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.IMPORTANCEMycoplasma pneumoniae, a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designatedcommunity-acquiredrespiratorydistresssyndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated withM. pneumoniaeinfection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminateM. pneumoniae-associated airway and extrapulmonary pathologies.

scholarly journals Mycoplasma pneumoniae CARDS toxin exploits host cell endosomal acidic pH and vacuolar ATPase proton pump to execute its biological activities

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kumaraguruparan Ramasamy ◽  
Sowmya Balasubramanian ◽  
Alejandra Kirkpatrick ◽  
Daniel Szabo ◽  
Lavanya Pandranki ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mycoplasma Pneumoniae ◽  
Mammalian Cells ◽  
Biological Activities ◽  
Electrical Potential ◽  
Retrograde Transport ◽  
The United States ◽  
Community Acquired Pneumonia ◽  
Amino Terminal ◽  
Cards Toxin

AbstractMycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalized children in the United States. It is also responsible for a spectrum of other respiratory tract disorders and extrapulmonary manifestations in children and adults. The main virulence factor of M. pneumoniae is a 591 amino acid multifunctional protein called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. The amino terminal region of CARDS toxin (N-CARDS) retains ADP-ribosylating activity and the carboxy region (C-CARDS) contains the receptor binding and vacuolating activities. After internalization, CARDS toxin is transported in a retrograde manner from endosome through the Golgi complex into the endoplasmic reticulum. However, the mechanisms and criteria by which internalized CARDS toxin is transported and activated to execute its cytotoxic effects remain unknown. In this study, we used full-length CARDS toxin and its mutant and truncated derivatives to analyze how pharmacological drugs that alter pH of intracellular vesicles and electrical potential across vesicular membranes affect translocation of CARDS toxin in mammalian cells. Our results indicate that an acidic environment is essential for CARDS toxin retrograde transport to endoplasmic reticulum. Moreover, retrograde transport facilitates toxin clipping and is required to induce vacuole formation. Additionally, toxin-mediated cell vacuolation is strictly dependent on the function of vacuolar type-ATPase.

scholarly journals Alternative Splicing of the HumanRab6AGene Generates Two Close but Functionally Different Isoforms

2000 ◽  
Vol 11 (11) ◽  
pp. 3819-3833 ◽  
Author(s):  
Arnaud Echard ◽  
Frank J.M. Opdam ◽  
Hubert J.P.C. de Leeuw ◽  
Florence Jollivet ◽  
Paul Savelkoul ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Alternative Splicing ◽  
Amino Acid ◽  
Retrograde Transport ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Binding Properties ◽  
Rab Protein ◽  
Gtp Binding ◽  
Functional Differences

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A′, which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A′ (Rab6A′ Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A′ Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A′ is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A′ interacts with two Rab6A partners, GAPCenA and “clone 1,” but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A′ are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.

scholarly journals Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity

Microbiology ◽  
2020 ◽  
Vol 166 (7) ◽  
pp. 629-640 ◽  
Author(s):  
Monica Feng ◽  
Andrew C. Schaff ◽  
Mitchell F. Balish
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Type Species ◽  
Cell Envelope ◽  
Bacterial Pathogen ◽  
Community Acquired Pneumonia ◽  
Atypical Cell ◽  
Content Type ◽  
Link Type ◽  
Cards Toxin

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H2S and H2O2, but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H2S and H2O2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

scholarly journals Molecular Detection and Characterization of Mycoplasma pneumoniae Among Patients Hospitalized With Community-Acquired Pneumonia in the United States

2015 ◽  
Vol 2 (3) ◽  
Author(s):  
Maureen H. Diaz ◽  
Alvaro J. Benitez ◽  
Kristen E. Cross ◽  
Lauri A. Hicks ◽  
Preeta Kutty ◽  
...  
Keyword(s):  
United States ◽  
Mycoplasma Pneumoniae ◽  
Variable Number Tandem Repeat ◽  
The United States ◽  
Variable Number ◽  
Community Acquired Pneumonia ◽  
Molecular Characteristics

Abstract Background.  Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP). The molecular characteristics of M pneumoniae detected in patients hospitalized with CAP in the United States are poorly described. Methods.  We performed molecular characterization of M pneumoniae in nasopharyngeal/oropharyngeal swabs from children and adults hospitalized with CAP in the Centers for Disease Control and Prevention Etiology of Pneumonia in the Community (EPIC) study, including P1 typing, multilocus variable-number tandem-repeat analysis (MLVA), and macrolide susceptibility genotyping. Results.  Of 216 M pneumoniae polymerase chain reaction-positive specimens, 40 (18.5%) were obtained from adults and 176 (81.5%) from children. P1 type distribution differed between adults (64% type 1 and 36% type 2) and children (84% type 1, 13% type 2, and 3% variant) (P < .05) and among sites (P < .01). Significant differences in the proportions of MLVA types 4/5/7/2 and 3/5/6/2 were also observed by age group (P < .01) and site (P < .01). A macrolide-resistant genotype was ide.jpegied in 7 (3.5%) specimens, 5 of which were from patients who had recently received macrolide therapy. No significant differences in clinical characteristics were ide.jpegied among patients with various strain types or between macrolide-resistant and -sensitive M pneumoniae infections. Conclusions.  The P1 type 1 genotype and MLVA type 4/5/7/2 predominated, but there were differences between children and adults and among sites. Macrolide resistance was rare. Differences in strain types did not appear to be associated with differences in clinical outcomes. Whole genome sequencing of M pneumoniae may help ide.jpegy better ways to characterize strains.

Racial and Sex Disparities in Trauma Outcomes Based on Geographical Region

2020 ◽  
pp. 000313482096006
Author(s):  
William Q. Duong ◽  
Areg Grigorian ◽  
Cyrus Farzaneh ◽  
Jeffry Nahmias ◽  
Theresa Chin ◽  
...  
Keyword(s):  
United States ◽  
The United States ◽  
Trauma Patients ◽  
Improvement Program ◽  
Trauma Outcomes ◽  
Risk Of Death ◽  
Hospital Access ◽  
Risk Of Mortality ◽  
Sex Disparities ◽  
Male Patients

Objectives Disparities in outcomes among trauma patients have been shown to be associated with race and sex. The purpose of this study was to analyze racial and sex mortality disparities in different regions of the United States, hypothesizing that the risk of mortality among black and Asian trauma patients, compared to white trauma patients, will be similar within all regions in the United States. Methods The Trauma Quality Improvement Program (2010-2016) was queried for adult trauma patients, separating by U.S. Census regions. Multivariable logistic regression analyses were performed for each region, controlling for known predictors of morbidity and mortality in trauma. Results Most trauma patients were treated in the South (n = 522 388, 40.7%). After risk adjustment, black trauma patients had a higher associated risk of death in all regions, except the Northeast, compared to white trauma patients. The highest associated risk of death for blacks (vs. whites) was in the Midwest (odds ratio [OR] 1.30, P < .001). Asian trauma patients only had a higher associated risk of death in the West (OR 1.39, P < .001). Male trauma patients, compared to women, had an increased associated risk of mortality in all four regions. Discussion This study found major differences in outcomes among different races within different regions of the United States. There was also both an increased rate and associated risk of mortality for male patients in all regions. Future prospective studies are needed to identify what regional differences in trauma systems including population density, transport times, hospital access, and other trauma resources explain these findings.

scholarly journals H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?

2005 ◽  
Vol 79 (17) ◽  
pp. 11412-11421 ◽  
Author(s):  
Chang-Won Lee ◽  
David E. Swayne ◽  
Jose A. Linares ◽  
Dennis A. Senne ◽  
David L. Suarez
Keyword(s):  
United States ◽  
Amino Acid ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Basic Amino Acid ◽  
The United States ◽  
Influenza Viruses ◽  
Single Point Mutation ◽  
Highly Pathogenic

ABSTRACT In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

COI gene geographic variation of Gypsy moth (Lepidoptera: Lymantriidae) and a TaqMan PCR diagnostic assay

DNA Barcodes ◽  
2014 ◽  
Vol 2 (1) ◽  
Author(s):  
Lu Qian ◽  
Yulin An ◽  
Junxian Song ◽  
Mei Xu ◽  
Jianlin Ye ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gypsy Moth ◽  
Diagnostic Tool ◽  
Genetic Difference ◽  
Science And Technology ◽  
The United States ◽  
Coi Gene ◽  
Taqman Assay ◽  
Base Substitution ◽  
Separate Branch

AbstractGypsy moth, an important forest/urban pest worldwide, is separated into the European and Asian subspecies, and has important quarantine significance. Diagnostic technique that can accurately and quickly distinguish subspecies is lacking. This study aimed to evaluate genetic difference between the subspecies, and subsequently to develop a reliable and high throughput molecular based diagnostic tool for distinguishing the subspecies. COI genes of 25 gypsy moth samples from China, Russia, Mongolia, Japan and the United States were sequenced. DNASTAR analysis revealed that gypsy moth COI gene was 1531bp long. The UPGM phylogenetic tree constructed based on the COI gene indicated that European subspecies (U.S. population) and Asian subspecies were distinctively divided into two branches. Japanese populations had a far distantly relationship with other Asian populations forming a separate branch. There was a single base substitution (base transformation only) at 14 consistent locations between Asian and American populations, but 13 of them coded the same amino acid. A MGB proper and TaqMan assay was designed base on the base substitution at 406th bp that coded a different amino acid. This allele typing assay took only 4 hours and could accurately distinguish gypsy moth subspecies of Europe and Asia. The study enriches the knowledge basis of genetic differentiation of gypsy moth subspecies. And more importantly the TaqMan assay is the first report of such diagnostic tool that could deliver rapid and accurate results and suitable for routine quarantine inspections to distinguish Asian and European gypsy moth subspecies. This study was supported by the Ministry of Science and Technology of the People’s Republic of China (Science and technology supporting project: 2012BAK11B03; International cooperation project: 2009DFA31950) and Jiangsu Entry and Exit Inspection and Quarantine Bureau (2014KJ45).

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