Targeting HuR-Vav3 mRNA interaction prevents Pseudomonas aeruginosa adhesion to the cystic fibrosis airway epithelium

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Abstract

Cystic fibrosis (CF) is characterized by chronic bacterial infections leading to progressive bronchiectasis and respiratory failure. Pseudomonas aeruginosa (Pa) is the predominant opportunistic pathogen infecting the CF airways. The guanine nucleotide exchange factor Vav3 plays a critical role in Pa adhesion to the CF airways by inducing luminal fibronectin deposition that favors bacteria trapping. Here we report that Vav3 overexpression in CF is caused by upregulation of the mRNA-stabilizing protein HuR. We found that HuR accumulates in the cytoplasm of CF airway epithelial cells, binds to and stabilizes Vav3 mRNA. Interestingly, disruption of HuR-Vav3 mRNA interaction improved the CF epithelial integrity, inhibited the formation of the fibronectin-made bacterial docking platforms and prevented Pa adhesion to the CF airway epithelium. These findings indicate that targeting HuR represents a promising anti-adhesive approach in CF to prevent initial stages of Pa infection in a context of emergence of multidrug resistant pathogens.

Authors

Mehdi Badaoui, Cyril Sobolewski, Alexandre Luscher, Marc Bacchetta, Thilo Köhler, Christian van Delden, Michelangelo Foti, Marc Chanson

Escherichia coli catheter-associated urinary tract infections are associated with distinctive virulence and biofilm gene determinants

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Abstract

Urinary catheterization facilitates urinary tract colonization by Escherichia coli and increases infection risk. Here we aimed to identify strain-specific characteristics associated with the transition from colonization to infection in catheterized patients. In a single-site study population, we compared E. coli isolates from patients with catheter-associated asymptomatic bacteriuria (CAASB) to those with catheter-associated urinary tract infection (CAUTI). CAUTI isolates were dominated by a phylotype B2 subclade containing the multidrug resistant ST131 lineage relative to CAASB isolates, which were phylogenetically more diverse. A distinctive combination of virulence-associated genes was present in the CAUTI-associated B2 subclade. Catheter-associated biofilm formation was widespread among isolates and did not distinguish CAUTI from CAASB strains. Preincubation with CAASB strains could potently inhibit catheter colonization by multiple ST131 CAUTI isolates. Comparative genomic analysis identified a group of variable genes associated with high catheter-biofilm formation present in both CAUTI and CAASB strains. Among these, ferric citrate transport (Fec) system genes were experimentally associated with enhanced catheter biofilm formation using reporter and fecA deletion strains. Together, these results are consistent with a variable role for catheter biofilm formation in promoting CAUTI by ST131-like strains or resisting CAUTI by lower risk strains that engage in niche exclusion.

Authors

Zongsen Zou, Robert F. Potter, William H. McCoy 4th, John A. Wildenthal, George L. Katumba, Peter J. Mucha, Gautam Dantas, Jeffrey P. Henderson

Pseudomonas aeruginosa utilises the host‐derived polyamine spermidine to facilitate antimicrobial tolerance

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Abstract

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.

Authors

Chowdhury M. Hasan, Sian Pottenger, Angharad E. Green, Adrienne A. Cox, Jack S. White, Trevor Jones, Craig Winstanley, Aras Kadioglu, Megan H. Wright, Daniel R. Neill, Joanne L. Fothergill

Immunogenetics associated with severe coccidioidomycosis

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Abstract

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, <1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled DCM patients and performed whole-exome sequencing. In an exploratory set of 67 DCM patients, two had haploinsufficient STAT3 mutations, while defects in β-glucan sensing and response were seen in 34/67 (50.7%) cases. Damaging CLEC7A (n=14) and PLCG2 (n=11) variants were associated with impaired production of β-glucan-stimulated TNF-α from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Authors

Amy P. Hsu, Agnieszka Korzeniowska, Cynthia C. Aguilar, Jingwen Gu, Eric Karlins, Andrew J. Oler, Gang Chen, Glennys V. Reynoso, Joie Davis, Alexandria Chaput, Tao Peng, Ling Sun, Justin B. Lack, Derek J. Bays, Ethan R. Stewart, Sarah E. Waldman, Daniel A. Powell, Fariba M. Donovan, Jigar V. Desai, Nima Pouladi, Debra A. Long Priel, Daisuke Yamanaka, Sergio D. Rosenzweig, Julie E. Niemela, Jennifer Stoddard, Alexandra F. Freeman, Christa S. Zerbe, Douglas B. Kuhns, Yves A. Lussier, Kenneth N. Olivier, Richard C. Boucher, Heather D. Hickman, Jeffrey Frelinger, Joshua Fierer, Lisa F. Shubitz, Thomas L. Leto, George R. Thompson III, John N. Galgiani, Michail S. Lionakis, Steven M. Holland

Hyperglycemia exacerbates dengue virus infection by facilitating poly(A)-binding protein-mediated viral translation

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Abstract

Diabetes mellitus (DM) is highly comorbid with severe dengue diseases; however, the underlying mechanisms are unclear. DM patients display a 1.61-fold increased risk of developing dengue hemorrhagic fever. In search of host factors involved in DENV infection, this study utilizes high glucose (HG) treatment and shows that HG increases viral protein expression and virion release but has no effects on the early stages of viral infection. Following HG stimulation, DEN-Luc-transfected assay and cellular replicon-based assay display increased viral translation, while using the glucose uptake inhibitor phloretin blocks this effect. HG treatment increases the translational factor poly(A)-binding protein (PABP) in a glucose transporter-associated PI3K/AKT-regulated manner. Silencing PABP significantly decreases HG-prompted virion production. HG enhances the formation of the PABP-eIF4G complex, which is regulated by protein-disulfide isomerase. Hyperglycemia increases PABP expression, mortality, viral protein expression, and viral loads in streptozotocin-induced DM mice. Overall, hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation.

Authors

Ting-Jing Shen, Chia-Ling Chen, Tsung-Ting Tsai, Ming-Kai Jhan, Chyi-Huey Bai, Yu-Chun Yen, Ching-Wen Tsai, Cheng-Yi Lee, Po-Chun Tseng, Chia-Yi Yu, Chiou-Feng Lin

CLEC5A is critical in Pseudomonas aeruginosa-induced acute lung injury

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Abstract

Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with lipopolysaccharides of P. aeruginosa, and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa-induced pneumonia.

Authors

Pei-Shan Sung, Yu-Chun Peng, Shao-Ping Yang, Cheng-Hsun Chiu, Shie-Liang Hsieh

SARS-CoV-2 reinfections during the Delta and Omicron waves

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Abstract

BACKGROUND. Increased reinfection rates with SARS-CoV-2 have recently been reported, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. In this study, we used the Johns Hopkins SARS-CoV-2 genomic surveillance data to evaluate the frequency of sequencing validated, confirmed and inferred reinfections between March 2020 and July 2022. METHODS. Patients who had two or more positive SARS-CoV-2 tests in our system with samples sequenced as a part of our surveillance efforts were identified as the cohort for our study. SARS-CoV-2 genomes of patients’ initial and later samples were compared. RESULTS. A total of 755 patients (920 samples) had a positive test at least 90 days after the initial test with a median time between tests of 377 days. Sequencing was attempted on 231 samples and was successful in 127. Successful sequencing spiked during the Omicron surge and showed higher median days from initial infection compared to failed sequences. A total of 122 (98%) patients showed evidence of reinfection, 45 of which had sequence validated reinfection and 77 had inferred reinfections (later sequence showed a clade that was not circulating when the patient was initially infected). Of 45 sequence validated reinfections, 43 (96%) were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%) were vaccinated prior to the second infection, 6 (13%) were Immunosuppressed, and only 2 (4%) were hospitalized. CONCLUSIONS. Sequence validated reinfections increased with the Omicron variant but were generally associated with mild infections.

Authors

C. Paul Morris, Raghda E. Eldesouki, Amary Fall, David C. Gaston, Julie M. Norton, Nicholas D. Gallagher, Chun Huai Luo, Omar Abdullah, Eili Y. Klein, Heba H. Mostafa

Germline IgM predicts T-cell immunity to Pneumocystis

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Abstract

Pneumocystis is the most common fungal pulmonary infection in children under 5. In children with primary immunodeficiency, Pneumocystis often presents at 3-6 months that coincides with the nadir of maternal IgG and where IgM is the dominant immunoglobulin isotype. Since B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal specific IgMs in human and mice and that these IgM specificities would predict T cell antigens. We detected fungal specific IgMs in human and mouse serum and utilized immunoprecipitation to determine if any antigens were similar across donors. We then assessed T cell responses to these antigens. We found anti-Pneumocystis IgM in wild-type mice as well as Aicda-/- mice and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide specific lung T cell responses after P. murina infection. After mice were immunized with two of the antigens, adoptive transfer of vaccine elicited CD4+ T cells showed effector activity suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline encoded IgM B-cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.

Authors

Kristin Noell, Guixiang Dai, Dora Pungan, Anna Ebacher, Janet E. McCombs, Samuel J. Landry, Jay K. Kolls

Ebola virus protein VP40 stimulates IL-12- and IL-18-dependent activation of human natural killer cells

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Abstract

Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein (GP) and matrix protein VP40 provides rapid, NK cell-mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells (PBMCs) with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3-CD56+ NK cells, along with concomitant increase in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14- cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12 and IL-18 dependent manner that involves CD14+ and CD14- accessory cells. These novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.

Authors

Hung Le, Paul Spearman, Stephen N. Waggoner, Karnail Singh

Multicenter analysis of neutrophil extracellular trap dysregulation in adult and pediatric COVID-19

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Abstract

Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLL), otherwise known as “COVID toes”, remains unclear. Studying multinational cohorts, we found that, in CLL, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs post-disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19-affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased levels of NETs when compared to other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.

Authors

Carmelo Carmona-Rivera, Yu Zhang, Kerry Dobbs, Tovah E. Markowitz, Clifton L. Dalgard, Andrew J. Oler, Dillon R. Claybaugh, Deborah Draper, Meng Truong, Ottavia M. Delmonte, Francesco Licciardi, Ugo Ramenghi, Nicoletta Crescenzio, Luisa Imberti, Alessandra Sottini, Virginia Quaresima, Chiara Fiorini, Valentina Discepolo, Andrea Lo Vecchio, Alfredo Guarino, Luca Pierri, Andrea Catzola, Andrea Biondi, Paolo Bonfanti, Maria Cecilia Poli Harlowe, Yazmin Espinosa, Camila A. Astudillo, Emma Rey-Jurado, Cecilia Vial, Javiera De la Cruz, Ricardo Gonzalez, Cecilia Pinera, Jacqueline W. Mays, Ashley Ng, Andrew Platt, Beth A. Drolet, John Moon, Edward W. Cowen, Heather Kenney, Sarah E. Weber, Riccardo Castagnoli, Mary K. Magliocco, Michael Austin Stack, Gina A. Montealegre Sanchez, Karyl Barron, Danielle L. Fink, Douglas B. Kuhns, Stephen M. Hewitt, Lisa M. Arkin, Daniel S. Chertow, Helen C. Su, Luigi D. Notarangelo, Mariana J. Kaplan