Pseudomonas aeruginosa is an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis. P. aeruginosa strain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources. Genomic analysis predicted a total of 5,682 genes, with 5,434 (95.63%) being identical across all 10 strains. Phenotypic analyses revealed comparable growth phenotypes in rich media and biofilm formation profiles. Limited differences were observed in antibiotic susceptibility profiles and immunostimulatory potential, measured using heat-killed whole-cell preparations in four immortalized cell lines followed by quantification of interleukin-6 (IL-6) and IL-1β secretion. However, variability was observed in the profiles of secreted molecular products, most notably, in rhamnolipid, pyoverdine, pyocyanin, Pseudomonas quinolone signal (PQS), extracellular DNA, exopolysaccharide, and outer membrane vesicle production. Many of the observed phenotypic differences did not correlate with subline-specific genetic changes, suggesting alterations in transcriptional and translational regulation. Taken together, these results suggest that individually maintained sublines of PAO1, even when acquired from the same parent subline, are continuously undergoing microevolution during culture and storage that results in alterations in phenotype, potentially affecting the outcomes of in vitro phenotypic analyses and in vivo pathogenesis studies. IMPORTANCE Laboratory-adapted strains of bacteria are used throughout the world for microbiology research. These prototype strains help keep research data consistent and comparable between laboratories. However, we have observed phenotypic variability when using different strains of Pseudomonas aeruginosa PAO1, one of the major laboratory-adopted research strains. Here, we describe the genomic and phenotypic differences among 10 PAO1 strains acquired from independent sources over 15 years to understand how individual maintenance affects strain characteristics. We observed limited genomic changes but variable phenotypic changes, which may have consequences for cross-comparison of data generated using different PAO1 strains. Our research highlights the importance of limiting practices that may promote the microevolution of model strains and calls for researchers to specify the strain origin to ensure reproducibility.
SARS-CoV-2 is a viral respiratory pathogen responsible for the current global pandemic and the disease that causes COVID-19. All current WHO approved COVID-19 vaccines are administered through the muscular route. We have developed a prototype two-dose vaccine (BReC-CoV-2) by combining the Receptor Binding Domain (RBD) antigen, via conjugation to Diphtheria toxoid (EcoCRM®). The vaccine is adjuvanted with Bacterial Enzymatic Combinatorial Chemistry (BECC), BECC470. Intranasal (IN) administration of BreC-CoV-2 in K18-hACE2 mice induced a strong systemic and localized immune response in the respiratory tissues which provided protection against the Washington strain of SARS-CoV-2. Protection provided after IN administration of BReC-CoV-2 was associated with decreased viral RNA copies in the lung, robust RBD IgA titers in the lung and nasal wash, and induction of broadly neutralizing antibodies in the serum. We also observed that BReC-CoV-2 vaccination administered using an intramuscular (IM) prime and IN boost protected mice from a lethal challenge dose of the Delta variant of SARS-CoV-2. IN administration of BReC-CoV-2 provided better protection than IM only administration to mice against lethal challenge dose of SARS-CoV-2. These data suggest that the IN route of vaccination induces localized immune responses that can better protect against SARS-CoV-2 than the IM route in the upper respiratory tract.
The SARS-CoV-2 pandemic is impacting the global population. This study was designed to assess the interplay of antibodies with the cytokine response in SARS-CoV-2 patients. We demonstrate that significant levels of anti-SARS-CoV-2 antibody to receptor binding domain (RBD), nucleocapsid, and spike S1 subunit of SARS-CoV-2 develop over the first 10 to 20 days of infection. The majority of patients produced antibodies against all three antigens (219/255 SARS-CoV-2+ patient specimens, 86%), suggesting a broad response to viral proteins. Antibody levels to SARS-CoV-2 antigens were different based on patient mortality, sex, blood type, and age. Analyses of these findings may help explain variation in immunity between these populations. To better understand the systemic immune response, we analyzed the levels of 20 cytokines by SARS-CoV-2 patients throughout infection. Cytokine analysis of SARS-CoV-2+ patients exhibited increases in proinflammatory markers (interleukin 6 [IL-6], IL-8, IL-18, and gamma interferon [IFN-γ]) and chemotactic markers (IP-10 and eotaxin) relative to healthy individuals. Patients who succumbed to infection produced decreased IL-2, IL-4, IL-12, RANTES, tumor necrosis factor alpha (TNF-α), GRO-α, and MIP-1α relative to patients who survived infection. We also observed that the chemokine CXCL13 was particularly elevated in patients who succumbed to infection. CXCL13 is involved in B cell activation, germinal center development, and antibody maturation, and we observed that CXCL13 levels in blood trended with anti-SARS-CoV-2 antibody levels. Furthermore, patients who succumbed to infection produced high CXCL13 and had a higher ratio of nucleocapsid to RBD antibodies. This study provides insights into SARS-CoV-2 immunity implicating the magnitude and specificity of response in relation to patient outcomes. IMPORTANCE The SARS-CoV-2 pandemic is continuing to impact the global population, and knowledge of the immune response to COVID-19 is still developing. This study assesses the interplay of different parts of the immune system during COVID-19 disease. We demonstrate that COVID-19 patients produce antibodies to three proteins of the COVID-19 virus (SARS-CoV-2) and identify many other immunological proteins that are involved during infection. The data suggest that one of these proteins (CXCL13) may be a novel biomarker for severe COVID-19 that can be readily measured in blood. This information combined with our broad-scale analysis of immune activity during COVID-19 provides new information on the immunological response throughout the course of disease and identifies a novel potential marker for assessing disease severity.
Delivery of cargo to target cells is fundamental to bacterial competitiveness. One important but poorly understood system, ubiquitous among Gram-negative organisms, involves packaging cargo into outer membrane vesicles (OMVs). These biological nanoparticles are involved in processes ranging from toxin delivery to cell-cell communication. Despite this, we know comparatively little about how OMVs are formed. Building upon the discovery that the Pseudomonas Quinolone Signal (PQS) stimulates OMV biogenesis in Pseudomonas aeruginosa, we proposed a model where PQS interacts with the outer membrane to induce curvature and ultimately OMV formation. Though this model is well supported in P. aeruginosa, it remained unclear whether other organisms produce similar compounds. Here we describe the development of a tightly controlled experimental system to test the interaction of bacterially-produced factors with target cells. Using this system, we show that multiple species respond to PQS by increasing OMV formation, that PQS accumulates in the induced vesicles, and that other bacteria secrete OMV-promoting factors. Analysis of induced vesicles indicates that recipient-mediated mechanisms exist to control vesicle size and that relatedness to the producer organism can dictate susceptibility to OMV-inducing compounds. This work provides evidence that small molecule induced OMV biogenesis is a widely conserved process and that cross-talk between systems may influence OMV production in neighboring bacteria.
The C. elegans nervous system mediates protective physiological and behavioral responses amid infection. However, it remains largely unknown how the nervous system responds to reactive oxygen species (ROS) activated by pathogenic microbes during infection. Here, we show superoxide dismutase-1 (SOD-1), an enzyme that converts superoxide into less toxic hydrogen peroxide and oxygen, functions in the gustatory neuron ASER to mediate C. elegans pathogen avoidance response. When C. elegans first encounters pathogenic bacteria P. aeruginosa, SOD-1 is induced in the ASER neuron. After prolonged P. aeruginosa exposure, ASER-specific SOD-1 expression is diminished. In turn, C. elegans starts to vacate the pathogenic bacteria lawn. Genetic knockdown experiments reveal that pathogen-induced ROS activate sod-1 dependent behavioral response non cell-autonomously. We postulate that the delayed aversive response to detrimental microbes may provide survival benefits by allowing C. elegans to temporarily utilize food that is tainted with pathogens as an additional energy source. Our data offer a mechanistic insight into how the nervous system mediates food-seeking behavior amid oxidative stress and suggest that the internal state of redox homeostasis could underlie the behavioral response to harmful microbial species.
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