Triggering Toll-Like Receptor 5 signaling during pneumococcal superinfection prevents the selection of antibiotic resistance.

Toll-like receptor 5 (TLR5) signaling plays a key role in antibacterial defenses. We previously showed that respiratory administration of flagellin, a potent TLR5 agonist, in combination with amoxicillin improves the treatment of primary pneumonia or superinfection caused by amoxicillin-sensitive or -resistant Streptococcus pneumoniae. Here, the impact of adjunct flagellin therapy on antibiotic dose/regimen and the selection of antibiotic-resistant S. pneumoniae was investigated using superinfection with isogenic antibiotic-sensitive and -resistant bacteria and population dynamics analysis. Our findings demonstrate that flagellin allows for a 200-fold reduction in the antibiotic dose, achieving the same therapeutic effect observed with antibiotic alone. Adjunct treatment also reduced the selection of antibiotic-resistant bacteria in contrast to the antibiotic monotherapy. Finally, we developed a mathematical model that captured the population dynamics and estimated a 20-fold enhancement immune-modulatory factor on bacterial clearance. This work paves the way for the development of host-directed therapy and refinement of treatment by modeling.

Preventive nasal administration of flagellin restores antimicrobial effect of gentamicin and protects against a multidrug-resistant strain of Pseudomonas aeruginosa.

The increasing prevalence of multidrug-resistant Pseudomonas aeruginosa (PA) is a significant concern for chronic respiratory disease exacerbations. Host-directed drugs, such as flagellin, an agonist of toll-like receptor 5 (TLR5), have emerged as a promising solution. In this study, we evaluated the prophylactic intranasal administration of flagellin against a multidrug-resistant strain of PA (PAMDR) in mice and assessed the possible synergy with the antibiotic gentamicin (GNT). The results indicated that flagellin treatment before infection decreased bacterial load in the lungs, likely due to an increase in neutrophil recruitment, and reduced signs of inflammation, including proinflammatory cytokines. The combination of flagellin and GNT showed a synergistic effect, decreasing even more the bacterial load and increasing mice survival rates, in comparison to mice pre-treated only with flagellin. These findings suggest that preventive nasal administration of flagellin could restore the effect of GNT against MDR strains of PA, paving the way for the use of flagellin in vulnerable patients with chronic respiratory diseases.

A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection.

Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved lafB gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against S. pneumoniae serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection.

Amoxicillin treatment of pneumococcal pneumonia impacts bone marrow neutrophil maturation and function.

Pneumonia caused by Streptococcus pneumoniae is a leading cause of death worldwide. A growing body of evidence indicates that the successful treatment of bacterial infections results from synergy between antibiotic-mediated direct antibacterial activity and the host's immune defenses. However, the mechanisms underlying the protective immune responses induced by amoxicillin, a ß-lactam antibiotic used as the first-line treatment of S. pneumoniae infections, have not been characterized. A better understanding of amoxicillin's effects on host-pathogen interactions might facilitate the development of other treatment options. Given the crucial role of neutrophils in the control of S. pneumoniae infections, we decided to investigate amoxicillin's impact on neutrophil development in a mouse model of pneumococcal superinfection. A single therapeutic dose of amoxicillin almost completely eradicated the bacteria and prevented local and systemic inflammatory responses. Interestingly, in this context, amoxicillin treatment did not impair the emergency granulopoiesis triggered in the bone marrow by S. pneumoniae. Importantly, treatment of pneumonia with amoxicillin was associated with a greater mature neutrophil count in the bone marrow; these neutrophils had specific transcriptomic and proteomic profiles. Furthermore, amoxicillin-conditioned, mature neutrophils in the bone marrow had a less activated phenotype and might be rapidly mobilized in peripheral tissues in response to systemic inflammation. Thus, by revealing a novel effect of amoxicillin on the development and functions of bone marrow neutrophils during S. pneumoniae pneumonia, our findings provide new insights into the impact of amoxicillin treatment on host immune responses.

Treatment of Bacterial Infections with ß-Lactams: Cooperation with Innate Immunity.

ß-Lactams are the most widely prescribed antibiotics used for the control and treatment of bacterial infections. The direct effect of ß-lactams on bacteria is well studied worldwide. In the context of infections and as a consequence of their direct activity against the pathogen, ß-lactams also regulate antibacterial immune responses. This knowledge has led to the theorem that the effectiveness of ß-lactam treatment results from the synergy between the drug and the immune response. Key players in this immune response, with an essential role in the clearance of live and dead bacteria, are the myeloid cells. In this review, we summarize the data that shed light on how ß-lactams interact with myeloid cells during bacterial infection treatment.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Immunostimulatory Effect of Flagellin on MDR-Klebsiella-Infected Human Airway Epithelial Cells.

Pneumonia caused by multi-drug-resistant Klebsiella pneumoniae (MDR-Kpneu) poses a major public health threat, especially to immunocompromised or hospitalized patients. This study aimed to determine the immunostimulatory effect of the Toll-like receptor 5 ligand flagellin on primary human lung epithelial cells during infection with MDR-Kpneu. Human bronchial epithelial (HBE) cells, grown on an air-liquid interface, were inoculated with MDR-Kpneu on the apical side and treated during ongoing infection with antibiotics (meropenem) and/or flagellin on the basolateral and apical side, respectively; the antimicrobial and inflammatory effects of flagellin were determined in the presence or absence of meropenem. In the absence of meropenem, flagellin treatment of MDR-Kpneu-infected HBE cells increased the expression of antibacterial defense genes and the secretion of chemokines; moreover, supernatants of flagellin-exposed HBE cells activated blood neutrophils and monocytes. However, in the presence of meropenem, flagellin did not augment these responses compared to meropenem alone. Flagellin did not impact the outgrowth of MDR-Kpneu. Flagellin enhances antimicrobial gene expression and chemokine release by the MDR-Kpneu-infected primary human bronchial epithelium, which is associated with the release of mediators that activate neutrophils and monocytes. Topical flagellin therapy may have potential to boost immune responses in the lung during pneumonia.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the ß-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by a reduced availability of cell wall precursors at the site of cell wall synthesis due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier that is responsible for transporting these precursors across the cell membrane. The data suggest that, whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

Streptococcus pneumoniae is a bacterium that can cause pneumonia, meningitis and other life-threatening illnesses in humans. Currently, many S. pneumoniae infections are treated with the antibiotic amoxicillin, which kills the bacteria by weakening a structure known as the cell wall that surrounds each bacterium. However, more and more S. pneumoniae cells are becoming resistant to amoxicillin, making it harder to treat such infections. We need new ways to effectively treat S. pneumoniae infections in humans. One potential strategy would be to combine amoxicillin with another drug that boosts the activity of amoxicillin so that it is able to kill the resistant bacteria. Two drugs that both target the same process in cells are more likely to boost each other's activity. Therefore, Dewachter et al. decided to search for another drug that also weakens the cell wall of S. pneumoniae. The team first developed a new screening approach called sCRilecs-seq to silence individual genes in single S. pneumoniae cells. By looking at many cells that each had a different gene that was no longer active, the team were able to identify several genes that when silenced resulted in the cells becoming longer than normal cells (a sign the bacteria may have weak cell walls). Further experiments revealed that the cell walls of these bacteria were weaker than normal cells due to a shortage in a cell wall building material known as undecaprenyl phosphate. Dewachter et al. then demonstrated that combining an existing drug known as clomiphene ­ which is known to inhibit undecaprenyl phosphate production and is currently used to treat infertility in humans ­ together with amoxicillin is able to effectively kill S. pneumoniae that are resistant to amoxicillin alone. Clomiphene also boosted the activity of amoxicillin against S. pneumoniae that remain sensitive to the antibiotic. Before this new drug combination may be used to help treat S. pneumoniae infections in human patients, further experiments will be needed to find out the optimum dose of clomiphene to use with amoxicillin. In the future, the new screening approach developed by Dewachter et al. may also prove useful to other researchers studying a wide range of biological questions.

Live attenuated Bordetella pertussis vaccine candidate BPZE1 transiently protects against lethal pneumococcal disease in mice.

BPZE1 is a live attenuated vaccine against infection by Bordetella pertussis, the causative agent of whooping cough. It was previously shown that BPZE1 provides heterologous protection in mouse models of disease caused by unrelated pathogens, such as influenza virus and respiratory syncytial virus. Protection was also observed in mouse models of asthma and contact dermatitis. In this study, we demonstrate that BPZE1 also displays protection against an unrelated bacterial pathogen in a mouse model of invasive pneumococcal disease mediated by Streptococcus pneumoniae. While a single administration of BPZE1 provided no protection, two doses of 106 colony-forming units of BPZE1 given in a three-week interval protected against mortality, lung colonization and dissemination in both BALB/c and C57BL/6 mice. Unlike for the previously reported influenza challenge model, protection was short-lived, and waned within days after booster vaccination. Formaldehyde-killed BPZE1 protected only when administered following a live prime, indicating that priming requires live BPZE1 for protection. Protection against mortality was directly linked to substantially decreased bacterial dissemination in the blood and was lost in MyD88 knock-out mice, demonstrating the role of the innate immune system in the mechanism of protection. This is the first report on a heterologous protective effect of the live BPZE1 vaccine candidate against an unrelated bacterial infection.

TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium.

Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.

The Biosynthetic Monophosphoryl Lipid A Enhances the Therapeutic Outcome of Antibiotic Therapy in Pneumococcal Pneumonia.

Alternative treatment strategies against bacterial infections are required to decrease the use of antibiotics. This study tested the hypothesis that stimulation of the innate immune receptor Toll-like receptor 4 can be combined with antibiotics to improve the treatment of invasive pneumonia. The efficacy of the biosynthetic monophosphoryl lipid A (MPLA), a clinically approved Toll-like receptor 4 activator, was tested in a mouse model of Streptococcus pneumoniae respiratory infection. Interestingly, administration of amoxicillin or MPLA decreased 400- to 11 000-fold the bacterial load in the lungs and spleen but did not enhance survival compared to mock treatment. The single administration of a combination of MPLA and amoxicillin further reduced 10- to 18-fold the bacterial colonization and invasion and significantly improved protection against lethal disease. The combined administration of MPLA and amoxicillin in a context of infection was associated with transient increase of the serum concentrations of amoxicillin and pro-inflammatory cytokines and chemokines as well as the expression of immune genes in lung tissue. Remarkably, the systemic and lung immune activation extended beyond amoxicillin elimination, suggesting a two-step and cooperative anti-infective effect, i.e., rapid antibiotic-mediated alteration of bacteria and a long-lasting impact through mucosal and systemic immunity. Our proof-of-concept study demonstrated for the first time that boosting Toll-like receptor 4 signaling can synergize with antibiotics in order to increase the efficacy of therapy of bacterial pneumonia, thereby in fine reducing the dose or regimen of antibiotics.

Airway Administration of Flagellin Regulates the Inflammatory Response to Pseudomonas aeruginosa.

Excessive lung inflammation and airway epithelial damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. TLR5 (Toll-like receptor 5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of the innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pretreatment reduced the NF-κB nuclear translocation and the expression of proinflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pigz model. These results provide evidence that flagellin treatment contributes to a better regulation of the inflammatory response in inflammatory lung diseases such as CF.

Chronic Pseudomonas aeruginosa Lung Infection Is IL-1R Independent, but Relies on MyD88 Signaling.

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1-deficient mice control chronic P. aeruginosa RP73 infection and IL-1ß Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

A Model-Based Pharmacokinetic/Pharmacodynamic Analysis of the Combination of Amoxicillin and Monophosphoryl Lipid A Against S. pneumoniae in Mice.

Combining amoxicillin with the immunostimulatory toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) represents an innovative approach for enhancing antibacterial treatment success. Exploiting pharmacokinetic and pharmacodynamic data from an infection model of Streptococcus pneumoniae infected mice, we aimed to evaluate the preclinical exposure-response relationship of amoxicillin with MPLA coadministration and establish a link to survival. Antibiotic serum concentrations, bacterial numbers in lung and spleen and survival data of mice being untreated or treated with amoxicillin (four dose levels), MPLA, or their combination were analyzed by nonlinear mixed-effects modelling and time-to-event analysis using NONMEM® to characterize these treatment regimens. On top of a pharmacokinetic interaction, regarding the pharmacodynamic effects the combined treatment was superior to both monotherapies: The amoxicillin efficacy at highest dose was increased by a bacterial reduction of 1.74 log10 CFU/lung after 36 h and survival was increased 1.35-fold to 90.3% after 14 days both compared to amoxicillin alone. The developed pharmacometric pharmacokinetic/pharmacodynamic disease-treatment-survival models provided quantitative insights into a novel treatment option against pneumonia revealing a pharmacokinetic interaction and enhanced activity of amoxicillin and the immune system stimulator MPLA in combination. Further development of this drug combination flanked with pharmacometrics towards the clinical setting seems promising.

The Use of Translational Modelling and Simulation to Develop Immunomodulatory Therapy as an Adjunct to Antibiotic Treatment in the Context of Pneumonia.

The treatment of respiratory tract infections is threatened by the emergence of bacterial resistance. Immunomodulatory drugs, which enhance airway innate immune defenses, may improve therapeutic outcome. In this concept paper, we aim to highlight the utility of pharmacometrics and Bayesian inference in the development of immunomodulatory therapeutic agents as an adjunct to antibiotics in the context of pneumonia. For this, two case studies of translational modelling and simulation frameworks are introduced for these types of drugs up to clinical use. First, we evaluate the pharmacokinetic/pharmacodynamic relationship of an experimental combination of amoxicillin and a TLR4 agonist, monophosphoryl lipid A, by developing a pharmacometric model accounting for interaction and potential translation to humans. Capitalizing on this knowledge and associating clinical trial extrapolation and statistical modelling approaches, we then investigate the TLR5 agonist flagellin. The resulting workflow combines expert and prior knowledge on the compound with the in vitro and in vivo data generated during exploratory studies in order to construct high-dimensional models considering the pharmacokinetics and pharmacodynamics of the compound. This workflow can be used to refine preclinical experiments, estimate the best doses for human studies, and create an adaptive knowledge-based design for the next phases of clinical development.

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection.

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

The Toll-Like Receptor 5 agonist flagellin prevents Non-typeable Haemophilus influenzae-induced infection in cigarette smoke-exposed mice.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. The major bacterial cause of COPD exacerbations is non-typeable Haemophilus influenzae (NTHi). 25 to over 80% of cases are associated with NTHi. This susceptibility to infection involves a defective production of interleukin (IL)-22 which plays an important role in mucosal defense. Prophylactic administration of flagellin, a Toll-like receptor 5 (TLR5) agonist, protects healthy mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbations. Mice chronically exposed to cigarette smoke (CS), which presented COPD symptoms, were infected with NTHi and intraperitoneally treated with recombinant flagellin following a prophylactic or therapeutic protocol. Compared with control, cigarette smoke-exposed mice treated with flagellin showed a lower bacterial load in the airways, the lungs and the blood. This protection was associated with an early neutrophilia, a lower production of pro-inflammatory cytokines and an increased IL-22 production. Flagellin treatment decreased the recruitment of inflammatory cells and the lung damages related to exacerbation. Morover, the protective effect of flagellin against NTHi was altered by treatment with anti-IL-22 blocking antibodies in cigarette smoke-exposed mice and in Il22-/- mice. The effect of flagellin treatment did not implicated the anti-bacterial peptides calgranulins and defensin-ß2. This study shows that stimulation of innate immunity by a TLR5 ligand is a potent antibacterial treatment in CS-exposed mice, suggesting innovative therapeutic strategies against acute exacerbation in COPD.

Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential.

The GM-CSF Released by Airway Epithelial Cells Orchestrates the Mucosal Adjuvant Activity of Flagellin.

The TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin's activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4+ T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4+ T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4+ T cells and thus drives flagellin's adjuvant effect.

Synthetic gene-regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood, and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND gates and IMPLY gates. We demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors. Indeed, we were able to rewire gene expression of the capsule operon, the main pneumococcal virulence factor, to be externally inducible (YES gate) or to act as an IMPLY gate (only expressed in absence of inducer). Importantly, we demonstrate that these synthetic gene-regulatory networks are functional in an influenza A virus superinfection murine model of pneumonia, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.