Camilla Riva

Cystic Fibrosis-Niche Adaptation of Pseudomonas aeruginosa Reduces Virulence in Multiple Infection Hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

BACKGROUND Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects. METHODS The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models. RESULTS We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation. CONCLUSIONS The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection. GENERAL SIGNIFICANCE Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.

Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections.

Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host. This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease. This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa.

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a−/− or IL-17ra−/− mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-β1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection.

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge

ABSTRACT Trimethoprim-sulfamethoxazole (SXT) is a possible alternative for the treatment of community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) due to the susceptibility of most MRSA strains to the drug. However, after long-term treatment with SXT, thymidine-dependent (TD) SXT-resistant small-colony variants (SCVs) emerge. In TD-SCVs, mutations of thymidylate synthase ([TS] thyA) occur. Until now, it has never been systematically investigated that SXT is triggering the induction and/or selection of TD-SCVs. In our study, we performed induction, reversion, and competition experiments in vitro and in vivo using a chronic mouse pneumonia model to determine the impact of SXT on the emergence of TD-SCVs. SCVs were characterized by light and transmission electron microscopy (TEM) and auxotrophism testing. Short-term exposure of S. aureus to SXT induced the TD-SCV phenotype in S. aureus SH1000, while selection of TD-SCVs with thyA mutations occurred after long-term exposure. In reversion experiments with clinical and laboratory TD-SCVs, all revertants carried compensating mutations at the initially identified mutation site. Competition experiments in vitro and in vivo revealed a survival and growth advantage of the ΔthyA mutant under low-thymidine availability and SXT exposure although this advantage was less profound in vivo. Our results show that SXT induces the TD-SCV phenotype after short-term exposure, while long-term exposure selects for thyA mutations, which provide an advantage for TD-SCVs under specified conditions. Thus, our results further an understanding of the dynamic processes occurring during SXT exposure with induction and selection of S. aureus TD-SCVs.

Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia

ABSTRACT The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa. We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections.

Staphylococcus aureus Impacts Pseudomonas aeruginosa Chronic Respiratory Disease in Murine Models

Background Staphylococcus aureus and Pseudomonas aeruginosa are key bacterial pathogens of the respiratory tract in patients with cystic fibrosis (CF). Although P. aeruginosa chronic bronchial infection is associated with a poorer prognosis, the consequences of S. aureus colonization on CF outcomes are controversial. Methods In this paper, murine models of infection resembling traits of the CF human airways disease have been revisited using an infection schedule that mimics the sequence of events of pulmonary disease in CF patients. First, mice were infected with S. aureus, embedded in agar beads; this was followed by P. aeruginosa infection and analysis of bacterial load, leukocyte infiltration, and lung tissue damage. Results We reveal that (1) S. aureus promotes severe lesions including abscess formation, (2) S. aureus increases the risk of subsequent chronic P. aeruginosa respiratory infection, and (3) once the chronic infection has been established, P. aeruginosa influences most of the inflammatory responses independent of S. aureus. Conclusions Our findings established the significance of S. aureus colonization per se and the impact on the subsequent P. aeruginosa infection. This would point towards a thorough assessment for the need of treatment against S. aureus.

Synthesized Heparan Sulfate Competitors Attenuate Pseudomonas aeruginosa Lung Infection

Several chronic respiratory diseases are characterized by recurrent and/or persistent infections, chronic inflammatory responses and tissue remodeling, including increased levels of glycosaminoglycans which are known structural components of the airways. Among glycosaminoglycans, heparan sulfate (HS) has been suggested to contribute to excessive inflammatory responses. Here, we aim at (i) investigating whether long-term infection by Pseudomonas aeruginosa, one of the most worrisome threat in chronic respiratory diseases, may impact HS levels, and (ii) exploring HS competitors as potential anti-inflammatory drugs during P. aeruginosa pneumonia. P. aeruginosa clinical strains and ad-hoc synthesized HS competitors were used in vitro and in murine models of lung infection. During long-term chronic P. aeruginosa colonization, infected mice showed higher heparin/HS levels, evaluated by high performance liquid chromatography-mass spectrometry after selective enzymatic digestion, compared to uninfected mice. Among HS competitors, an N-acetyl heparin and a glycol-split heparin dampened leukocyte recruitment and cytokine/chemokine production induced by acute and chronic P. aeruginosa pneumonia in mice. Furthermore, treatment with HS competitors reduced bacterial burden during chronic murine lung infection. In vitro, P. aeruginosa biofilm formation decreased upon treatment with HS competitors. Overall, these findings support further evaluation of HS competitors as a novel therapy to counteract inflammation and infection during P. aeruginosa pneumonia.

WS10.5 Pseudomonas aeruginosa adaptation as a potential risk factor to the progression of cystic fibrosis airway disease in mice and humans

Objectives CF is characterized by loss of pulmonary function and tissue injury. As the disease progresses, P. aeruginosa adapts to the host and dramatically modifies its phenotype; however, it is still unclear if and how bacterial adaptation influences pathogenesis and disease development. Methods Initially-acquired and CF-adapted P. aeruginosa clonal variants were challenged in C57Bl/6 and CF ko mice after their inclusion in agar beads. Mice were evaluated for bacterial count, lung histopathology, markers of inflammation and tissue damage. Patho-adaptive traits of P. aeruginosa isolated from CF patients airways were correlated with clinical scores. Results Different to P. aeruginosa initially-acquired strains, CF-adapted variants caused long-term chronic infection in mice by shaping the immune response and activating pathways relevant to tissue damage and remodelling. At an advanced stage of a P. aeruginosa infection, murine airways displayed hallmarks of remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several markers of tissue damage. In mice, these pathologic traits are only partially dependent on the CFTR genetic background. In CF patients, we observed correlation between P. aeruginosa patho-adaptive traits and inflammation, remodelling processes and lung functions suggesting exploitable disease markers. Conclusion We conclude that bacterial adaptation may be a potential risk factor in the progression of airway disease and a critical success determinant for reproducing the human disease in mice. Supported by Italian CF Research Foundation.