Marcella Facchini

Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity

Although antibiotic resistance represents a public health emergency, the pipeline of new antibiotics is running dry. Repurposing of old drugs for new clinical applications is an attractive strategy for drug development. We used the bacterial pathogen Pseudomonas aeruginosa as a target for the screening of antivirulence activity among marketed drugs. We found that the antimycotic agent flucytosine inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major P. aeruginosa virulence factors, namely pyoverdine, PrpL protease, and exotoxin A. Flucytosine administration at clinically meaningful dosing regimens suppressed P. aeruginosa pathogenicity in a mouse model of lung infection. The in vitro and in vivo activity of flucytosine against P. aeruginosa, combined with its desirable pharmacological properties, paves the way for clinical trials on the anti-P. aeruginosa efficacy of flucytosine in humans.

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

ABSTRACT Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities.

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.

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.

Assessing Pseudomonas aeruginosa Virulence and the Host Response Using Murine Models of Acute and Chronic Lung Infection

Murine models of acute and chronic lung infection have been used in studying Pseudomonas aeruginosa for assessing in vivo behavior and for monitoring of the host response. These models provide an important resource for studies of the initiation and maintenance of bacterial infection, identify bacterial genes essential for in vivo maintenance and for the development and testing of new therapies. The rat has been used extensively as a model of chronic lung infection, whereas the mouse has been a model of acute and chronic infection. Intratracheal administration of planktonic bacterial cells in the mouse provides a model of acute pneumonia. Bacteria enmeshed in agar beads can be used in the rat and mouse to reproduce the lung pathology of cystic fibrosis patients with advanced chronic pulmonary disease. Here, we describe the methods to assess virulence of P. aeruginosa using prototype and clinical strains in the Sprague-Dawley rat and the C57BL/6NCrlBR mouse by monitoring several measurable read-outs including weight loss, mortality, in vivo growth curves, the competitive index of infectivity, and the inflammatory response.

Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1

Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host–cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.

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.

Targeting the bacterial cytoskeleton of the Burkholderia cepacia complex for antimicrobial development: A cautionary tale

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H2O2-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1β from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

WS7.1 Pre-clinical evaluation of novel antibiotic POL7001 against Pseudomonas aeruginosa in lung infection models

Objectives: The discovery, development, and clinical exploitation of antibiotics with a new mode of action combined with efficient pulmonary drug delivery systems is a top priority in the battle against untreatable chronic infections in cystic fibrosis (CF) patients. POL7001 is a novel Protein Epitope Mimetic (PEM) antibiotic with potent activity against Pseudomonas aeruginosa (Pa) (Srinivas et al, Science 2010). POL7001 showed a potent in vitro activity against a large panel of Pa CF multi-drug resistant strains. To evaluate CF lung infections as potential clinical application, the therapeutic efficacy of POL7001 in mouse models was investigated. Methods: Both Pa acute and chronic airway infection were established, and mice were treated by subcutaneous (s.c.) or pulmonary administrations (i.t.). Body weight, bacterial count and inflammation in lungs were evaluated at different time points. Comparison to clinically approved antibiotics was included. Results: High antibacterial activity of POL7001, in particular after i.t. administration, was demonstrated. Leukocyte recruitment (in particular neutrophils) in the airways was reduced after POL7001 i.t. administration. Pharmacokinetic studies confirmed that POL7001 reached favorable concentrations in the lung after i.t. administration, with rather low systemic exposure. Conclusion: The efficacy of POL7001 was superior to ciprofloxacin, one of the most effective clinically-approved antibiotics and used as an internal positive control in our pre-clinical studies. Based on these promising results, POL7001 was selected for further pre-clinical profiling.

S104 Targeting the bacterial cytoskeleton of CF pathogens for antimicrobial development–A cautionary tale?

Background Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult due to the inherent multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. We have previously shown that the novel compound Q22, which is related to A22 and inhibits the bacterial cytoskeletal protein MreB, inhibits growth of BCC bacteria. Aims We aimed to further analyse the phenotypic effects of Q22 treatment on BCC virulence traits to assess its feasibility as an antimicrobial. Methods BCC bacteria were grown in the presence of Q22 and a broad phenotypic analysis was performed, including resistance to H2O2 induced oxidative stress, changes in inflammatory potential of cell surface components and in vivo drug toxicity studies. The influence of Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. Compound Q22 was also assessed for toxicity in both zebrafish and mouse infection models. Results BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2 induced oxidative stress with some strains showing increased resistance upon Q22 treatment. An increased response in pro inflammatory activity elected by whole Q22 treated bacterial lysate was observed for cytokines TNFa and IL-1b but this was variable between strains. Further dissection of this response is under investigation. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. Conclusions In the case of BCC bacteria destabilisation of the bacterial cytoskeleton using compounds such as Q22 can lead to unexpected increases of in vitro virulence-related traits. These changes appear to vary depending on strain and species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in vivo environment of CF infection. Work supported by Newcastle-Upon-Tyne Hospitals Special Trustees & Italian CF Research Foundation (FFC).