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Mycobacterium abscessuscauses severe, virtually incurable disease in young patients with cystic fibrosis. Little is known inM. abscessusabout the roles of small regulatory RNAs (sRNA) in gene expression regulation. Here, we show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough colony morphology, increased pro-inflammatory signaling and virulence inin-vivoinfection models, and increased resistance to clinically relevant antibiotics. Examination of clinical isolate cohorts revealed some isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of two mutations found in clinical isolates. Approximate 230 genes were differentially expressed in the B11 deletion mutant. Strains with the clinical B11 mutations showed similar expression trends to the deletion mutant but of a lesser magnitude, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with a model of translational repression via base-pairing of B11 to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences in their RBS, consistent with B11 functioning as a negative regulator through direct binding of target mRNAs. Intriguingly, the genes upregulated in the absence of B11 included components of the ESX-4 secretion system, known to be critical forM. abscessusvirulence. One of these genes had a B11-complementary sequence at its RBS, and fusing the UTR of this gene to a reporter was sufficient to make the reporter suppressible by B11. Taken together, our data show that B11 may act as either a negative or positive regulator with pleiotropic effects on gene expression and clinically important phenotypes inM. abscessus. The presence of hypomorphic B11 mutations in clinical strains supports the idea that lower B11 activity may be advantageous forM. abscessusin some clinical contexts. To our knowledge, this is the first report of the role of an sRNA inM. abscessus.

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of neutrophils, lymphocytes and aggregates of vacuolated foamy cells, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

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.

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.

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.

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.

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.

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 Δ thyA 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.

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.

The pathophysiological mechanisms driving exaggerated inflammation and tissue damage, associated to un-resolved airway infections, in the context of chronic lung diseases remain to be elucidated. In the host-pathogen interplay, two-component defense responses, resistance and tolerance, are increasingly described. Recent reports prompt the hypothesis that chronic lung disease associated to persistent infections, such as Pseudomonas aeruginosa , may be mediated by IL-17 immunity. Thus, we dissected the IL17 pathway during long term chronic infection by P. aerginosa in murine models. When C57Bl/6 mice were infected with P. aeruginosa embedded in agar beads for one month, we found that IL-17A is sustained and secreted by CD4+ T cells at the advanced phases. To directly address the role of IL-17, we infected IL-17a − / −, IL-17ra − / − and congenic controls comparing their host response. 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 markers of tissue damage (pro-MMP-9, elastin degradation, TGF- β 1), cytokines levels (KC, IL-33) and tissue innate infiltrating cells, proving alteration of host tolerance, through tissue damage control. Blockade of IL-17 activity, by an anti-IL-17A mAb starting from ten days post-infection did not affect host resistance in terms of bacterial load and incidence of infection, but increase mainly host tolerance, reducing exaggerated neutrophils infiltration, and levels of KC, pro-MMP-9, TGF- β 1. Overall, the IL-17A cytokine represents a potential host-based intervention to ameliorate lung physiology without compromising host resistance against pathogens during chronic airway infection.

Introduction Cystic Fibrosis (CF) lung disease is characterized by transient airway P. aeruginosa infections and excessive neutrophil-dominated inflammation early in life followed by permanent chronic infection that causes persistent respiratory symptoms and decline in lung functions.As the disease progresses, strain variants can be distinguished from the strain initially acquired.However, the contribution of P. aeruginosa adaptation to lung disease is not completely understood.Comparing sequential strains isolated from a CF patient in multihost pathogenesis system, including non-mammalian and mammalian hosts, our previous works showed that P. aeruginosa early strains were lethal, while late adapted clonal isolates were attenuated in acute virulence but retained their capacity to persist in murine lungs 1,2,3 . Methods To dissect the contribution of P. aeruginosa patho-adaptive strains during the progression of lung disease, we report the host-response in murine models of acute and chronic P. aeruginosa infection.As a model system in studying long term and persistent CF infections, the agar beads mouse model was used in a time course for up to three months in C57Bl/6 mice and Cftrtm1UNCTgN (FABPCFTR) mice.Bacterial count, cytokines profile, assessed by bioplex assay, and lung pathology, assessed by H& E, Masson’s and AB-PAS staining, were evaluated. Results During acute infection, cytokines and chemokines amounts in the whole lungs were lower after infection with late P. aeruginosa patho-adaptive strains compared with early strain, confirming that late strains have evolved the capacity to reduce innate immune system detection.After one month of chronic infection with P. aeruginosa late strains, cytokines/chemokines involved in leukocytes recruitment (IL-1 β , IL-6, KC, MIP-1 α and MIP-2) were lower than after acute infection.Impressively, MCP-1, IL-17A, IFN- γ and TNF- α , correlated to tissue damage control, were maintained during acute and chronic infection, indicating a continuous and elevated triggering of specific pathways.Furthermore, high TGF- β production were found in the whole lung, confirming that late adapted strains may modulate tissue damage control during chronic infection.Preliminary results in CF mice showed similar trends to wt mice.Moreover cytokines mainly correlated with Th17 lymphocytes pathways, like CD40L, IL-17F, IL-23p19, IL-22, MIP-3 α and IL-21, were tested in the lung homogenates by bioplex.Results show that these markers are activated after one month of chronic infection in CF and wt mice, indicating that late adapted strains may modulate tissue damage control through this pathway.Immune cellular phenotype of lymphocytes involved in this chronic model of infection is under investigation.In addition, histopathological examination of lung tissue sections showed that late adapted strains induced mucin-positive goblet cells metaplasia, collagen deposition and apoptosis, typical hallmarks of damage in the airway of CF patients. Conclusion Our findings suggest that during chronic infection, P. aeruginosa patho-adaptive variants attenuate cytokines involved in innate immune system detection, while modulate those mainly linked to tissue damage control, favoring bacterial survival and persistence. Supported by Italian CF Research Foundation (FFC#20_2011)

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.