Project description:Objectives: Therapies being safe, effective and not vulnerable to develop resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by the use of host-directed drugs to counteract microbial infections. This study sought to identify cellular genes and pathways differentially expressed during airways epithelial infection by nontypable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). Based on the proposed relationship between NTHi intracellular location and persistence, the antimicrobial potential of a panel of drugs perturbing host pathways used by NTHi to enter epithelial cells was investigated. Methods: We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing A549 cell whole genome expression profiling, and identified a panel of host target candidates which were pharmacologically modulated. Interfering drugs were tested for their bactericidal effect, cytotoxicity, effect on the interplay NTHi-epithelial cell, and effect on NTHi respiratory infection in vivo, by assessing lung bacterial loads in a murine intranasal infection model. Results: The sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi; the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts both intracellularly and in the lungs of infected mice. Conclusions: PDE4 inhibition is currently prescribed in COPD; resveratrol is a geroprotector attractive for COPD treatment by preventing lung aging. This work provides evidence for the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection. Haemophilus influenzae clinical strain NTHi375 induced gene expression in A549 human type II pneumocyte cell line was measured at 2 h post-infection. Non infected cells were used as a control. Four independent experiments were performed for each condition.
Project description:Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.
Project description:The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is an important cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) that requires efficient treatments. A previous screening for host genes differentially expressed upon NTHi infection identified sirtuin-1, which encodes a NAD-dependent deacetylase protective against emphysema and is activated by resveratrol. This polyphenol concomitantly reduces NTHi viability, therefore highlighting its therapeutic potential against NTHi infection at the COPD airway. In this study, resveratrol antimicrobial effect on NTHi was shown to be bacteriostatic and did not induce resistance development in vitro. Analysis of modulatory properties on the NTHi-host airway epithelial interplay showed that resveratrol modulates bacterial invasion but not subcellular location, reduces inflammation without targeting phosphodiesterase 4B gene expression, and dampens ? defensin-2 gene expression in infected cells. Moreover, resveratrol therapeutics against NTHi was evaluated in vivo on mouse respiratory and zebrafish septicemia infection model systems, showing to decrease NTHi viability in a dose-dependent manner and reduce airway inflammation upon infection, and to have a significant bacterial clearing effect without signs of host toxicity, respectively. This study presents resveratrol as a therapeutic of particular translational significance due to the attractiveness of targeting both infection and overactive inflammation at the COPD airway.
Project description:Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection in vivo. In vitro, collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and Haemophilus autotransporter protein (Hap). Similar observations were made in vivo specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.
Project description:Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae.
Project description:Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases.
Project description:Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.
Project description:Antimicrobial peptides (AMPs) contribute to host innate immune defense and are a critical component to control bacterial infection. Nontypeable Haemophilus influenzae (NTHI) is a commensal inhabitant of the human nasopharyngeal mucosa, yet is commonly associated with opportunistic infections of the upper and lower respiratory tracts. An important aspect of NTHI virulence is the ability to avert bactericidal effects of host-derived antimicrobial peptides (AMPs). The Sap (sensitivity to antimicrobial peptides) ABC transporter equips NTHI to resist AMPs, although the mechanism of this resistance has remained undefined. We previously determined that the periplasmic binding protein SapA bound AMPs and was required for NTHI virulence in vivo. We now demonstrate, by antibody-mediated neutralization of AMP in vivo, that SapA functions to directly counter AMP lethality during NTHI infection. We hypothesized that SapA would deliver AMPs to the Sap inner membrane complex for transport into the bacterial cytoplasm. We observed that AMPs localize to the bacterial cytoplasm of the parental NTHI strain and were susceptible to cytoplasmic peptidase activity. In striking contrast, AMPs accumulated in the periplasm of bacteria lacking a functional Sap permease complex. These data support a mechanism of Sap mediated import of AMPs, a novel strategy to reduce periplasmic and inner membrane accumulation of these host defense peptides.
Project description:Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.
Project description:The microbiota of the upper respiratory tract (URT) protects the host from bacterial pathogenic colonization by competing for adherence to epithelial cells and by immune response regulation that includes the activation of antimicrobial and (anti-)inflammatory components. However, environmental or host factors can modify the microbiota to an unstable community that predisposes the host to infection or inflammation. One of the URT diseases most often encountered in children is otitis media (OM). The role of pathogenic bacteria like Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the pathogenesis of OM is well documented. Results from next-generation-sequencing (NGS) studies reveal other bacterial taxa involved in OM, such as Turicella and Alloiococcus Such studies can also identify bacterial taxa that are potentially protective against URT infections, whose beneficial action needs to be substantiated in relevant experimental models and clinical trials. Of note, lactic acid bacteria (LAB) are members of the URT microbiota and associated with a URT ecosystem that is deemed healthy, based on NGS and some experimental and clinical studies. These observations have formed the basis of this review, in which we describe the current knowledge of the molecular and clinical potential of LAB in the URT, which is currently underexplored in microbiome and probiotic research.