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Background The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. Objectives In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. Methods Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. Results Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. Conclusions These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Background: Respiratory syncytial virus (RSV) causes exacerbations of asthma and preschool wheeze (PSW). However, the anti-viral and repair responses of the bronchial epithelium in children with severe therapy-resistant asthma (STRA) and PSW are poorly understood. Methods: Children with STRA (age 12 [6-16] years), PSW (age 2 [1-5] years) and non-asthmatic controls (age 7 [2-14] years) underwent bronchoscopy with endobronchial brushings and biopsies. Anti-viral, wound injury responses were quantified in biopsies and primary bronchial epithelial cells (PBECs) in response to RSV, poly(I:C), house dust mite (HDM) or IL-33 using RT-qPCR, Luminex and live cell imaging. Collagen deposition and tissue expression of epithelial growth factor receptor (EGFR), IL-33 and receptor ST2 were investigated in bronchial biopsies. Results: PBECs from STRA and PSW had increased TLR3 gene expression and increased secretion of anti-viral and pro-inflammatory cytokines (IFN-γ, IL-6 and IL-13) in response to RSV compared to controls. Exposure of PBECs to concomitant TLR3 agonist poly(I:C) and HDM resulted in a significant reduction in epithelial cell proliferation in PSW compared to controls. Wound-healing was also impaired in PSW compared to controls at baseline and following IL-33 stimulation. In addition, tissue EGFR expression was significantly reduced in PSW and correlated with collagen deposition in endobronchial biopsies. Conclusions: Despite increased anti-viral responses, preschool children with severe wheeze had impaired airway epithelial proliferative responses following damage. This might be connected to the low expression of EGFR in PSW which may affect epithelial function and contribute to asthma pathogenesis. (Less)

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. We have identified a new mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identified an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular tetrahydrofolate and related compounds. ThfT likely modifies the substrate specificity of an ECF transporter to acquire the end products of folate biosynthesis. As ThfT-mediated resistance is undetectable by routine surveillance methods, our study highlights the need to understand antibiotic susceptibility during infection to reduce inappropriate antibiotic use and treatment failures.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation. Specifically, high-throughput phenotyping of 530 knockout mouse lines identified 140 monogenic “hits” (>25%), most of which had never hitherto been implicated in immunology. Furthermore, they were conspicuously enriched in genes for which humans show poor tolerance to loss-of-function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with one another and with specific physiologic traits. By limiting the freedom of individual immune parameters, such linkages impose genetically regulated “immunological structures”, whose integrity was found to be associated with immunocompetence. Hence, our findings provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.

Asthma exacerbations are associated with human rhinovirus (HRV) infections, and more severe exacerbations are associated with HRV-C. We have previously shown that the HRV-C-specific antibody response is low in healthy adult sera and that most of the antibody to HRV-C is cross-reactive with HRV-A.To compare the antibody response to each HRV species in asthmatic and nonasthmatic children in whom the type of HRV infection was known.Total and specific IgG₁ binding to HRV viral capsid protein antigens of HRV-A, -B, and -C were tested in the plasma from nonasthmatic children (n = 47) and children presenting to the emergency department with asthma exacerbations (n = 96). HRV, found in most of the children at the time of their exacerbation (72%), was analyzed using molecular typing.Asthmatic children had higher antibody responses to HRV. The titers specific to HRV-A, and to a lesser extent HRV-B, were higher than in nonasthmatic controls. The species-specific responses to HRV-C were markedly lower than titers to HRV-A and HRV-B in both asthmatic and nonasthmatic children (P.001). The titers both at presentation and after convalescence were not associated with the HRV genotype detected during the exacerbation.The higher total anti-HRV antibody titers of asthmatic children and their higher anti-HRV-A and -B titers show their development of a heightened antiviral immune response. The low species-specific HRV-C titers found in all groups, even when the virus was found, point to a different and possibly less efficacious immune response to this species.

Background: Human rhinoviruses (HRV) are associated with upper and lower respiratory illnesses, including severe infections causing hospitalization in both children and adults. Although the clinical significance of HRV infections is now well established, no detailed investigation of the immune response against HRV has been performed. The purpose of this study was to assess the IgG1 antibody response to the three known HRV species, HRV-A, -B and -C in healthy subjects. Methods: Recombinant polypeptides of viral capsid protein 1 (VP1) from two genotypes of HRV-A, -B and -C were expressed as glutathione S-transferase (GST) fusion proteins and purified by affinity and then size exclusion chromatography. The presence of secondary structures similar to the natural antigens was verified by circular dichroism analysis. Total and species-specific IgG1 measurements were quantitated by immunoassays and immunoabsorption using sera from 63 healthy adults. Results: Most adult sera reacted with the HRV VP1 antigens, at high titres. As expected, strong cross-reactivity between HRV genotypes of the same species was found. A high degree of cross-reactivity between different HRV species was also evident, particularly between HRV-A and HRV-C. Immunoabsorption studies revealed HRV-C specific titres were markedly and significantly lower than the HRV-A and HRV-B specific titres (P,0.0001). A truncated construct of HRV-C VP1 showed greater specificity in detecting anti-HRV-C antibodies. Conclusions: High titres of IgG1 antibody were bound by the VP1 capsid proteins of HRV-A, -B and -C, but for the majority of people, a large proportion of the antibody to HRV-C was cross-reactive, especially to HRV-A. The improved specificity found for the truncated HRV-C VP1 indicates species-specific and cross-reactive regions could be defined.