Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.

Sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP from a rod insert vaginal ring

Administration of biomacromolecular drugs in effective quantities from conventional vaginal rings is hampered by poor drug permeability in the polymers from which rings are commonly constructed. Here, we report the formulation development and testing of rod insert rings for sustained release of the candidate antiretroviral peptides T-1249 and JNJ54310516-AFP (JNJ peptide), both of which have potential as HIV microbicides. Rod inserts were prepared comprising antiviral peptides T-1249 or JNJ peptide in combination with a hydrophilic excipient (sodium chloride, sodium glutamate, lactose or zinc acetate) dispersed at different loadings within a medical grade silicone elastomer. The inserts were tested for weight change and swelling when immersed in simulated vaginal fluid (SVF). Dye migration into the inserts was also assessed visually over 28 days. In vitro release of T-1249 and JNJ peptide from rings containing various insert types was tested. Weight change and degree of swelling of rods immersed in SVF was dependent on the type and concentration of excipient present. The rods displayed the following rank order in terms of weight change: sodium glutamate > zinc acetate ≈ sodium chloride > lactose. The weight change and degree of swelling of the inserts did not correlate with the level of dye uptake observed. In vitro release of T-1249 was improved through addition of lactose, sodium chloride and sodium glutamate, while release of JNJ peptide was improved through addition of sodium chloride or sodium glutamate. Sustained release of hydrophobic peptides can be achieved using a rod insert ring design formulated to include a hydrophilic excipient. Release rates were dependent upon the type of excipient used. The degree of release improvement with different inserts partially reflects their ability to imbibe surrounding fluid and swell in aqueous environments.

Reduced risk of pancreatic cancer associated with asthma and nasal allergies

OBJECTIVE: Studies indicate an inverse association between ductal adenocarcinoma of the pancreas (PDAC) and nasal allergies. However, controversial findings are reported for the association with asthma. Understanding PDAC risk factors will help us to implement appropriate strategies to prevent, treat and diagnose this cancer. This study assessed and characterised the association between PDAC and asthma and corroborated existing reports regarding the association between allergies and PDAC risk.

DESIGN: Information about asthma and allergies was collated from 1297 PDAC cases and 1024 controls included in the PanGenEU case-control study. Associations between PDAC and atopic diseases were studied using multilevel logistic regression analysis. Meta-analyses of association studies on these diseases and PDAC risk were performed applying random-effects model.

RESULTS: Asthma was associated with lower risk of PDAC (OR 0.64, 95% CI 0.47 to 0.88), particularly long-standing asthma (>=17 years, OR 0.39, 95% CI 0.24 to 0.65). Meta-analysis of 10 case-control studies sustained our results (metaOR 0.73, 95% CI 0.59 to 0.89). Nasal allergies and related symptoms were associated with lower risk of PDAC (OR 0.66, 95% CI 0.52 to 0.83 and OR 0.59, 95% CI 0.46 to 0.77, respectively). These results were supported by a meta-analysis of nasal allergy studies (metaOR 0.6, 95% CI 0.5 to 0.72). Skin allergies were not associated with PDAC risk.

CONCLUSIONS: This study shows a consistent inverse association between PDAC and asthma and nasal allergies, supporting the notion that atopic diseases are associated with reduced cancer risk. These results point to the involvement of immune and/or inflammatory factors that may either foster or restrain pancreas carcinogenesis warranting further research to understand the molecular mechanisms driving this association.

Petal-rod - A Novel Method of Lightweighting

Lightweighting the preform to reduce material cost is possibly reaching its limit; key areas of the bottle however may deserve closer attention. The lack of material stretching at the bottle base insists on increased thickness to compensate for the impaired strength in this area. To improve this phenomenon, design of a novel stretch-rod that can mechanically stretch the preform tip during the ISBM process has been developed.

PETAL-ROD is a device that can be retrofitted to existing ISBM machines with minimum interruption to production rate, with the added benefit that material characteristics similar to the sidewall of the bottle can be achieved in the area of the base. Coupled with an optimized preform design, material weight saving can be improved.